EPA600/8-91/049cF
August 1993
Air Quality Criteria for
Oxides of Nitrogen
Volume III of
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
Office of Research and Development
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Printed on Recycled Paper
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DISCLAIMER
This document has been reviewed in accordance with U S Environmental Protection
Agency pokey and approved for publication Mention of trade names or commercial
products does not constitute endorsement or recommendation for use
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PREFACE
The U S Environmental Protection Agency (EPA) promulgates the National Ambient
Air Quality Standards (NAAQS) on the basis of scientific information contained in criteria
documents In 1971, the first air quality criteria document for nitrogen oxides (NOX) was
issued by the National Air Pollution Control Administration, a predecessor of EPA On the
basis of scientific information contained in that document, NAAQS were promulgated for
nitrogen dioxide (NO^ at levels of 0.053 ppm (100 jug/m3), averaged over 1 year The last
full-scale NOX criteria document revision was completed by EPA in 1982, leading to an
Agency decision in 1985 to reaffirm the annual average NO2 NAAQS of 0 053 ppm The
present, revised criteria document, Air Quality Criteria for Oxides of Nitrogen, assesses the
current scientific basis for periodic reevaluation of the NO2 NAAQS in accordance with the
provisions identified in Sections 108 and 109 of the Clean Air Act
Key chapters in this document evaluate the latest scientific data on (a) health effects of
NOX measured in laboratory animals and exposed human populations and (b) effects of NOX
on agricultural crops, forests, and ecosystems, as well as (c) NOX effects on visibility and
nonbiological materials Other chapters describe the nature, sources, distribution,
measurement, and concentrations of NOX in the environment These chapters were prepared
and peer reviewed by experts from various state and Federal government offices, academia,
and private industry for use by EPA to support decision making regarding potential risks to
public health and the environment Although the document is not intended to be an
exhaustive literature review, it is intended to cover all the pertinent literature through early
1993
The Environmental Criteria and Assessment Office of EPA's Office of Health and
Environmental Assessment acknowledges with appreciation the contributions provided by the
authors and reviewers and the diligence of its staff and contractors in the preparation of this
document at the request of EPA's Office of Air Quality Planning and Standards
m-iii
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Air Quality Criteria for Oxides of Nitrogen
TABLE OF CONTENTS
Volume I Page
1 EXECUTIVE SUMMARY OF AIR QUALITY CRITERIA FOR
OXIDES OF NITROGEN . 1-1
2 INTRODUCTION 2-1
3 GENERAL CHEMICAL AND PHYSICAL PROPERTIES OF
OXIDES OF NITROGEN AND OXIDES OF NITROGEN-DERIVED
POLLUTANTS . 3-1
4 AMBIENT AND INDOOR SOURCES AND EMISSIONS OF
NITROGEN OXIDES . . 4-1
5 TRANSPORT AND TRANSFORMATION OF NITROGEN
OXIDES . 5-1
6 SAMPLING AND ANALYSIS FOR NITROGEN OXIDES
AND RELATED SPECIES 6-1
7 AMBIENT AND INDOOR CONCENTRATIONS OF NITROGEN
OXIDES 7-1
8 ASSESSING TOTAL HUMAN EXPOSURE TO NITROGEN
DIOXIDE 8-1
Volume n
9 EFFECTS OF NITROGEN OXIDES ON VEGETATION 9-1
10 THE EFFECTS OF NITROGEN OXIDES ON NATURAL
ECOSYSTEMS AND THEIR COMPONENTS 10-1
11 EFFECTS OF NITROGEN OXIDES ON VISIBILITY 11-1
12 EFFECTS OF NITROGEN OXIDES ON MATERIALS 12-1
ffl-v
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Air Quality Criteria for Oxides of Nitrogen
TABLE OF CONTENTS (cont'd)
Volume HI Page
13 STUDIES OF THE EFFECTS OF NITROGEN COMPOUNDS
ON ANIMALS 13-1
14. EPIDEMIOLOGY STUDIES OF OXIDES OF NITROGEN 14-1
15. CONTROLLED HUMAN EXPOSURE STUDIES OF NITROGEN
OXIDES . 15-1
16 HEALTH EFFECTS ASSOCIATED WITH EXPOSURE TO
NITROGEN DIOXIDE . 16-1
APPENDIX A- GLOSSARY OF TERMS AND SYMBOLS A-l
m-vi
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TABLE OF CONTENTS
LIST OF TABLES . . IH-xii
LIST OF FIGURES . . . HI-xvii
AUTHORS . . m-xix
CONTRIBUTORS AND REVIEWERS HI-xxi
CLEAN AIR SCIENTIFIC ADVISORY COMMITTEE IH-xxvii
PROJECT TEAM FOR DEVELOPMENT OF AIR QUALITY CRITERIA
FOR OXIDES OF NITROGEN HI-xxix
13 STUDIES OF THE EFFECTS OF NITROGEN COMPOUNDS
ON ANIMALS . . 13-1
13 1 INTRODUCTION . . 13-1
13 2 NITROGEN DIOXIDE . . . 13-1
13 2 1 Respiratory Tract Transport and Absorption 13-1
1321 I Introduction 13-1
13 2 12 Principles of Gas Uptake and
Dosimetry Models 13-3
13 2 1 3 Dosimetry of Nitrogen Dioxide 13-6
13 2 2 Respiratory Effects 13-13
13 2 2 1 Host Defense Mechanisms 13-13
13 2 2 2 Lung Biochemistry 13-55
13 2 2 3 Pulmonary Function 13-78
13224 Morphologic Studies 13-89
13 2 3 Extrapulmonary Effects 13-124
13 2 3 1 Body Weight 13-125
13 2 3 2 Hematologic Changes 13-129
13 2 3 3 Hepatic Function 13-137
13 23 4 Effects on the Kidney and on Urine
Content 13-142
13 2 3 5 Cardiovascular Effects . 13-144
13 2 3.6 Effects on the Central Nervous System
and Behavioral Effects 13-145
13 2 3 7 Reproductive, Developmental, and
Rentable Mutagemc Effects 13-150
13 2 3 8 Potential Carcinogenic or
Cocarcinogemc Effects 13-152
13 3 EFFECTS OF MDITURES CONTAINING
NITROGEN DIOXIDE 13-163
13 4 NITRIC OXIDE 13-185
13 5 NITRIC ACID AND NITRATES 13-193
13 5 1 Nitnc Acid 13-193
13 5 2 Nitrates 13-194
13 6 SUMMARY . . 13-195
1361 Animal-to-Human Dosimetnc Extrapolation Estimates 13-196
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TABLE OF CONTENTS (cont'd)
13 6 2 Biochemical and Cellular Mechanisms 13-197
13 6 3 Effects on Host Defenses 13-198
13 6 4 Influence of Concentration., Duration, and
Exposure Regimen 13-200
13 6 5 Impact of Exposure Duration 13-202
13 6 6 Effects of Pollutant Mixtures 13-205
REFERENCES . . 13-207
14. EPIDEMIOLOGY STUDIES OF OXIDES OF NITROGEN 14-1
141 INTRODUCTION . 14-1
14 2 METHODOLOGICAL CONSIDERATIONS 14-2
14 2 1 Measurement Error 14-2
14 2 2 Misclassification of Health Outcomes 14-4
14.2 3 Adjustments for Covanates 14-4
14 2 4 Selection Bias 14-5
14 2 5 Internal Consistency 14-5
14.2 6 Plausibility of Effects 14-6
14.3 STUDIES OF RESPIRATORY ILLNESS 14-6
14.3 1 Indoor Studies . 14-10
14 3 1 1 United Kingdom Studies 14-10
14 3 1 2 United States Six Cities Studies 14-18
14 3 1 3 Iowa Study 14-23
14 3 1 4 Dutch Studies 14-24
14 3 1 5 Ohio Study 14-26
14 3 1 6 Tayside Study 14-27
14 3 1 7 Albuquerque Study 14-28
14 3 1 8 Chestnut Ridge Study 14-33
14 3 1 9 Swiss Study 14-34
14 3 1 10 Connecticut Study 14-35
14.3 1 11 Maryland Study 14-37
14 3 1 12 German Study 14-37
14 3 1 13 Canadian Studies 14-38
14 3 1 14 North Carolina Study 14-38
14 3 1 15 United States and Canadian Skating
Rink Exposures 14-40
14 3.2 Outdoor Studies 14-40
14 3 2 1 Six City Studies 14-41
14 3.2 2 Swiss Study 14-42
14323 German Studies 14-43
14 3 2 4 Los Angeles Student Nurses Data . 14-44
14 3 2 5 Chestnut Ridge Study 14-45
14 3 2 6 Finland Studies 14-45
14 3 2 7 California Seventh-Day Adventist Study 14-46
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TABLE OF CONTENTS (cont'd)
14 3 2 8 Chattanooga Studies 14-47
14329 Glendora, California, Study 14-49
14 4 STUDIES OF PULMONARY FUNCTION . 14-49
14 4 1 Indoor Studies . . 14-50
14 4 2 Outdoor Studies . 14-52
14 5 OUTCOMES RESULTING FROM OCCUPATIONAL
EXPOSURES 14-53
14 6 SYNTHESIS OF THE EVIDENCE 14-55
14 6 1 Health Outcome Measures . 14-55
14 6 2 Biologically Plausible Hypothesis 14-63
14 6 3 Publication Bias 14-64
14 6 4 Quantitative Analysis 14-65
14 6 5 Summary of Synthesis of Evidence 14-77
14 7 CONCLUSIONS 14-78
14 8 SUMMARY 14-79
REFERENCES . 14-85
APPENDK 14A 14-A1
15 CONTROLLED HUMAN EXPOSURE STUDIES OF
NITROGEN OXIDES . . 15-1
15 1 INTRODUCTION 15-1
15 2 EFFECTS OF NITROGEN OXIDES IN HEALTHY
SUBJECTS . 15-10
15 2 1 Lung Function Effects of Nitrogen Dioxide 15-10
15 2 1 1 Concentrations Above 1 0 ppm 15-10
15 2 12 Concentrations Below 1 0 ppm 15-23
15 2 1 3 Respiratory Symptom and Sensory Effects
of Nitrogen Dioxide Exposure 15-26
15 2 1 4 Mucociliary Clearance After Nitrogen
Dioxide Exposure . 15-26
15 2 2 Effects of Nitric Oxide 15-26
15 2 3 Effects of Nitrogen Dioxide Gas or Gas/Aerosol
Mixtures on Lung Function in Normal Subjects 15-27
15 2 4 Summary . . 15-35
15 3 THE EFFECTS OF NITROGEN OXIDE EXPOSURE
IN SENSITIVE SUBJECTS . . . 15-36
15 3 1 The Effects of Nitrogen Dioxide on Asthmatics 15-37
15 3 1 1 Effects of Nitac Acid Vapor on
Asthmatics 15-57
15 3 2 Effects of Nitrogen Dioxide on Patients with
Chronic Obstructive Lung Disease 15-58
15 3 3 Summary . 15-62
15 4 EFFECTS OF NITROGEN DIOXIDE EXPOSURE
ON AIRWAY RESPONSIVENESS 15-66
m-ix
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TABLE OF CONTENTS (cont'd)
Page
15 4 1 Healthy Subjects 15-67
15 4 2 Asthmatic Subjects 15-67
15 5 EFFECTS OF NITROGEN DIOXIDE OR NITRIC ACID
EXPOSURE ON BLOOD, URINE, AND
BRONCHOALVEOLAR LAVAGE FLUID BIOCHEMISTRY 15-76
15 5 1 Biochemical Effects in Blood 15-76
15 5 2 Bronchoalveolar Lavage Fluid Biochemistry . 15-78
15 5.3 Urine Biochemistry 15-79
15 6 EFFECTS OF NITROGEN DIOXIDE OR NITRIC ACID
VAPOR EXPOSURE ON HUMAN PULMONARY HOST
DEFENSE RESPONSES 15-79
15.7 EFFECTS OF NITRATES ON HUMAN LUNG FUNCTION 15-85
15 8 CONCLUSIONS AND DISCUSSION 15-88
REFERENCES . 15-94
16. HEALTH EFFECTS ASSOCIATED WITH EXPOSURE
TO NITROGEN DIOXIDE 16-1
161 INTRODUCTION 16-1
16 2 KEY HEALTH EFFECTS OF NITROGEN DIOXIDE 16-1
16.2 1 Airway Responsiveness in Asthmatics and
Short-Term (One- to Three-Hour) Exposure
to Nitrogen Dioxide 16-1
16 2 2 Respiratory Morbidity in Children Associated
with Exposure to Nitrogen Dioxide 16-4
1623 Biological Bases Relating Nitrogen Dioxide Exposure
to Respiratory Morbidity Effects of Nitrogen
Dioxide on the Respiratory Host Defense System 16-5
16 2 4 Emphysema and Exposure to Nitrogen Dioxide 16-9
16.3 CONCENTRATION-RESPONSE RELATIONSHIPS HEALTH
EFFECTS OF EXPOSURE TO NITROGEN DIOXIDE 16-11
1631 Clinical Studies 16-11
16 3.2 Epidemiological Studies 16-12
16 3 3 Animal Toxicological Studies 16-13
16 4 SUBPOPULATIONS POTENTIALLY AT RISK FOR
NITROGEN DIOXIDE HEALTH EFFECTS 16-15
16.5 NITROGEN DIOXIDE LEVELS, EXPOSURES,
AND ESTIMATES 16-19
16 5 1 Ambient and Indoor Nitrogen Dioxide Levels 16-19
16 5 2 Patterns of Potential Exposure to Nitrogen
Dioxide and Related Health Effects 16-22
16 5 2 1 Patterns of Exposure 16-23
16 5 2 2 Long-Term Exposure Estimates 16-24
m-x
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TABLE OF CONTENTS (cont'd)
16 5 3 Nitrogen Dioxide Exposure Estimates 16-28
REFERENCES . 16-32
APPENDIX A GLOSSARY OF TERMS AND SYMBOLS A-l
m-xi
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LIST OF TABLES
Number Page
13-1 Effects of Nitrogen Dioxide on Mucociliary Activity 13-18
13-2 Effects of Nitrogen Dioxide on Alveolar Macrophages 13-24
13-3 Effects of Nitrogen Dioxide on the Immune System 13-35
13-4 Interaction of Nitrogen Dioxide with Infectious Agents 13-39
13-5 Effects of Nitrogen Dioxide on Lipid Metabolism 13-56
13-6 Effects of Nitrogen Dioxide on Lung Amino Acids, Proteins,
and Enzymes 13-63
13-7 Effects of Nitrogen Dioxide on Antioxidant Metabolism and
Influence of Antioxidants 13-72
13-8 Effects of Nitrogen Dioxide on Pulmonary Function 13-79
13-9 Effects of Acute Exposure to Nitrogen Dioxide on
Lung Morphology 13-90
13-10 Effects of Subchronic Exposure to Nitrogen Dioxide on
Lung Morphology 13-91
13-11 Effects of Chronic Exposure to Nitrogen Dioxide on
Lung Morphology 13-95
13-12 Effects of Nitrogen Dioxide on the Development of Emphysema 13-121
13-13 Extrapulmonary Effects of Nitrogen Dioxide Body Weight 13-126
13-14 Effects of Nitrogen Dioxide on Red Blood Cells and
Hemoglobin 13-130
13-15 Effects of Nitrogen Dioxide on Leukocytes and Platelets 13-132
13-16 Effects of Nitrogen Dioxide on Red Blood Cell Membranes 13-135
13-17 Effects of Nitrogen Dioxide on Serum Proteins and
Clinical Chemistries 13-138
13-18 Effects of Nitrogen Dioxide on the Liver 13-139
m-xii
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LIST OF TABLES (cont'd)
Number
13-19 Effects of Nitrogen Dioxide on the Kidney and
on Urine Contents 13-143
13-20 Effects of Nitrogen Dioxide on the Central Nervous
System and Behavior . . 13-146
13-21 Effects of Nitrogen Dioxide on Reproduction, Development,
and Rentable Mutagenesis 13-151
13-22 Effects of Nitrogen Dioxide on Carcinogenesis or
Cocarcinogenesis . 13-153
13-23 Toxicologic Interactions to Simple Mixtures Containing
Nitrogen Dioxide 13-164
13-24 Effect of Nitnc Oxide on Respiratory Tract Morphology 13-187
14-1 Respiratory Symptom Rates of United Kingdom Children by
Gender, Social Class, and Cooking Type . 14-12
14-2 Hasselblad et al (1992) Multiple Logistic Analysis of Data
from the Meha et al (1977) Study . 14-12
14-3 Unadjusted Rates of One or More Respiratory Symptoms Among
United Kingdom Children by Gender, Social Class, and
Cooking Type 14-14
14-4 Hasselblad et al (1992) Multiple Logistic Analysis of Data
from Meha et al (1979) Study 14-14
14-5 Unadjusted Rates of One or More Respiratory Symptoms
Among United Kingdom Boys and Girls by Bedroom Levels of
Nitrogen Dioxide . 14-15
14-6 Unadjusted Rates of One or More Respiratory Symptoms Among
United Kingdom Boys and Girls by Bedroom Levels of
Nitrogen Dioxide 14-17
14-7 Nitrogen Dioxide Concentrations by Season and Stove
Type in Portage, Wisconsin . . 14-19
14-8 Odds Ratios and 95% Confidence Intervals for the Effect of
an Additional 0 015 ppm Nitrogen Dioxide on the Symptom
Prevalence 14-21
m-xiii
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Number
14-9
14-10
14-11
14-12
14-13
14-14
14-15
14-16
14-17
14-18
14-19
14-20
14-21
LIST OF TABLES (cont'd)
Odds Ratios and 95 % Confidence Intervals for the Effect of
Ordered Nitrogen Dioxide Exposures on the Prevalence of
Lower Respiratory Symptoms
Odds Ratios and 95 % Confidence Intervals for the Effect of an
Additional 0 015 ppm Nitrogen Dioxide on the Prevalence of Lower
Respiratory Symptoms by Sampling Location and Season
Analysis of Iowa City School Children Respiratory Symptoms by
Gas Stove Type and Parental Smoking
Dutch Study Estimated and Measured Personal Nitrogen Dioxide
Exposure for a Single Weekly Average
Frequency and Prevalence of Reported Respiratory Symptoms for
Different Categories of Mean Indoor Nitrogen Dioxide
Concentrations in a Population of 775 Dutch Children
6 to 12 Years Old
Regression Coefficients for Multiple Logistic Analyses of
Respiratory Illness in Tayside Children
Odds Ratios for Effect of Nitrogen Dioxide Exposure on
Incidence of Respiratory Illness
Effects of Outdoor Nitrogen Dioxide Exposure on
Respiratory Disease
Adjusted Annual Respiratory Symptom Duration and
Nitrogen Dioxide Levels by Region
Health Outcome and Nitrogen Dioxide Exposure Measures Used
in Selected Indoor Nitrogen Dioxide Epidemiology Studies
Summary of Odds Ratios from Indoor Studies of the Effects of
Nitrogen Dioxide Increased by 0 015 ppm
United States Environmental Protection Agency Combined
Analyses of Indoor Studies on Respiratory Illness Effects
of Nitrogen Dioxide Increased by 0 015 ppm
Covanate Treatment and Other Factors in Selected Nitrogen
Dioxide Epidemiology Studies in Meta-Analysis
14-22
14-23
14-24
14-25
14-26
14-28
14-32
14-41
14-43
14-57
14-71
14-72
14-74
m-xiv
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LIST OF TABLES (cont'd)
Number
14-22 Summary of Odds Ratios of the Effects of Nitrogen Dioxide,
Health Outcome, and Exposure Estimates foi Infant
Epidemiology Studies 14-76
15-1 Responses of Healthy Subjects to Nitrogen Dioxide Exposure 15-11
15-2 Exposure of Healthy Subjects to Nitrogen Dioxide Mixtures 15-29
15-3 Classification of Asthma by Seventy of Disease . 15-38
15-4 Characteristics of Asthmatic Subjects Exposed to Nitrogen
Dioxide 15-40
15-5 Exposure Conditions and Responses in Asthmatics Exposed to
Nitrogen Dioxide 15-43
15-6 Subject Characteristics for Patients with Chronic Obstructive
Pulmonary Disease Exposed to Nitrogen Dioxide 15-59
15-7 Exposure Conditions and Responses in Chronic Obstructive
Pulmonary Disease Patients Exposed to Nitrogen Dioxide 15-60
15-8 Airway Resistance and Forced Expiratory Volume in One Second
Changes in Asthmatics Exposed to Nitrogen Dioxide 15-63
15-9 Changes in Airway Responsiveness Associated with Nitrogen
Dioxide Exposure . 15-68
15-10 Fraction of Nitrogen Dioxide-Exposed Subjects with Increased
Airway Responsiveness . 15-75
15-11 Exposure Conditions and Responses in Subjects Exposed to
Nitrates . . 15-86
16-1 Key Human Health Effects of Exposure to Nitrogen
Dioxide—Clinical Studies 16-12
16-2 Key Human Health Effects of Exposure to Nitrogen
Dioxide—Epidemiological Studies 16-14
16-3 Key Animal Toxicological Effects of Exposure to Nitrogen
Dioxide . 16-16
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LIST OF TABLES (cont'd)
Number
16-4 Estimates of the Resident Population of Children and Young
Adults of the United States, by Age and Sex, July 1, 1989 16-18
16-5 United States Environmental Protection Agency Analysis of
Variability in Two-Week Ambient Averages of One-Hour
Nitrogen Dioxide Data at 10 Selected Locations 16-26
16-6 Average Annual Nitrogen Dioxide Means and Standard Errors
of Estimates for 40 Sites in the United States by Annual
Average as Derived by the United States Environmental
Protection Agency from the Aerometac Information Retrieval
System (1991) . 16-27
16-7 Average Annual Nitrogen Dioxide Means and Standard
Errors of Estimates for 100 Homes Based on Data
of Lambert (1991) as Derived by the U S Environmental
Protection Agency 16-27
16-8 Nitrogen Dioxide Exposure Estimates in Parts per Million
Derived by the United States Environmental Protection Agency as
Function of Outdoor Nitrogen Dioxide Concentration and Percent
Time Outdoors, Where Indoor/Outdoor Ratio Equals 0 59 and
Assuming a Baseline Concentration of 0 005 ppm 16-29
16-9 Nitrogen Dioxide Exposure Estimates in Parts per Million
Derived by the United States Environmental Protection Agency at
Selected Outdoor Nitrogen Dioxide Concentrations, Indoor/Outdoor
Concentration Ratios, and Percentages of Tune Spent Outdoors,
Compared with a Nitrogen Dioxide Exposure of 0 005 ppm 16-31
m-xvi
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LIST OF FIGURES
Number Page
13-1 Mortality enhancement for mice exposed to nitrogen dioxide
at various concentrations and for various durations prior
to challenge with streptococci 13-48
14-1 Distribution of tune at risk by bedroom nitrogen dioxide
concentration . . 14-31
14-2 Nitrogen dioxide ambient and indoor concentrations in four
Swiss regions with 95 % confidence range 14-34
14-3 Total personal exposure to nitrogen dioxide versus nitrogen
dioxide levels in Connecticut residences 14-36
14-4 For the Melia et al (1979) study, a graph of the marginal
likelihood function of the odds ratios for combined gender
(boys and girls) of the respiratory illness outcome measures
developed by the United States Environmental Protection
Agency 14-60
14-5 United States Environmental Protection Agency meta-analysis of
indoor epidemiologic studies of nitrogen dioxide exposure effects
on respiratory disease in children 5 to 12 years old 14-70
15-1 Percent change (post-air versus post-nitrogen dioxide) in
forced expiratory volume in one second versus nitrogen dioxide
dose in parts per million tunes liters in asthmatics . 15-65
15-2 Percent change ([post-nitrogen dioxide - post-air]/post-air)
in resistance versus nitrogen dioxide dose in parts per
million tunes liters in asthmatics 15-65
ffi-xvu
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AUTHORS
Chapter 13 Studies of the Effects of Nitrogen Compounds on Animals
Ms Beverly Comfort
Environmental Criteria and Assessment Office
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Donald Gardner
ManTech Environmental Technology, Inc
P O Box 12313
Research Triangle Park, NC 27709
Dr Judith A Graham
Environmental Criteria and Assessment Office
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Jerry last
California Primate Research Center
University of California
Davis, CA 95616
Dr Susan Loscutoff
2594 W Ellery Street
Fresno, CA 93711
Dr John Overton
Health Effects Research laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Richard Schlesinger
New Yoik University
IxMig Meadow Road
Tuxedo, NY 10987
Dr Jeffrey L Tepper
ManTech Environmental Technology, Inc
PO Box 12313
Research Triangle Park, NC 27709
Dr Walters Tyler
V M Anatomy
University of California
Davis, CA 95616
Chapter 14. Epidemiology Studies of Oxides of Nitrogen
Dr Victor Hasselblad
Center for Health Policy Research
Duke University
Durham, NC 27713
Dr Dennis J Kotchmar
Environmental Criteria and Assessment Office
Office of Health and Environmental
Assessment
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Chapter 15 Controlled Human Exposure Studies of Nitrogen Oxides
Dr Lawrence J Folinsbee
Health Effects Research laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
m-xrx
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AUTHORS (cont'd)
Chapter 16 Health Effects Associated with Exposure to Nitrogen Dioxide
Dr. Donald Gardner
ManTech Environmental Technology, Inc
P.O Box 12313
Research Triangle Park, NC 27709
Dr. Victor Hasselblad
Center for Health Policy Research
Duke University
Durham, NC 27713
Dr. Dennis J Kotchmar
Environmental Criteria and Assessment Office
Office of Health and Environmental
Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Lawrence J Folinsbee
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Judith A Graham
Environmental Criteria and Assessment Office
U S Environmental Protection Agency
Research Triangle Park, NC 27711
m-xx
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CONTRIBUTORS AND REVIEWERS
Chapters 13-15
Dr Ursula Ackermann-Liebnch
Department of Social and Preventative
Medicine
University of Basel
St Albanvorstadt 19
4052 Basel
Switzerland
41-61-21-60-67
Dr Ester Azoulay-Dupms
INSERM Unit 13-Hospital Claude Bernard
10 Avenue de la Porte
D'Aubervilleis
75019 Pans
France
Dr Michael A Berry
Environmental Criteria and Assessment Office
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr IrwinH Billick
Gas Research Institute
8600 West Bryn Mawr Avenue
Chicago, IL 60631
Dr Gary R Burleson
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Robert Chapman
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Mr David Coffin
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Albert Collier
535 Clinical Sciences Bldg C #7220
University of North Carolina
Chapel Hill, NC 27599-7220
>
Dr Steven D Colome
Integrated Environmental Services
University Towers, Suite 1090
4199 Campus Drive
Irvine, CA 92715
Ms Beverly Comfort
Environmental Criteria and Assessment Office
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr James Crapo
Division of Allegy, Critical Care and
Respiratory Medicine
Box 3177
Duke University Medical Center
Durham, NC 27710
Dr Douglas Dockery
Harvard School of Public Health
Department of Environmental Science and
Physiology
665 Huntington Avenue
Boston, MA 02115
Di D L Dungworth
Department of Veterinary Pathology
University of California
Davis, CA 95616
Di Lawrence J. Folinsbee
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
m-xxi
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CONTRIBUTORS AND REVIEWERS (cont'd)
Dr. Donald Gardner
ManTech Environmental Technology, Inc
P.O. Box 12313
Research Triangle Park, NC 27709
Dr Judith A Graham
Environmental Criteria and Assessment Office
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Lester D Grant
Environmental Criteria and Assessment Office
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Elaine Grose
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Jack Hackney
Environmental Health Services-Room 51
Rancho Los Amigos Hospital
7601 Imperial Highway
Downey, CA 90242
Dr. Victor Hasselblad
Center for Health Policy Research
Duke University
Durham, NC 27713
Dr. Gary Hatch
Health Effects Research Laboratory
U.S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Carl Hayes
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Milan Hazucha
Center for Environmental Medicine and
Lung Biology
CB #7310 Trailer #4 Medical Research
Building C
University of North Carolina
Chapel Hill, NC 27599
Dr Donald Horstman
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Ms Pamela Johnson
Office of Air Quality Planning and Standards
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Howard Kehrl
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr HillelKoren
Health Effects Research Laboratory
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr Dennis J Kotchmar
Environmental Criteria and Assessment Office
U S Environmental Protection Agency
Research Triangle Park, NC 27711
Dr PaulKotin
4505 South Yosemite #339
Denver, CO 80237
Dr Jerry Last
California Primate Research Center
University of California
Davis, CA 95616
HI-xxii
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CONTRIBUTORS AND REVIEWERS (cont'd)
Dr Brian Leaderer
Pierce Foundation Laboratory
Yale University School of Medicine
290 Congress Avenue
New Haven, CT 06519
Dr Thomas Lindvall
Institute of Environmental Medicine
Doktorsnngen 18
Tomtebodavagen 30
The Karolinska Institute
S-104 01 Stockholm, Sweden
Dr Susan Loscutoff
2594 W. Ellery Street
Fresno, CA 93711
Dr Joe L Mauderly
Lovelace Inhalation Toxicology Research Institute
PO Box 5890
Albuquerque, NM 87185
Dr Daniel Menzel
Department of Community and Environmental
Medicine
University of California
Irvine, CA 92717
Dr Fred Miller
Box 3177
Duke University
Department of Medicine
Durham, NC 27710
Dr Paul E Morrow
University of Rochester Medical Center
School of Medicine and Dentistry
Box EHSC
Rochester, NY 14642
Mr Lucas Neas
Harvard School of Public Health
6555 Huntington Avenue
Boston, MA 02115
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Chapter 16
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13. STUDIES OF THE EFFECTS OF NITROGEN
COMPOUNDS ON ANIMALS
13.1 INTRODUCTION
This chapter discusses the effects of the oxides of nitrogen (NOX) in experimental
animals Previous reviews of the literature have appeared in a criteria document (U S
Environmental Protection Agency, 1982) A World Health Organization summary has also
been published (World Health Organization, 1977)
Most of the data presented in this chapter relate to the effects of nitrogen dioxide (NO^)
on experimental animals because the vast majority of the NOX literature is on NO2 The
results of the few comparative NOX studies show that NO2 appears to be the most toxic of
the NOX species, but definitive conclusions on relative quantitative potency must await new
information on major NOX species The majority of the literature describes the effects of
NO2 on the respiratory tract, however, extrapulmonary effects also have been observed and
are included here A broad range of NO2 concentrations have been evaluated, but only
studies conducted at less than 18,800 jug/m (10 ppm) NO2 are discussed, with emphasis on
those studies at exposure concentrations of 9,400 jwg/m (5 0 ppm) or less, with the exception
of studies on dosimetry and emphysema
Discussions of the available literature on the effects of other NOX compounds and
mixtures containing NO2 are also included in this chapter These sections are short because
of the general lack of information in these areas
13.2 NITROGEN DIOXIDE
13.2.1 Respiratory Tract Transport and Absorption
13.2.1.1 Introduction
*
Dosimetry refers to measurement or estimation of the quantity of a chemical absorbed
by target sites such as the pulmonary region tissue or, more locally, the tissue of the.
centnacmar region Dosimetry allows exposure-response data into be transformed to dose-
response relationships However, to quantitatively extrapolate animal data to humans,
13-1
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knowledge of dosimetry and species sensitivity must both be considered Even when two
species receive an identical local tissue/cellular dose, cellular sensitivity and repair/defense
mechanisms determine the extent and type of injury produced These mechanisms are likely
to vary among humans and animals because of dissimilarities in pharmacokinetics, genetic
makeup, metabolic rates, detoxification systems, and/or other factors At present, knowledge
of dosimetry is more advanced than knowledge of species sensitivity, inhibiting quantitative
animal-to-human extrapolation of effective NO2 concentrations Nevertheless, knowledge of
interspecies differences and similarities in dosimetry alone is crucial to the process of risk
assessment, as will be discussed
The compound most directly responsible for toxic effects may be the inhaled gas, here
NO2, or chemical reaction products or metabolites Complete identification of the actual
toxic agents and their integration into dosimetry is a complex issue that has not been fully
resolved Thus, most dosimetry investigations, which are difficult enough, are concerned
with the dose of the primary inhaled chemical In the context of inter- or intraspecies
dosimetric extrapolation, a further confounding factor can be the units of dose (e g , mass
retained per breath, mass retained per breath per body weight, mass retained per breath per
respiratory tract surface area) That is, when comparing dose between species, what is the
relevant measure of dose*? This question, like the previous issue, has not been answered,
units are often dictated by the type of experiment and/or by the choice of the investigators
Theoretical (modeling) and experimental studies are used to obtain information on dose
Experiments have been conducted to obtain direct measurements of absorbed NO2 in the total
respiratory tract, the upper respiratory tract (region proximal to the trachea! entrance), and in
the lower respiratory tract (region distal to tracheal entrance) However, obtaining
experimental absorption data for smaller regions or locations, such as specific airways or in
the centnacinar region where lesions due to NO2 occur (see Section 13 2 2 4), is extremely
difficult, and may not be possible in the near future because of technical limitations
Nevertheless, experimentation is important for determining dose, assessing hypotheses and
concepts, and validating mathematical models that may be of use in predicting dose to
specific sites
Theoretical studies are based on the use of mathematical models developed for the
purposes of simulating the uptake and distribution of gases that are absorbed in the tissues
13-2
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and fluids of the respiratory tract, usually at the generational level Because the factors
affecting the transport and absorption of gases are general to all mammals, a model that uses
appropnate species and/or disease-specific anatomical and ventilatory parameters can be used
to describe absorption by different-sized, aged, or diseased members of the species Models
may also be used to identify areas needing additional research, to make niter- and
intraspecies dose comparisons, to compare and reconcile data from different experiments, to
predict dose in conditions not possible or feasible experimentally, to better understand the
processes involved in toxic effects, and to design experiments
The amount of NO2 acting at a given site is dependent on the nearby airway luminal or
airspace concentration and the transport of the gas to the site In the movement of the gas
from the airway lumen or airspace to the cellular components of the respiratory tract, NO2
first comes in contact with the liquid or fluid that hues the cells or tissues of the respiratory
tract (mainly consisting of mucus and penciliary fluid in the upper respiratory tract and
tracheobronchial region and of surfactant film and serous fluid in the pulmonary region)
Nitrogen dioxide reacts chemically with the constituents of fluids and tissues The reactions
with the chemical components of the liquid lining are likely to reduce the quantity of the
inhaled gas reaching the tissue However, reactions in the lining may also produce products
that, in turn, may increase toxicity above that produced by the direct action of NO2 alone
13.2.1.2 Principles of Gas Uptake and Dosimetry Models
To further our understanding of the absorption of gases, mathematical models have
been developed to simulate the processes involved and to predict absorption by various
regions and sites within the respiratory tract
Animal species-specific information that characterizes respiratory tract morphology and
anatomy and transport processes, including chemical reactions, is needed for mathematical
modeling Anatomical information needed includes data about the physical dimensions and
geometry of the structural elements of the respiratory tract (e g , airways, alveoli), the liquid
linings, the underlying tissues, and the capilliary blood system In the air phase or air
compartment (airway lumens and airspaces), the processes of convection, molecular
diffusion, turbulence, dispersion, and the loss or gam of gaseous species to and from the
respiratory tract walls must be taken into account Factors to be considered in lung fluids
13-3
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and tissues include the biochemical constituents, chemical reactions, solubility, molecular
diffusion, convection due to mucociliary action, and capillary blood flow A detailed
discussion of these factors can be found in Overton (1984) and Ultman (1988)
Although variations in physiological processes and structures occur between species,
laboratory animals and humans are very similar, enabling dosimetry models to be developed
based on the valid concept of a general mammalian respiratory tract structure and
physiological processes that apply to both humans and laboratory animals As a result, only
one dosimetry model is needed for the simulation of uptake in several species or subjects
However, species- or subject-dependent structural and physiological data in a form useable
v
by such a model is required to simulate the absorption of NO2 by a specific animal or
human
There is only one reported theoretical investigation of NO2 dosimetry, it was discussed
originally in Miller et al (1982) and later in Overton (1984) The dosimetry model used for
this investigation was one developed for modeling of ozone (O3) uptake, however, because
NC>2, like 03, is highly reactive in the respiratory tract tissues and fluids and is not very
soluble, this model is considered to be valid for NO2 (Miller et al, 1982) Because there
are very few NO2 dosimetry modeling results and the principles of O3 uptake are general and
apply to NO2, the following is a discussion of the general aspects and the factors that have
been considered for dosimetry models of O3
For dosimetry models, lung dimensions and form usually are accounted for by using
airway or anatomical models based on upper respiratory tract, tracheobronchial, and
pulmonary region data The upper respiratory tract can be modeled as a series of segments
along the path through the upper respiratory tract airways, segment dimensions (length,
diameter, surface areas, etc ) are often based on cast data (e g , Schreider and Raabe [1981]
for the rat and Patra et al [1986] for a human child) In the lower respiratory tract, the
complex and numerous branching airways are represented by a sequence of sets of right
circular cylinders (e g , Weibel [1963] for humans and Yeh et al [1979] for rats) Each set
corresponds to a generation and each cylinder represents an airway or alveolar duct This
type of lower respiratory tract model simplifies the development of dosimetry models in that
all paths from the trachea to the last generation airway or duct are assumed identical Thus,
13-4
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a descnption of transport and absorption in one airway in a given generation is the same as
any other airway in that generation (Overton, 1984)
Although gas transport in the airways is a three-dimensional problem, the use of the
stylized anatomical models have lead to the development of methods that allow the
descnption of transport in terms of a one-dimensional, time-dependent partial differential
equation (Overton, 1984) The change with respect to tune of the average cross-sectional
concentration of the inhaled gas in an airway is related to axial dispersion, convection, and
wall loses due to physical absorption and chemical reactions Transfer to the liquid lining is
often described in terms of mass transfer coefficients and the liquid-phase and gas-phase
interfacial concentrations, related by Henry's law constant In respiratory tract fluids and
tissues, only chemical reactions and transport perpendicular to the interfaces between the air,
liquid, and tissue compartments have been taken into account Chemical reactions in the
tissue and fluids have been modeled in several ways, depending on assumptions about the
type and nature of the reactions that effectively describe the reaction processes Reactions
have been modeled as instantaneous or first-order and time-dependent or steady state,
depending on the investigators and the state of understanding Dosimetry models also
account for ventilation, which plays an important role in delivered dose, some models
include the effects of expansion and contraction to account for the fact that dimensions vary
during the breathing cycle (Overton et al , 1987)
Postlethwait and Bidani (1990) exposed ventilated, isolated rat lungs to 18,800 to
118,000 /tg/m (10 to 63 ppm) NO2 for up to 1 h and found evidence for both linear and
nonlinear chemical reaction processes NO2 uptake (mass of NO2 retained) was proportional
to inhalation rates from 2 to 14 /*g NO2/min, but saturated with greater NO2 inhalation rates
For the lower rates, reactions can be modeled as first order kinetics Evidence for linear
reactions was also observed in an in vitro experiment in which Postlethwait et al (1990)
•2
exposed rat bronchoalveolar lavage fluid to NO2 (< 19,700 /*g/m , 10 5 ppm) How this
relates to in vivo conditions is not clear The authors suggested that the renewal of
biochemical compounds plays a role in NO2 uptake and that it is the rate of this renewal that
is limiting the uptake A further understanding of the nonlineanty aspects is needed to judge
if linearity is a reasonable assumption for environmental conditions or if nonlineanty must be
taken into account (e g , renewal)
13-5
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In another in vitro experiment by Postlethwait and coworkers (Postlethwait et al,
1991), rat bronchoalveolar lavage fluid was used to explore whether or not unreacted NO2
can penetrate the epithelial liquid lining to underlying sites The investigators concluded that
3
inhaled NO2 (^18,800 /*g/m , 10 ppm) does not penetrate the epithelial liquid lining to
underlying sites In addition to implying a very high chemical reactivity in this layer, the
investigators suggested that cytotoxicity is the result of chemical reaction products formed in
the liquid lining
In light of the work of Postlethwait and coworkers, it is of interest to note that for the
theoretical NO2 dosimetry investigation of Miller et al (1982), NO2 chemical reactions were
modeled as instantaneous and only as occurring in the liquid lining Further, a renewal
process was assumed On the other hand, the tissue was assumed to be very reactive and
NO2 was predicted to reach and react with the tissue throughout most of the lower
respiratory tract Uptake saturation would not have been predicted by the model and, in
contrast to Postlethwait and Bidam (1990) and Postlethwait et al (1990), the model predicted
nonlinear uptakes at low NO2 concentrations and linear uptakes at high NO2 concentrations
(Miller et al, 1978)
The word "uptake" is often used in conjunction with gas dosimetry Generally, the
meaning of this word depends on context and should be defined to reduce ambiguity, if the
meaning is not obvious Uptake can denote a measure of the quantity of gas absorbed (e g ,
100 g) in a region or at a specific site Unless otherwise stated, the terms "fractional
uptake" and "percent uptake" refer, respectively, to the fraction or the percent of the inhaled
NO2 retained by the specified respiratory tract region
13.2.1.3 Dosimetry of Nitrogen Dioxide
Based on the findings of NO2-induced lesions in the respiratory tract of experimental
animals (Section 13 2 2 4), it appears that NO2 is absorbed along the entire respiratory tract
Upper Respiratory Tract Absorption
The upper respiratory tract uptake of NO2 has been experimentally estimated in dogs,
rats, and rabbits Using unidirectional flow, Yokoyama (1968) measured NO2 uptake in the
isolated upper airways of dogs and rabbits exposed to 7,520 to 77,100 /*g/m3 (4 to 41 ppm)
13-6
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The upper respiratory tract of the two species was observed to remove 42 1 % (standard
deviation = 14 9%) of the NO2 drawn through the noses The authors did not discuss the
relative humidity of the air If it were not sufficiently high, the continuous airflow would
dehydrate the mucous membrane, possibly affecting the uptake properties of the upper
respiratory tract Cavanagh and Morns (1987) exposed the isolated upper respiratory tract of
naive and previously exposed rats (76,000 Mg/m3, 40 4 ppm NO2) under unidirectional flow
and found the uptakes to be 28 and 25%, respectively The relative humidity of the
"inhaled" air was maintained so as to be equivalent to 92% at 37° C The reported uptake
difference between the naive and previously exposed rats may not be significant because a
multivanable analysis of variance, and not an analysis of variance, test should have been
used to analyze the data
Kleinman and Mautz (1991) exposed six tracheostomized dogs to 1,880 or 9,400 /jg/m3
(1 0 or 5 0 ppm) NO2 and measured uptake during inhalation Ventilation rates were varied
from approximately 2 to 14 L/min by adding carbon dioxide (COj) to the inspired air
During mouth breathing, fractional uptake was not dependent on concentration and the mean
fractional uptake decreased from 65% to 30% as the ventilation rate increased for 2 to
8 L/min However, for nasal breathing, exposure to 1,880 /xg/m3 NO2 resulted in
-------
experimentally determined K for NO2 is over 240 times as large as those used for modeling
ozone uptake Further work is needed to determine mass transfer coefficients of NO2
Postlethwait and Mustafa (1981) measured the uptake of NO2 by an isolated ventilated
perfused rat lung The isolated lungs were exposed for 90 nun to 9,400 ji*g/m (5 0 ppm)
NO2 at a ventilation rate of 45 mL/min Thirty-six percent of the ventilated NO2 was
retained In a later similar experiment with the exposure concentration ranging from
•5
7,520 to 37,600 /tg/m (4 to 20 ppm) and the minute volume ranging from 45 to
130 mL/min for different groups of lungs, Postlethwait and Mustafa (1989) found that the
quantity of NO2 retained was related linearly to the inhaled quantity of NO2, the percent
uptake ranged from 60 to 72 % with an average of 65 % These results differ considerably
from the first experiment and no reasons for the differences in results were given Although
the tidal volumes in these experiments are realistic, the breathing frequencies are generally
much lower than for rats breathing normally Similar experiments should be performed
using more realistic ventdatory parameters
In addition to measuring upper respiratory tract uptakes in dogs, Klernman and Mautz
(1991) also measured lower respiratory tract uptake They found that the fractional uptake of
NO2 by dog lungs ventilated through a tracheostomy was about 90% of the NO2 inspired by
the lungs This fractional uptake was basically independent of ventilation rates from 3 to
16 L/min, but was somewhat higher at lower ventilation rates From 1 to 4 L/min, some of
their fractional uptakes were greater than 100%, which is not possible The investigators
hypothesized that the greater than 100% values were related to an experimental error in the
dead space correction equation used for estimation of fractional uptake How this type of
error effects other measurements is not clear, but the effect of instrument dead space on
estimates of fractional uptake should decrease as tidal volume increases
Results from simulations of NO2 uptake in the lower respiratory tract were described
by Miller et al (1982) and Overton (1984) The model used for this investigation was the
same as the dosrmetry model described in Miller et al (1978) for O3, but with the diffusion
coefficient and Henry's law constant appropnate to NO2, however, values of the latter
constant and the chemical reactions were considered uncertain The investigation was mainly
a sensitivity study of the effects of Henry's law constant and reaction rates for which the
upper limit of the latter was assumed to be the reaction rate of O3 For humans, the results
13-8
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indicate that NO2 is absorbed throughout the lower respiratory tract, but the major dose to
tissue would be delivered in the centriacinar region, thai is, the junction between the
conducting and respiratory airways Simulations also predicted that peak tissue levels would
occur in this same anatomical region of the rat, guinea pig, and rabbit These findings are
consistent with the site of morphological effects (Section 13224) Beyond this region there
is a rapid fall in the NO2 dose delivered to tissue Depending on the tracheal concentration
and tidal volume, the model predicted that 75 to 95 % oi the NO2 entering the trachea could
be retained in the lower respiratory tract tissues and fluids However, these predictions are
dependent on the investigator's choices of values for the uncertain parameters The results
also predict that exercise will increase the amount of NO2 delivered to and absorbed in the
pulmonary region over that at rest, and will reduce percent uptake in the tracheobronchial
region (Miller et al., 1982, Overton, 1984)
Total Respiratory Tract Absorption
Total respiratory tract uptake has been measured in healthy and diseased humans
Healthy humans were exposed by Wagner (1970) to a nitric oxide (NO)/NO2 mixture
containing 550 to 13,500 /ig/m (0 29 to 7 2 ppm) NO2 for brief (but unspecified) periods
Of the inhaled NO2, 81 to 90% was absorbed during normal respiration, this increased to
91 to 92% with maximal ventilation Bauer et al (1986) exposed adult asthmatics to
o
560 /wg/m (0 3 ppm) NO2 for 30 nun The exposed subjects inhaled NO2 by mouthpiece for
20 mm at rest, then exercised for 10 min on a bicycle eigometer (30 L/min). The inspired
and expired NO2 concentrations were measured, showing that the average uptake was 72 % at
rest, whereas the average uptake was 87% during exercise, a statistically significant increase
The effects of NO2 exposure on pulmonary function in humans following this exposure
regime are reported in Chapter 15 of this document
Russell et al (1991) exposed rats to NO2 labeled with oxygen-15 ([^OJ-NO^ and
determined the distribution of this compound in the upper and lower respiratory tract They
found that the combined nasopharynx and larynx contained 94% of the radiolabeled NO2
retained by the respiratory tract In the lower respiratory tract, the trachea and the five lung
lobes each contained from 0 6 to 1 5 % of the retained [ O]-NO2
13-9
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Kleimnan and Mautz (1991) also measured the fractional uptake of the entire
respiratory tract of female beagle dogs exposed to 9,400 /*g/m3 (5 0 ppm) while standing at
rest or exercising on a treadmill The dogs were not tracheostimized, but breathed through a
small respiratory mask During rest at the end of exercise and during continuous rest, the
fractional uptake of the entire respiratory tract was measured to be about 78% The
fractional uptake was 94% during the exercise exposures
Effect of Exercise on Respiratory Tract Dosimetry
Experiments indicate that increased ventilation decreases percent uptake in the upper
respiratory tract, and increases the percent uptake in the lower and total respiratory tract
In all cases, the effect of increased ventilation is an increase in the quantity of NO2 absorbed
by the individual regions The reduction of percent uptake in the upper respiratory tract due
to increased ventilation results in a greater proportion of inhaled NO2 being deliveied to the
lower respiratory tract Also, the switch from oronasal to oral breathing at high exercise
levels is expected to increase the delivery of NO2 to the lower respiratory tract because the
percent of NO2 removed by the mouth is less than that removed by the nasal cavity
Aharonson et al (1974) predicted a negative correlation between percent upper
respiratory tract uptake and ventilation using a model The model analyzed data from
experiments on the uptake of vapors by the nose The model was based on assumptions of
quasi-steady-state flow, mass balance, that the flux of a trace gas at the air-mucus interface is
proportional to the gas-phase concentration of the trace gas and a local mass trace gas, and a
local mass transfer coefficient Miller et al (1985) and Overton et al (1987) illustrated the
effects of ventilation on the lower respiratory tract uptake of O3 The theoretical work of
Miller et al (1985) on uptake in humans shows that exercise has a minimal effect in the
tracheobronchial region on tissue dose (i e , quantity of O3 absorbed by unit area of tissue
per unit tune) and a pronounced increase in the dose to the pulmonary region tissues As the
exercise level was increased, the maximum tissue dose (at the first respiratory bronchiole for
the resting state) shifted distaUy several generations and increased by a factor of 19 (over that
of the resting state and at the generation of the shifted maximum) for the highest simulated
exercise level Furthermore, this dose increase was more than twice the ratio of the exercise
to rest minute volumes On the whole, the pulmonary region absorbed 13 times as much at
13-10
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the highest ventilation rate than at the lowest ventilation rate In contrast, the
tracheobronchial region dose only increased by a factor of 1 4 for the same ventilation rate
increase The simulation results for rats (Overton et al, 1987) are in agreement with these
predictions Because the upper respiratory tract delivers a greater proportion of inhaled NO2
to the lower respiratory tract during exercise than at rest, the factors quoted above are
expected to be conservative Thus, the modeling results predict that exercise (with respect to
the resting state) delivers a disproportionately greater quantity of the inhaled mass to the
pulmonary region and an even greater disproportionate quantity to the more distal pulmonary
region surfaces Qualitatively, similar conclusions for NO2 are reasonable
Systemic Dosimetry
Once deposited, NO2 dissolves in lung fluids, and various chemical reactions occur,
giving rise to products that are found in the blood and other body fluids Svorcova and Kaut
(1971) suggested that inhaled NO2 entered the bloodstream based upon estimates of
radiolabelled nitrate and nitrite levels in the blood and urine of rabbits following exposure to
2
45,120 jttg/m (24 ppm) NO2 for 4 h The initial transformation products following NO2
absorption have been the subject of some speculation, howevei
The distribution of NO2 radiolabeled with nitrogen-13 (560 to 1,710 /*g/m3 [0 3 to
0 91 ppm] inhaled for 7 to 9 mm) in rhesus monkeys was investigated by Goldstein et al
(1977b) They concluded that inhaled NO2 was distributed throughout the lungs, and that it
probably reacts with the water molecules in the fluids of the respiratory tract to form nitrous
acid (HONO) and nitric acid (HNO3), the authors suggested that the acids were responsible
for subsequent toxic effects Based upon the absorption of NO2 by isolated ventilated
perfused rat lungs, Postlethwait and Mustafa (1981) proposed that the mam reaction of NO2
was not with lung water, but with readily oxidizable tissue components (e g , proteins and
lipids) to produce nitrite These investigators found that 70% of absorbed NO2 appeared as
nitrite in perfusate and lung tissue, and that the concentration of nitrite produced increased
with time during exposure They also hypothesized that nitrite in the blood may then be
oxidized to nitrate by interaction with hemoglobin in red blood cells In a similar
experiment, Postlethwait and Mustafa (1989) exposed isolated perfused rat lungs, to various
'j
concentrations (7,520 to 37,600 /*g/m , 4 to 20 ppm) and minute volumes (45 to 130 L/min)
13-11
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and found that the amount of nitrite detected in the perfusate was proportional (56%) to the
amount of NO2 retained by the lung Saul and Archer (1983) provided support for this
pathway using an in vivo system in which rats were exposed for 24 h to NO2 concentrations
of 2,260 to 16,540 pg/m3 (1 2 to 8 8 ppm) They also concluded that the main reaction
pathway of absorption in the lungs was the reaction of NO2 with oxidizable tissue
components to produce nitrite This product may then serve as a precursor for other
chemical reactions at extrarespiratory sites
The current data base indicates that once NO2 is absorbed in lung fluids, the subsequent
reaction products are rapidly taken up and then translocated via the bloodstream For
example, intratracheally instilled nitrite has been shown to be rapidly absorbed into blood
from the lungs (Parks et al, 1981) Oda et al (1979, 1980b), after exposing rats to
20,900 to 113,000 /ig/m3 (11 1 to 59 9 ppm) NO2 labeled with mtrogen-15 for 0 5 to
53.9 h, found increased levels of labeled nitrogen in the lungs, kidneys, plasma, and urine,
as well as an increased level of nitrite in plasma In a later study, Oda et al (1981) noted a
concentration-dependent increase in both nitrite and nitrate levels in the blood of mice during
1-h exposures to 9,400 to 75,200 /*g/m3 (5 to 40 ppm) NO2 The blood levels of nitrite and
nitrate declined rapidly after exposures ended, with the decay half-times of a few minutes for
nitrite and about 1 h for nitrate The shorter tune for the former was ascribed to its rapid
reaction (oxidation) with hemoglobin, producing nitrate and methemoglobin (Oda et al,
1981; Kosaka et al, 1979, Case et al, 1979), although such measurements were not made
Free nitrate in blood is generally excreted in unne (Greene and Hiatt, 1954, Hawksworth and
Hill, 1971)
Summary
The important physical, chemical, and biological factors involved in the uptake of NO2
by the respiratory tract were reviewed These factors must be taken into account in order to
interpret and understand experimental dosimetry results and to develop models that simulate
NO2 uptake for interspecies extrapolation purposes With respect to dosimetry, the following
has been observed Total respiratory tract uptake in humans ranged from 72 to 92%
depending on the investigation and the breathing state (Wagner, 1970, Bauer et al, 1986)
The percent total uptake was found to increase with increasing exercise level Upper
13-12
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respiratory tract uptakes have been measured in dogs, rabbits, and rats (Yokoyama, 1968,
Cavanagh and Moms, 1987, Kleinman and Mautz, 1991, Russell et al, 1991) Uptake
values ranged from as low as 25% to as high as 94% depending on the study, species, air
flow rate, and mode of breathing (nasal or oral) Percent upper respiratory tract uptakes
were found to decrease with increasing ventilation, uptakes via nasal breathing were
determined to be significantly greater than oral breathing uptakes For the lower respiratory
tract, Kleinman and Mautz (1991) found that the fractional uptake of NO2 by dog lungs
ventilated through a tracheostomy was about 90% of the inspired dose Uptake values of
36 and 72% have been reported for isolated, ventilated, perfused rat lungs (Postiethwait and
Mustafa, 1981, Postiethwait and Mustafa, 1989) Experimental evidence indicated that NO2
chemically reacted with lung tissue, but did not penetrate directly to blood However, the
reaction products, labeled nitrogen compounds, have been found throughout the body of
experimental animals (Oda et al, 1979, 1980b)
There has been very little modeling of the uptake of NO2 The results of the only
simulation study predicted that the maximum NO2 tissue dose in humans, rats, guinea pigs,
and rabbits occurred in the vicinity of the centnacinar region where morphological lesions
are commonly observed (Miller et al, 1982, Overton, 1984) Modeling has been used to
estimate the effect of increasing ventilation on the distribution of absorbed fy, which is
similar to NO2, in humans (Miller et al , 1985, Overton et al, 1987) These simulations,
the qualitative results of which were expected to apply to NO2, predicted that increasing
ventilation had little effect on uptake in the tracheobronchial region, but greatly enhanced
pulmonary region uptake
13.2.2 Respiratory Effects
13.2.2.1 Host Defense Mechanisms
The respiratory tract defenses encompass many interrelated responses, however, for
simplicity, they can be divided into two major components the physical defense mechanisms
and the cellular defense mechanisms The physical defense mechanisms in the upper and
lower airways (mucociliary system) begin with aerodynamics (Newhouse et al, 1976)
There is a large amount of turbulence experienced by the incoming airstream, causing the
nasopharyngeal removal of many of the large particles (> 10 pm) Smaller particles can also
13-13
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be deposited on the mucociliary escalator of the tracheobronchial region Particles deposited
on the mucus layer are removed by ciliary action that directs overlying mucus, particles, and
absorbed gases toward the pharynx where they are swallowed or expectorated (Breeze and
Wheeldon, 1977, Green, 1970, Newhouse et al, 1976, Proctor, 1977) This mucociliary
clearance is the first line of defense of the conducting airways
The second component of the pulmonary defense system is the cellular defense
mechanisms (phagocytic and unmunologic reactions), which operate mostly in the pulmonary
region of the lung Large mononuclear cells, alveolar macrophages (AMs), are the first line
of cellular defense (Hocking and Golde, 1979) The role of AMs in host defense is diverse
and varied, including such important activities as detoxifying and/or removing inhaled
particles, maintaining sterility against inhaled microorganisms, interacting with lymphoid
cells in a variety immunologic reactions, and removing damaged or dying cells from the
alveoli through phagocytosis (Pels and Cohn, 1986) Alveolar macrophages migrate
throughout the pulmonary region and it is this mechanism that allows contact with foreign
material entering the lungs Once phagocytosis has occurred, the particle is encased in a
phagocytic vesicle and fuses with lysosomes to form phagolysosomes, the prime subcellular
compartment for the killing of engulfed bacteria (Nathan et al, 1980, Silverstein et al ,
1977) During the phagocytosis process, AMs release oxygen radicals and enzymes either
into the phagosome or into the external milieu (Johnston et al, 1978) Oxygen radicals and
enzymes are believed to be essential for bacterial killing by phagocytes Both oxidation and
decarboxylation of bacterial membranes appear to be the major mechanisms by which oxygen
radicals induce bacterial killing (Klebanoff, 1968, Strauss et al, 1970)
Polymorphonuclear leukocytes (PMNs), another phagocytic cell, are present in a small
percentage in normal lungs In response to a variety of insults, there can be an influx of
PMNs from blood into the lung by chemotaxis Once recruited to the lung, PMNs then
ingest and kill opsomzed microbes and other foreign substances by mechanisms that are
similar to those described for AMs (Sibille and Reynolds, 1990) In contrast to AMs, PMNs
contain substantial amounts of myeloperoxidase stored in the primary granules and,
therefore, are an important source of hydroxide radicals, considered to be major bactericidal
agents (Klebanoff, 1982)
13-14
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In most cases, optimal phagocytosis of microorganisms by PMNs and AMs requires the
presence of opsomns Opsonins are immunoglobulins that have the capacity to enhance
phagocytosis of microorganisms When the phagocytic response is not sufficient to
effectively remove particles from the respiratory tract, immunologic (humoral and cell-
mediated immunity) responses are provided by the lymphocytes The humoral part of this
system primarily involves the B cells that function in the synthesis and secretion of antibodies
(immunoglobulin A PgA], immunoglobulin G PgGJ, immunoglobulin M [IgM]) into the
blood and body fluids Secretory IgA is found in the upper respiratory tract and
tracheobronchial region and its primary role is to inhibil microbial attachment to the
conducting airway surfaces Immunoglobulin G is the predominant class of immunoglobulins
of the lower respiratory tract These immunoglobulins act to enhance macrophage
functioning Immunoglobulin M is also present in the lower respiratory tract, but in low
concentrations The interactions of these defense mechanisms are complicated and not
completely understood
The cell-mediated component primarily involves T lymphocytes, which are the effectors
of cellular defense These cells are responsible for delayed hypersensitivity and defense
against viral, fungal, bacterial, and neoplastic disease Alveolar macrophages may be
responsible for the initiation of humoral and cellular immune responses by the ingestion of
particles or soluble antigens and the "processing" and "presenting" of these antigens for
specific antigen-reactive B and T cells This process stimulates the B and T cells to
proliferate and differentiate into effectors of humoral (antibody production) and cell-mediated
(cytotoxic lymphocytes) immunity It is these responses that are important in acute and
chronic infections and form the basis for antimicrobial immunity (Harada and Repine, 1985)
Alveolar macrophages also secrete alpha, beta, and gamma interferon and platelet
activating factor The interferons, protein substances produced by virus-invaded cells that
prevent the replication of the virus (Lefkowitz et al, 1984, Lefkowitz et al, 1983), have
potentially important implications in modulating the antiviral and immune activities of AMs
Recently, AMs were found to release a factor first thought to be related to the interferon
family and therefore initially named interferon beta 2 It was later determined to be a
cytokine (Wong and Clark, 1988) Cytokines are believed to play an immunoregulatory role
in the respiratory tract defenses, affecting inflammatory responses and tumoncidal activities
13-15
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(Dinarello et al, 1986) Platelet activating factors may play an important role in the
inflammatory response and are capable of PMN aggregation and of the release of arachidonic
acid metabolites from PMNs (Braquet and Rola-Pleszczynski, 1987)
Although antiviral pulmonary immune functions have not been adequately evaluated
after NO2 exposure, their proper functioning is essential in humoral and cell-mediated
immunity against viral disease These functions include (1) interferon production in
bronchoalveolar lavage (BAL) fluid, (2) AM function, (3) natural killer (NK) activity,
(4) cytotoxic T lymphocyte activity, and (5) antibody production (Burleson, 1987) A host
infected with virus manifests a cascade of immune responses, both specific and nonspecific
Local and circulating interferons (alpha, beta, and gamma) are produced in the first 24 h
after viral exposure Interferons have antiviral, antitumor, and immunoregulatory activities
Interferons serve as an immunoregulator to stimulate the immunological activity of both AMs
and lymphocytes exhibiting NK activity Interferon, AMs, and NK cells all contribute to the
nonspecific antiviral immunity of the host Cytotoxic T lymphocytes are the first specific
immunological response against viruses These cells can be activated by interferons and
therefore, are most relevant in defense against viral infections and tumors
Mucociliary Clearance
The effectiveness of the mucociliary system is dependent upon the integrity of the cilia
and respiratory epitheha, the chemicophysical properties of the mucus, and the rate of mucus
transport. Viral and bacterial infections and various chemicals can lead to over or under
secretion of mucus, alterations in mucous flow characteristics, and loss or paralysis of the
ciha The cilia can respond to insults in a number of ways changes in beat frequency,
cessation -of ciliary beating, and/or development of abnormal forms of cilia Substances that
produce such disruption or impairment of this defense system can result in an excess
accumulation of cellular secretions, increased acute bacterial and viral infections, chronic
bronchitis, and prolonged pulmonary complications possibly associated with the pathogenesis
of chronic obstructive pulmonary disease or bronchial cancer through the accumulation of
inhaled carcinogens (Schlesinger et al, 1987a)
There are numerous reports of significant loss of cilia and ciliated cells in the
bronchiolar epithelium A description of these histopathological changes can be found in
13-16
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Section 13 2 2 4, addressing the morphological effects of NO2 exposure This section and
Table 13-1 only summarize NO2 effects on mucociliarv activity
Clearance of marker substances deposited in the airways has been used to assess the
effects of NO2 on mucociliary clearance This method has merit as an index of overall
efficiency of mucociliary clearance The mucocilary clearance of inhaled tracer particles
deposited into the tracheobronchial tree of rabbits exposed 2 h/day for 2, 7, or 14 days to
0, 560, or 1,880 /tg/m3 (0, 0 3, or 1 0 ppm) NO2 did not alter the rate of removal
(Schlesinger et al, 1987a) Using a different approach discussed later in this section,
short-term physical clearance of bacteria was not affected at concentrations up to
27,800 fjig/m3 (14 8 ppm) (4 h) (Goldstein et al, 1973, Parkei et al, 1989)
Giordano and Morrow (1972) demonstrated significant impairment of tracheobronchial
clearance rates in rats following exposure to 11,280 /ug/m3 (6 0 ppm) NO2 for 6 weeks
This decrease in ciliary clearance was not accompanied by any observable abnormality of the
airways A 2-h exposure to 14,100 fcg/m (7 5 ppm) failed to alter the tracheal mucus
velocity in sheep, but exposure to twice that concentration for 2 h produced a significant
slowing of mucus movement (Abraham et al, 1980) Nitrogen dioxide-induced effects on
airway clearance appear to be both concentration and duration-dependent, however, it would
take prolonged exposure to high concentrations to induce alterations that would have
detrimental health effects Data would indicate that even a severely damaged airway
/
epithelium has the ability to maintain mucus transport (Abraham, 1984)
A few in vitro experiments have examined the effect of NO2 on isolated ciliated
epithelium cells and tracheal rings Kita and Omichi (1974) reported that exposure to NO2 at
concentrations greater than 9,400 /*g/m (5.0 ppm) resulted in a decreased rate of ciliary
beating Schiff (1977) isolated and exposed hamster tracheal ring cultures to 3,760 /wg/m3
(2 0 ppm) NO2 for 1 5 h/day, 5 days/week for 1 to 4 weeks After 14 days of exposure, the
NO2 produced decreased ciliary beating
Alveolar Macrophages
The effectiveness of AMs depends on the type, number, and viability of the cells The
cells must maintain an intact membrane, mobility, and phagocytic activity, and have
functioning enzyme systems Evidence from both in vivo and in vitro studies indicate that
13-17
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TABLE 13-1. EFFECTS OF NITROGEN DIOXIDE ON MUCOCELIARY ACTIVITY3
N02 Concentration
jig/m
560
1,880
3,760
9,400
11,280
14,100
w 28,200
oo 18,000
ppm
03
10
20
50
60
75
15
10
Exposure Gender
2 h/day, M
14 days
In vitro, NS
1 5 h/day,
5 days/week,
1-4 weeks
In vitro
7 days/week, F
6 weeks
2h NS
2h M
Species
Age (Strain)
NS Rabbit
(New
Zealand)
2 weeks Hamster
(Syrian
Golden)
N/A
NS Rat
(Long Evans)
NS Sheep
(NS)
NS Rabbit
(New
Zealand)
Effects
No effect on tracheobronchial
clearance
After 2-week exposure, ciliary
beating activity was decreased and
morphological changes were
observed
Decrease in ciliary beating activity
Decrease in mucociliary velocity,
functional impairment reversed by
1 week postexposure
Slowing of mucus velocity at
highest concentration
No effect on bronchial clearance
rate
Reference
Schlesinger et al (1987a)
Schiff (1977)
Kita and Onuchi (1974)
Giordano and Morrow (1972)
Abraham et al (1980)
Schlesinger et al (1987b)
"M = Male
NS = Not Stated
F = Female
-------
NO2 exposure alters these functions, thereby increasing the risk of the host to disease
Human clinical studies of AMs are discussed more fully in Section 15 6
In Vivo Exposure. Alveolar macrophages isolated from mice that had been
continuously exposed to 3,760 jng/m3 (2 0 ppm) NO2 for 33 weeks or exposed to 940 /*g/m3
(0 5 ppm) NO2 with a 1-h peak to 3,760 jttg/m3 (2 0 ppm) for 5 days/week for 21 weeks
showed distinctive morphological changes as compared to controls (Aranyi et al , 1976)
Examples of structural changes observed included the loss of surface processes, appearance
of fenestrae, bleb formation, and denuded surface areas Exposure to a lower level (i e ,
3 3
940 jitg/m [0 5 ppm] continuous or 188 /tg/m [0 1 ppm] continuous with a 3-h peak to
1,880 jwg/m [1 0 ppm]) for periods of up to 24 weeks did not result in any significant
identifiable morphological or biochemical changes Such morphological changes would be
expected to interfere with a number of cellular functions, such as chemotaxis and
phagocytosis
Studies have shown that exposure to NO2 at concentrations greater than 9,400 jttg/m3
(5 0 ppm) causes concentration-related increases in the number of AMs (Suzuki et al , 1986,
Rombout et al , 1986, Sherwin et al , 1968) A 12-week exposure to 2,256 or 7,520 /tg/m3
(1 2 or 4 0 ppm) NO2 increased the AM population by 30% over that of controls (Mochitate
et al , 1992) Alveolar macrophage accumulation has also been reported after 15 weeks of
3
exposure at lower concentrations, such as to either 9,400 /tg/m (5 0 ppm) NO2 or to
1,880 jig/m3 (1 0 ppm) with two daily 1 5-h peaks to 9,400 /*g/m3 (Gregory et al , 1983)
After a 1-day exposure to 5,000 jig/m3 (2 7 ppm) NO2, Rombout et al (1986) reported an
increase in the number of AMs in the terminal bronchioles and proximal alveoli of rats The
increase in AMs was associated with morphological changes in the respiratory tract, as
discussed in Section 13 22 4 addressing NO2-induced changes in lung morphology
No effect was seen at 1,000 or 2,500 /xg/m3 (0 53 or 1.33 ppm)
Chang et al (1986) and Crapo et al (1984) studied the response of 1-day-old and
6-week-old rats following exposure to 0 5 ppm (940 /ig/m3) NO2, 23 h/day for 6 weeks
<5
Another group of 6-week-old rats was exposed to 2 0 ppm (3,760 //cg/m ) for the same
duration Two daily 1-h peaks of three tunes the baseline level (i e , 2,820 and
11,280 jttg/m , 1 5 and 6 0 ppm) were applied Monday-Friday In these studies, the older
13-19
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rats were more responsive to NO2 than younger animals, showing a significant increase in
both number and volume of AMs The studies conducted by Azoulay-Dupuis et al (1983)
also demonstrated that in both the rat and the guinea pig, newborns were less affected by a
o
3-day exposure to 3,760 and 18,800 jwg/m (2 0 ppm and 10 ppm) NO2 than older animals
based on changes in lung structure and superoxide dismutase activity in isolated AMs
Mochitate et al (1986) reported a significant increase in the total number of AMs
fj
isolated from rats during 10 days of exposure to 7,520 ^g/m (4 0 ppm) NO2 At this
exposure concentration, AMs accounted for 97% of the total lavaged cells The number of
PMNs did not show any significant increase over this exposure period Alveolar
macrophages from exposed animals also exhibited increased metabolic activity as measured
by the activities of glucose-6-phosphate dehydrogenase, glutathione peroxidase, and pyruvate
kinase, which were significantly increased by 1 29-fold, 1 17-fold, and 1 2-fold,
respectively Simultaneously with these effects, the exposed AMs exhibited a significant
increase in the rate of synthesis of protein and DNA All responses peaked on Day 4 and all
values had returned to control levels by the tenth day
Suzuki et al (1986) found that NO2 exposure significantly increased the number of
AMs in BAL fluid from rats exposed to either 7,520 or 15,000 jttg/m3 (4 0 or 8 0 ppm)
In this study, the exposure continued for 10 days, but the effect became most significant at
the fifth day of exposure The viability of these isolated cells was decreased on Day 1 and
remained depressed for the remainder of the exposure penod There was no evidence that
either exposure caused an increase of PMNs However, Schlesinger (1987b) failed to find
any significant changes in the number or the viability of AMs in lung lavage from rabbits
exposed to 1,880 /*g/m3 (1 0 ppm) NO2, 2 h/day for 13 days
Rose et al (1989) reported a concentration-dependent decrease in AM phagocytosis in
the lower respiratory tract of mice exposed to 1,880 or 9,400 /xg/m3 (1 0 or 5 0 ppm) NO2
for 6 h on 2 consecutive days, but the decrease was only significant at 9,400 jwg/m3 Mice
were mtratracheally inoculated with gold-198 colloid following NO2 exposure When the
3
exposure was increased to 28,200 /ig/m (15 ppm) NO2, further effects on AM phagocytosis
were not noted The authors suggested that the lack of additional effects on AM
phagocytosis with increasing NO2 concentrations may be due to an influx of phagocytic cells
in the lower respiratory tract due to an inflammatory response to the NO2 Additional
13-20
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<>
studies at 9,400 jwg/m did not show an effect on the infectivity of murine cytomegalovirus
for AMs
The phagocytic activity of rat AMs was significantly depressed after 5 days of exposure
to 15,000 /xg/m3 (8 0 ppm) (Suzuki et al , 1986) A similar suppression was noted following
exposure to 7,520 /xg/m3 (4 0 ppm), but only after 7 days of exposure In all cases, the
phagocytic activity of these affected cells recovered to the control values at Day 10 of
exposure Suzuki et al (1986) proposed that the suppression of phagocytosis might be due to
the ability of NO2 to cause membrane lipid peroxidation The study by Dowell et al (1971)
adds support to this hypothesis As reported in Section 13 2 2 4 on lung morphology,
O
evidence of mitochondria! damage was noted in AMs fiom dogs exposed to 5,640
(3 0 ppm), and became most evident in dogs exposed to 13,160 /tg/m3 (7 0 ppm) NO2 The
authors described this effect as a manifestation of damage to the membrane function The
apparent recovery of the AMs after 10 days of exposure is thought to be due to the influx of
new AMs into the alveoli The morphological changes were also associated with changes in
lung biochemistry
Schlesinger (1987b) did not find any significant changes in the number or the viability
'j
of AMs in BAL fluid from rabbits exposed to 560 or 1 ,880 /tg/m (0 3 or 1 0 ppm) NO2,
2 h/day for 2, 6, or 13 days Although there were no effects on the numbers of AMs that
phagocytized latex spheres, 2 days of exposure to 560 /xg/m3 decreased the phagocytic
capacity (i e , number of spheres phagocytized per cell), the higher level of NO2 increased
this parameter After 6 or 13 days of exposure, phagocytosis was normal Lefkowitz et al
(1986) failed to find any depression in phagocytosis after mice were exposed for 7 days to
9,400 /tg/m3 (5 0 ppm) NO2
Alveolar macrophages isolated from humans exposed for 3 h to 1,130 jixg/m3 (0 6 ppm)
NO2 had a tendency (p < 0 07) to be less able to inactivate influenza virus than controls
cells (Frampton et al , 1989) However, when the concentration was reduced to 94 /tg/m3
(0 05 ppm) NO2 with three 15 min peaks to 3,760 ftg/rn3 (2 0 ppm) (same product of
concentration and time [C x T] as the 1,120 /xg/m3 regimen), the rate of viral inactivation
was unchanged Alveolar macrophages from humans exposed for 4 h to 3,760 /*g/m3
(2 0 ppm) exhibited a decrease in phagocytosis of Candida albicans (Devlin et al , 1992)
See Section 15 6 for a fuller discussion of these human studies
13-21
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Alveolar macrophages obtained by lavage from baboons exposed to 3,760
(2 0 ppm) NO2 for 8 h/day, 5 days/week for 6 mo had significantly unpaired responsiveness
to migration inhibitory factor produced by sensitized lymphocytes (Greene and Schneider,
1978) This substance affects the behavior of AMs by inhibiting free migration, which in
turn interferes with the functional capacity of these defense cells The random mobility of
AMs was significantly depressed in rabbits following a 2 h/day exposure for 13 days to
560 /*g/m3 (0 3 ppm) but not at 1,880 /tg/m3 (1 0 ppm) (Schlesinger, 1987b) Such effects
are important in the mediation of local immunologic responses in the lung and would be
expected to prolong the residence tune of the multitude of inhaled deposited particles in the
deep lung.
Vollmuth et al (1986) studied the clearance of strontmm-85 tagged polystyrene latex
spheres from the lungs of rabbits following a single 2-h exposure to 560, 1,880, 5,600, or
18,800 {Ag/m (0 3, 1 0, 3 0, or 10 ppm) NO2 An acceleration in clearance (decreased daily
retention) was evident immediately after exposure to the two lowest NO2 concentrations
A similar effect was observed by Schlesinger and Gearhart (1987) At the higher levels of
NO2, an acceleration in clearance was not evident until midway through the 14-day
postexposure period Repeated exposure to 1,880 or 18,800 /wg/m3 NO2, 2 h/day for
14 days produced a response similar to a single exposure at the same concentration,
indicating that, with repeat exposures, some attenuation may be produced after the initial
exposure (Vollmuth et al., 1986) However, in rats exposed chronically (7 h/day,
o
5 days/week, 18 to 22 mo) to 17,900 /*g/m (9 5 ppm) NO2, there was no effect on long-
term clearance of radiolabeled fused aluminosilicate particles (Mauderly et al , 1990)
Several studies have reported that NO2 exposure significantly decreases the ability of
AMs to produce superoxide anion radical, which may limit the antibacterial activity of these
cells. Amoruso et al (1981) presented evidence of such an effect in rats exposed to NO2
concentrations ranging from 2,444 to 31,960 /*g/m3 (1 3 to 17 ppm) The duration of the
NO2 exposure, other than a statement that it was an acute exposure, was not given In this
study, the author's objective was to compare the measured NO2 response with O3 effects,
and the data were expressed in terms of ppm-h, making it impossible to determine the
specific concentration and duration of exposure that elicited the effect Superoxide anion
radical reduction began after exposure to 18 3 ppm-h NO2 Production was reduced by 50%
13-22
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after 29 1 ppm-h, and at 51 ppm-h, the highest exposure tested, the production was
decreased by 85% Devlin et al (1992) also observed a decrease in the release of
o
superoxide amon from AMs of humans exposed (with exercise) for 4 h to 3,760 /*g/m
(2 0 ppm) Suzuki et al (1986) reported a marked decrease in the ability of rat AMs to
produce superoxide anion radical following a 10-day exposure to either 7,520 or
15,040 jitg/m3 (4 0 or 8 0 ppm) At the highest concentration, the depression was significant
only on exposure Days 3, 5, and 10
A number of animal studies have been performed to induce various structural,
functional, and biochemical changes in AMs by exposing the test animals to exceedingly high
concentrations (i e , greater than 9,400 /*g/m3, 5 0 ppm NO2). Some of these studies, along
with the studies conducted at lower exposure concentrations, are listed in Table 13-2 to
familiarize the reader with the broad spectrum of biological responses that AMs can exhibit
following NO2 exposure
In Vitro Exposure, Voism and co-workers (Voisui et al, 1977, Voisin and Aerts,
1984) have shown concentration-related effects after exposure for 30 nun to NO2
concentrations as low as 188 /-ig/m (0 1 ppm) In this system, the AMs, attached to
cellulose acetate fibers, were floated on top of a nutrient medium that diffused through the
filter, maintaining cell viability without submerging the cells These floating cells were then
exposed to NO2 In these studies, guinea pig AMs seemed to be sensitive to NO2, exhibiting
a reduction in phagocytic and normal bactericidal activity, reduced adenosine taphosphase
(ATP) content, and major morphological changes The seventy of these changes was related
to the NO2 concentration (Voisin et al, 1977) Increasing either the exposure concentration
to 9,400 /ig/m (5 0 ppm) or the exposure duration to 24 h resulted in complete destruction
of the cell (Voisin and Averts, 1984) As described more fully in Section 15 6, normal
human AMs exposed in vitro to high levels of NO2 (9,400, 18,800, or 28,200 /tg/m3, 5, 10,
or 15 ppm) for 3 h did not exhibit any change in cell visibility or the release of either
neutrophil chemotactic factor or interleukin-1 (Pinkston et al , 1988)
Robison et al (1990) exposed AMs from rats to 188, 1,880, 9,400, or 37,600 /tg/m3
(01, 1 0, 5 0, or 20 ppm) NO2 for 1 h in vitro to determine if NO2-induced infiltration of
PMNs in vivo was in response to the synthesis of leukotnene B4 (LTB4) by AMs Alveolar
13-23
-------
TABLE 13-2. EFFECTS OF NITROGEN DIOXIDE ON ALVEOLAR MACROPHAGES'
NO2 Concentration
/ 3
/tg/m
94 base +
3,760 peaks
1,130
188
1,880
9,400
37,600
940
£ 188 base +
^ 1,880 peak
*"•
3,760
940 base +
3,760 peak
ppm
0 05 base +
2 0 peaks
06
0 1
10
50
20
05
0 1 base +
1 Opeak
20
0 5 base +
2 Opeak
Species
Exposure Gender Age (Strain)
3 h base + NS NS Human
three 15-min
peaks
3h
Ih NS NS Rat
(Sprague-
Dawley)
(in vitro)
Continuous, NS NS Mouse
24 weeks
Continuous
base + 3-h
peak,
5 days/week,
24 weeks
Continuous,
33 weeks
Continuous
base + 1-h
peak,
5 days/week,
33 weeks
Effects
No effects at 0 05 ppm NO2 with peaks,
trend (p < 0 07) towards AMs losing
ability to inactivate influenza virus at
0 6 ppm
At 5 0 ppm increase in LTB4,
concentration-related decrease in SOD
production in AMs at > 1 0 ppm, increase
in LDH in AMs at 5 0 and 20 ppm
No effects on AM morphology at 0 5 ppm
continuous or 0 1 ppm base + peak
exposures
After 21 weeks of exposure to 2 0 ppm
continuous or 0 5 ppm base + peak,
morphological changes were identified,
such as loss of surface processes,
appearance of fenestrae, bleb formation,
and denuded surface areas
Reference
Framptonetal (1989)
Robisonetal (1990)
Aranyietal (1976)
-------
TABLE 13-2 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON ALVEOLAR MACROPHAGES"
NO2 Concentration
3
ug/m
560
1,880
560
1,880
560
1,880
5,640
18,800
560
1,880
940 base +
2,820 peak
3,760 base +
11, 300 peak
1,000
2,500
5,000
1,880
9,400
28,200
ppm
03
10
03
10
03
10
30
10
10
10
0 5 base +
1 5 peak
2 0 base +
6 Opeak
05
13
27
10
50
15
Exposure Gende:
2 h/day, M
2, 6, 13 days
2 h/day up to M
14 days
2h M
2 h/day,
14 days
Base M
22 h/day,
7 days/week
+ two 1-h
peaks,
5 days/week,
6 weeks
Continuous, M
28 days
6 h/day, NS
2 days
Species
r Age (Strain)
NS Rabbit
(New Zealand)
NS Rabbit
(New Zealand)
NS Rabbit
(New Zealand)
1 day Rat
and (Fischer 344)
6 weeks
6 weeks Rat
(Wistar)
4- Mouse
6 weeks (CD-I)
Effects
Decreased phagocytic ability of AMs at
0 3 ppm after 2 days of exposure, increased
at 1 0 ppm after 2 days of exposure, no
effect on cell number or viability, random
mobility reduced at 0 3 ppm only, no
effects after 6 days of exposure
Increase in alveolar clearance
Concentration-related acceleration in
clearance of particles from lung with the
greatest increase at two lowest
concentrations, effects from repeated
exposures similar to those seen after acute
exposures to same concentrations
Trend towards increase m number of AMs
and cell volume in younger animals,
increase in number of AMs and cell volume
in older rats
Increase m AMs m highest exposed group,
no effects noted in 2 lowest exposure
groups
Exposure-related decrease in AM
phagocytosis from 1 0-5 0 ppm, decrease
was not further affected by 15 ppm
Reference
Schlesinger (1987b)
Schlesinger and Gearhart
(1987)
Vollmuthetal (1986)
Crapo et al (1984)
Chang et al (1986)
Ramboutetal (1986)
Roseetal (1989)
-------
TABLE 13-2 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON ALVEOLAR MACROPHAGES"
N©2 Concentration
Atg/m
1,880
28,200
45,120
1,880
9,400
1,880 base +
9,400 peaks
,_ 2,440-
60 days
3-4 years
NS
6 weeks
NS
Species
(Strain)
Rat
(Long Evans)
Rat
(Fischer 344)
Rat
(Sprague-
Dawley)
Guinea pig
(Dunkin
Hartley)
Rat
(Wistar)
Baboon
Human
Rat
(Wistar)
Dog
(Beagle)
Effects
Stimulated clearance of particles from
lung at lowest concentration, but
decreased clearance rate at two highest
concentrations
Accumulation of AMs Superimposed
peak exposures produced changes that
may persist with continued exposures
Decreased production of superoxide
anion radical
Newborns were less affected than adults
when AMs were tested for SOD levels
Impaired AM responsiveness to
migration inhibitory factor
Decreased phagocytosis and superoxide
anion release
Increase in number of AMs
Enhanced swelling of AMs
Reference
Fenn and Leach (1977)
Gregory et al (1983)
Amorusoetal (1981)
Azoulay-Dupuis et al (1983)
Greene and Schneider (1978)
Devlin etal (1992)
Romboutetal (1986)
Dowelletal (1971)
-------
TABLE 13-2 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON ALVEOLAR MACROPHAGES"
NO2 Concentration
/tg/m ppm
6,770 3 6
22,700 12 1
7,520 4
19,000 10
47,000 25
7,520 4 0
7,520 4 0
15,000 8 0
9,400 50
Species
Exposure Gender Age (Strain)
Ih F NS Rat
2 h (Sprague-
Dawley)
(in vitro)
6h/day, M NS Rat
7, 14, or (Wistar)
21 days
10 days M 19-23 Rat
weeks (Wistar)
Up to NS NS Rat
10 days (Fischer 344)
7 days F NS Mouse
(CD-I)
Effects
Enhanced macrophage agglutination with
concanavalin A at both concentrations
tested
Changes in morphology at all
concentrations, increase in number of
AMs at 2: 10 ppm, phagocytic capacity
reduced after 14 and 21 days of exposure
to 25 ppm
Increase in number of AMs, no increase
in PMNs, mcreased metabolic activity,
protein, and DNA synthesis, all responses
peaked on Day 4 and returned to normal
on Day 10
Increase in number of AMs at both
concentrations, reaching a peak on Day 3
and 5, no increase in number of PMNs,
decrease in AM viability throughout
exposure period Suppression of
phagocytic activity after 7 days of
exposure to 4 ppm and after 5 days of
exposure to 8 ppm, returned to normal
value at 10 days Decrease in superoxide
radical production, but at 4 ppm, the
effect became significant on Days 3, 5,
and 10, at 8 ppm, the effect was
significant at all time periods tested
No effect on phagocytic activity
Reference
Goldstein et al (1977a)
Hooftmanetal (1988)
Mochitate et al (1986)
Suzuki etal (1986)
Lefkowitz et al (1986)
-------
TABLE 13-2 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON ALVEOLAR MACROPHAGES"
oo
N02 Concentration
3
9,400
28,200
9,400
18,800
28,200
9,400-
113,000
13,200
17,900
19,000
19,000
19,000
47,000
ppm
5
15
5
10
15
5-
60
70
95
10
10
10
25
Exposure
3 h after
infection with
paramfluenza
3 virus
3h
3h
24 h
7 h/day,
5 days/week,
18-22 mo
35 days
4h
24 h
Gender
NS
M
Fb
NS
NS
M
NS
F
M
Age
NS
NS
NS
NS
18
weeks
NS
NS
12-13
weeks
Species
(Strain)
Rabbit
(New Zealand)
Humans
(In vitro
exposure)
Rabbit
(New Zealand)
Rabbit
Rat
(Fischer 344)
Guinea pig
Mouse
(Swiss)
Rat
(Sprague-
Dawley)
Effects
AMs lost resistance to challenge with
rabbit pox virus after exposure to 15 ppm
No change in cell viability, release of
neutrophil chemotactic factor, or
interleukin-1
Inhibition of phagocy tic activity
Increased rosette formation in AMs
treated with hpase
No effect on long-term clearance of
radiolabeled tracer particles
63% increase in epithelial cells positive
for macrophage congregation
Increase in total pulmonary cells in
animals infected with some species of
bacteria
Decreased phagocytosis at 25 ppm only
Reference
Acton and Myrvik (1972)
Pinkstonetal (1988)
Gardner et al (1969)
Acton and Myrvik (1972)
Hadleyetal (1977)
Mauderlyetal (1990)
Sherwinetal (1968)
Jakab (1988)
Katz and Laskin (1976)
aNS = Not stated
AMs = Alveolar macrophages
LTB4 = Leukotnene 84
LDH = Lactate dehydrogenase
M = Male
F = Female
SOD = Superoxide dismutase
PMNs = Polymorphonuclear leukocytes
Only one female used in study
-------
macrophage production of LTB4 was not affected by exposure to 188, 1,880, or
37,600 jwg/m3, however, levels of LTB4 were elevated following exposure to 9,400 /xg/m3
fy
NO2 Products from AMs exposed to 9,400 or 37,600 /tg/m enhanced PMN chemotaxis
Superoxide production was decreased ui a concentration-related manner, starting with AMs
9,400 jug/m , 5 0 ppm) can significantly depress the immune
response, as determined by one or more tests available 1o assess the functional integrity of
13-29
-------
the specific components Unfortunately, there are only a very few studies conducted at near
ambient concentrations, and, independent of the concentrations tested, only a few of the
numerous immune parameters have been evaluated
Exposing sheep to 9,400 jwg/m (5 0 ppm) NO2, 1 5 h/day for 10 to 11 days showed
that such intermittent, short-term exposure may temporarily alter their pulmonary immune
responsiveness (Joel et al, 1982) One technique commonly used in determining the
production of antibody forming cells is to measure the number of plaque-forming cells
(PFCs) in the spleen or blood of immunized animals In this study, the authors assessed
immunological response by monitoring the daily output of PFCs in the efferent lymph of
caudal mediastmal lymph nodes Although the number of sheep used was small and the data
were not analyzed statistically, it would appear that in the ammals that were immunized with
horse red blood cells (a T-cell dependent antigen) 2 days, but not 4 days, after initiation of
NO2 exposure, the output of PFCs was below controls Blastogenic responses of T cells
from the efferent pulmonary lymph and blood also appeared depressed
Hfflam et al (1983) examined the effects of a 24-h exposure to 9,400, 18,800, and
o
48,900 /tg/m (5, 10, and 26 ppm) NO2 on cellular immunity in rats after intratracheal
immunization of the lung with sheep red blood cells (SRBCs) Cellular immunity was
evaluated by antigen-specific lymphocyte stimulation assays of pooled lymphoid cell
suspensions from either the thoracic lymph nodes or the spleen A concentration-response
effect with elevated cellular immunity was observed
Studies conducted by Fenters et al (1971, 1973) and Ehrkch and Fenters (1973) using
squirrel monkeys showed the impact of NO2 on the humoral immune response to
intratracheally delivered influenza vaccine In monkeys exposed for 493 days (16 mo) to
o
1,880 jig/m (1 0 ppm) NO2 and immunized with monkey-adapted virus (A/PR/8/34), the
serum neutralizing antibody titers were significantly increased earlier, and to a greater
degree, than in controls (Fenters et al , 1973, Ehrlich and Fenters, 1973) In monkeys
exposed to 9,400 /*g/m (5 0 ppm) NO2 for a total of 169 days and immunized with mouse-
adapted influenza virus (A/PR/8), serum neutralization titers were initially lower than
controls; no significant difference was observed by 133 days of exposure (Fenters et al,
1971; Ehrlich and Fenters, 1973) In all of these studies, the hemagglutination inhibition
antibody titers were not affected The authors discussed these differences, suggesting that
13-30
-------
the difference in the virus used for immunization played a role, along with exposure
<5
differences The authors also hypothesized that exposure to 1,880 /tg/m NO2 unproved the
establishment and survival of the monkey-adapted virus within the respiratory tract, resulting
in an increase in antibody production
The results of Holt et al (1979) suggest that both the nature of and rate of change in
immunological function from NO2 exposure can be both concentration- and tune-dependent
Tests were conducted at 7-week intervals to assess the functioning ability of the immune
system of mice exposed to 18,800 ftg/m (10 ppm) NO?, 2 h/day for periods of up to
30 weeks (Holt et al , 1979) Chronic exposure exhibited a general suppression in antibody
liters and ability of the T cells to function in a graft versus host reaction However, the
more acute exposures resulted in an enhancement of immunological responsiveness
A series of immunological studies designed to examine the effects of NO2 on the
humoral antibody response to SRBCs was reported by Fujimaki and Shimrzu (1981) and
Fujimaki (1989) They exposed mice for 12 h to 9,400, 37,600, and 75,200 /tg/m3 (5, 20,
and 40 ppm) NO2 and reported a significant suppression in PFCs (primary antibody
response) in response to SRBCs at the two highest levels of exposure When mice were
•5
exposed to 7,520 /ig/m (4 0 ppm) NO2 for 3, 7, 14, 01 56 days, no suppression in antibody
response was observed •
Using this same model, Fujimaki et al (1982) reported a similar effect in mice (i e ,
suppression of primary antibody PFC response in the spleen) after 4 weeks of continuous
<>
exposure to 752 and 3,000 /xg/m (0 4 and 1 6 ppm) NO2 At the higher concentration,
there were no significant differences observed in the activities of the T and B lymphocytes
Secondary antibody response was not affected at 752 /jg/m , but was slightly enhanced at
3,000 /tg/m3 exposure level However, Maigetter et al (1978) found that the normal
transformation response of mouse splenic T and B cells to phytohemagglutinin (PHA) and
bacterial lipopolysacchande (LPS), respectively, was suppressed following NO2 exposure
•3
Mice were exposed to 940 /tg/m (0 5 ppm) NO2, 24 h/day, 7 days/week for up to 1 year or
to a baseline concentration of NO2 (188 /*g/m , 0 1 ppm) for 24 h/day, 7 days/ week with 3-h
peaks 5 days/week to either 470, 940, or 1,880 ^g/m3 (0 25, 0 5, or 1 0 ppm) NO2 The
decrease in mitogemc responses of splenic lymphocytes to PHA and LPS was not
concentration- or duration-dependent in the base + peak exposure groups The decrease in
13-31
-------
T-cell mitogenesis was linearly related to the increased duration of continuous exposure to
940 jig/m3 NO2
Lefkowitz et al (1986) employed several methods to measure immunoactivity of mice
o
exposed to 9,400 /*g/m (5 0 ppm) NO2, 24 h/day for 6 days and injected with SRBCs after
the first day of exposure Nitrogen dioxide did not affect hemagglutination antibody liters or
cell-mediated immunity (blastogenesis of splenic T cells), but did significantly reduce the
number of splenic PFCs to SRBCs The authors stated (data were not shown) that mice
*a
exposed to 2,820 /*g/m (1 5 ppm) NO2 for 14 or 21 days also showed a 33 and 50%
decrease, respectively, in the number of PFCs
<>
Kosmider et al (1973b) exposed guinea pigs to 1,880 /*g/m (1 0 ppm) NO2 for 6 mo
and reported a significant reduction m all serum immunoglobulin fractions and complement
Decreased levels of these substances may lead to an increase in the frequency, duration, and
-a
severity of an infectious disease Mice exposed to a baseline of 940 jig/m (0 5 ppm) plus
peak of 3,760 jtg/m (2 0 ppm) NO2 for 3 mo had decreased serum levels of IgA and
exhibited nonspecific increases in serum IgM, IgG, and IgG2 (Ehrlich et al, 1975)
Effects on lymphocyte populations were tested in mice by Richters and Damji (1988)
The percentage of the total T-lymphocyte population was lower in the spleens of mice
exposed for 7 weeks (7 h/day, 5 days/week) to 470 /*g/m3 (0 25 ppm) NO2 The
percentages of mature helper/inducer T lymphocytes and T-cytotoxic/suppressor lymphocytes
were also lower in the spleen of exposed animals There were no statistically significant
changes in the percentages of NK cells or mature T cells Mice exposed to 670 /*g/m3
(0 35 ppm) for 7 h/day, 5 days/week for 12 weeks also showed a suppression in the
percentage of total matured T lymphocytes, but no statistically significant effect on any
specific subpopulation However, when the exposure was increased to 7,520 jwg/m3
(4.0 ppm) for 8 h, the percentage of total T lymphocytes, T-helper/inducer lymphocytes, and
T-cytotoxic/suppressor lymphocytes was significantly lower in mice (Damji and Richters,
1989). The most susceptible subpopulation was the large T-cytotoxic/suppressor
lymphocytes.
Richters and Damji (1990) reported similar findings m mice exposed to 470 /ng/m3
(0 25 ppm) NO2, 7 h/day, 5 days/week when the exposure tune was increased to up to
181 days The splenic T-helper/inducer (CD4+) lymphocytes were reduced, no effects were
13-32
-------
observed on T-cytotoxic/suppressor cells Spontaneously developing lymphomas in
NO2-exposed animals had progressed more slowly than those in control animals This was
attributed to the NO2-induced reduction in the T-helper/mducer lymphocytes
Conversely, Selgrade et al (1991) reported that exposure to an NO2 peak slowly rising
<5
to 2,820 /tg/m (1 5 ppm) for 6 h/day, 5 days/week, superimposed on a baseline
q
concentration of 940 /*g/m (0 5 ppm) for 22 h/day, 7 days/week for up to 78 weeks did not
affect the splenic or circulating T-cell response to mitogens in rats There were also no
NO2-related effects on the B-cell population There was a transient decrease in splenic NK
cell activity This effect was, however, only noted after 3 weeks of exposure
Mice that were vaccinated with influenza virus (A-2/Taiwan/l/64) after 3 mo of
continuous exposure to 3,760 /tg/m3 (2 0 ppm) or to 940 jwg/m3 (0 5 ppm) NO2 with a 1-h
<>
daily, 5 day/week peak exposure to 3,760 /*g/m (2 0 ppm) had mean serum neutralizing
antibody titers that were fourfold lower than clean air controls (Ehrlich et al , 1975) The
hemagglutination inhibition titers in these animals were unchanged This agrees with the
Fenters et al (1971) findings in squirrel monkeys exposed to 9,400 /*g/m3 (5 0 ppm) for
1 year and inoculated with A/PR/8/34 influenza virus
Immune response to muruie cytomegalovirus was not substantially affected by NO2,
even though NO2 increased susceptibility to infection (Rose et al , 1989) Mice were
exposed to 9,400 jwg/m3 (5 0 ppm) NO2 for 6 h/day on 2 days prior to viral intratracheal
inoculation and 4 days after inoculation Splenic lymphocyte response to PHA or circulating
specific antibody titers were not changed However, lymphocyte secondary proliferative
response to the viral antigen was decreased
Few studies have been undertaken to assess the effects of NO2 on interferon
production Mice exposed to either 9,400 or 47,000 jwg/m3 (5 or 25 ppm) NO2 for 3 to
7 days had serum levels of interferon similar to controls (Lefkowitz et al , 1984, Lefkowitz
et al , 1983)
An increase in certain immunological functions may also be detrimental to the host's
health by stimulating the immune system to react against the host's own tissue Balchum
et al (1965) identified such an effect when guinea pigs were exposed to 9,400 jug/m3
3
(5 0 ppm) or 28,200 jtg/m (15 ppm) NO2 There was a noticeable increase in the liter of
serum antibodies against lung tissue in all test animals exposed, starting after 160 h of
13-33
-------
exposure These antibody titers continued to increase with the increases in NO2
concentration and exposure duration
The effects of NO2 exposure on the immune system appear to be concentration- and
time-dependent Some studies suggest little effect, whereas others suggest suppression or
activation, depending not only on concentration, but also on length of exposure, species
tested, and specific end points measured Table 13-3 summarizes the reported
immunological effects following exposure to NO2
Interaction with Infectious Agents
Different experimental approaches using laboratory animals have been employed in an
effort to determine the functional efficiency of the host's pulmonary defenses following NO2
exposure. In the most commonly used infectivity model, animals are randomly selected for
exposure to either a pollutant, in this case NO2, or filtered air After exposure, the
treatment groups are combined and exposed for approximately 15 mm to an aerosol of a
viable agent, such as Streptococcus sp , Klebsiella pneumomae, Diplococcus pneumomae,
influenza A2/Taiwan virus, or A/PR/8 influenza virus The animals are then returned to
clean air for a holding period (usually 15 days) and the mortality rates in the NO2-exposed
and the air-exposed groups are compared If the normal pulmonary defenses are functioning
properly, the deposited viable microorganisms will be quickly killed and the lungs will
remain sterile, and only a small percentage (typically between 5 to 15%) of the control
animals will succumb to the laboratory infection However, if host defenses are
compromised by the chemical exposure, mortality rates will be higher (Ehrlich, 1963, 1966,
1980; Gardner et al, 1982, Coffin and Gardner, 1972, Henry et al , 1970)
A wide variety of mammalian species, including humans, share an array of defensive
mechanisms that are anatomically and physiologically integrated in the respiratory tract to
prevent and control most invading infectious organisms The infectivity model is an
excellent indicator of a weakened host defense system The effects seen in laboratory
animals represent alterations in host defenses Studies have shown that these responses are
valid across species, sensitive to a variety of chemicals, supported by mechanistic studies,
and capable of epidemiological confirmation Similar alterations in these basic defense
13-34
-------
TABLE 13-3. EFFECTS OF NITROGEN DIOXIDE ON THE IMMUNE SYSTEM3
w
NO2 Concentration
/ ^
/tg/m
470
470
670
752
3,000
940
188 base +
470, 940, or
1,880 peak
940 base +
2,820 peak
ppm
025
025
035
04
1 6
05
0 1 base +
0 25, 0 5, or
1 Opeak
0 5 base +
1 Speak
Exposure Gender
7 h/day, F
5 days/week,
7 weeks
7 h/day, F
5 days/week,
181 days
7 h/day, M
5 days/week,
12 weeks
Continuous, M
4 weeks
Continuous, M
up to 1 year
24 h/day,
7 days/week
base +
3 h/day,
5 days/week
peak for up
to 1 year
22 h/day, M
7 days/week
base +
6 h/day,
5 days/week
peak for up
to 78 weeks
Species
Age (Strain)
6 weeks Mouse
(AKR/cum)
5 weeks Mouse
(AKR/cum)
6 weeks Mouse
(C57BL/6J)
7 weeks Mouse
(BALB/c)
10-11 Mouse
weeks (CD-I)
10 Rat
weeks (Fischer 344)
Effects
Reduced percentage of total T-cell population
and trend towards reduced percentage of
certain T-cell subpopulations and NK cells
Reduced percentage of total T-cell population
and percentages of T-helper/rnducer cells on
Days 37 and 181
Trend towards suppression in total
percentage of T cells Percentages of other
T-cell subpopulations lower, but not
significantly
Decreased primary splenic PFC response at
both concentrations, increased secondary
antibody response at 1 6 ppm
Linear decrease in PHA-induced mitogenesis
with NC>2 duration
Variable suppression of splenic T- and B-cell
responsiveness to mitogens but not
concentration- or exposure duration-related
No effect on splenic or circulating B- and
T-cell response to mitogens, decrease in
splenic NK cell activity only after 3 weeks
exposure No histological changes in
lymphoid tissue
Reference
Richters and Damji (1988)
Richters and Damji (1990)
Richters and Damji (1988)
Fujimakietal (1982)
Maigetter et al (1978)
Selgradeetal (1991)
-------
TABLE 13-3 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON THE IMMUNE SYSTEM3
NO2 Concentration
/ig/trf5 ppm Exposure Gendej
940 base +05 base + 24 h/day, M
3,760 2 0 peak 5 days/week
peak base + 1 h
daily peaks
for 3 mo
1,880 1 0 Continuous, M
16 mo
1,880 10 6 mo M
V3 2,820 1 5 24 h/day, F
& 7, 14 or
21 days
7,520 40 Continuous, M
up to 56 days
7,520 40 8h F
9,400 50 15 h/day, M/F
10-11 days
9,400 50 4 or 7 h/day, NS
5 days/week,
5 5 mo
9,400 5 0 Continuous, M
169 days
Species
r Age (Strain)
6 Mouse
weeks (CD-I)
NS Monkey
(Squirrel)
NS Guinea pig
(NS)
NS Mouse
(CD-I and
C57BL)
8-10 Mouse
weeks (BALB/c)
6 Mouse
weeks (C57BL/6cwm)
NS Sheep
(Dorset)
NS Guinea pig
(New England)
NS Monkey
(Squirrel)
Effects
Vaccination with influenza A2/Taiwan virus followed exposure
Decrease in serum neutralizing antibody, hemagglutination
inhibition liters unchanged, before virus challenge, NO2
exposure decreased serum IgA and increased IgGj, IgM, and
IgG2, after virus, serum IgA unchanged and IgM increased
Monkeys challenged five times with monkey-adapted influenza
virus during NO2 exposure Hemagglutination inhibition
antibody liters not altered Compared to controls, NO2 caused
an earlier and greater increase in serum neutralization antibody
tilers to the virus
Intranasal challenge with K pneumoniae after exposure
Decreased hemolytic aclivily of complemenl, decrease in all
immunoeleclrophoretic fractions
Splenic PFCs reduced on Day 14 and 21 by 33 and 50%,
respectively, no effect on cell-mediated immune system or
hemagglutinating liters
No suppression of anlibody response to SRBCs
Alleralion in T lymphocyte subpopulation
Reduction in PFCs in lymph, depressed blastogenic response of
T cell from lymph and blood
Serum antibodies againsl lung tissue increased with
concentration and duration of exposure
Monkeys challenged 4 X with mouse-adapted influenza virus
Initial depression in serum neulrahzabon liters wilh return to
normal by Day 133, no effect on hemagglutin inhibition liter
Reference
Ehrhch et al
(1975)
Fenters et al
(1973)
Kosmider
etal (1973b)
Lefkowilz
etal (1986)
Fujimaki
(1989)
Damji and
Richters (1989)
Joel el al
(1982)
Balchum el al
(1965)
Fenters et al
(1971)
-------
TABLE 13-3 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON THE IMMUNE SYSTEM3
NO2 Concentration
/*g/m3
9,400
47,000
9,400
9,400
18,800
48,900
9,400
9,400
9,400
37,600
75,200
10,000
18,800
ppm
50
250
50
50
10
26
50
50
50
20
40
53
10
Exposure
7 days
3 days
24 h/day,
6, 7, or
15 days
24 h
Continuous,
6 mo
6 h/day,
6 days
12 h
Continuous,
19, 26, or
33 days
2 h/day,
30 weeks
Gender Age
F 8-12
weeks
F NS
M 24-28
weeks
NS NS
NS 4-6
weeks
M/F 6-9
weeks
F NS
F 6-8
weeks
Species
(Strain)
Mouse
(C57BL/6)
Mouse
(CD-I,
C57BL)
Rat
(Fischer 344)
Monkey
(Squirrel)
Mouse
(CD-I)
Mouse
(BALB/c)
Guinea pig
(BFA-ZH-
Kisslegg)
Mouse
(BALB/c)
Effects
No effect on serum interferon levels
No effect on serum antibody titers in mice exposed for
6 days (only exposure tested), decrease in splenic PFCs
was noted in some groups exposed for 7 or 15 days
- Concentration-related elevation of cellular immunity in
thoracic lymph nodes and spleen after immunizing the
lung with SRBCs
Depressed postvaccination serum neutralizing antibody
formation
Mice infected with munne cytomegalovirus after
second day of exposure No effect on circulating
specific antibody liter to virus or splenic lymphocyte
response to PHA Decrease in secondary prohferative
response of splenic lymphocytes to viral antigen
No effect on primary and secondary splenic PFC
response at 5 ppm, at higher levels, suppression in
primary antibody response
No effect on antibody production
Marked depression in ability of T cells to respond to
nonspecific stimuli, reduced ability to reject tumors
Reference
Lefkowitz et al (1983,
1984)
Lefkowitz et al (1986)
Hillametal (1983)
Ehrhch and Fenters (1973)
Roseetal (1989)
Fujimaki and Smmizu
(1981)
Fujimakietal (1981)
Hidekazu and Fujio (1980)
Antweiller et al (1975)
Holtetal (1979)
aF = Female
NK = Natural killer cells
M = Male
IgA = ImmunoglobulmA
IgG = Immunoglobulm G
IgM = Immunoglobulm M
NS = Not stated
PFCs = Plaque forming cells
SRBCs = Sheep red blood cells
-------
mechanisms that occur in animals could also occur in humans because they have equivalent
pulmonary defenses In the animal model frequently used, mortality is the sensitive response
indicator for alterations in host defense functioning However, with today's medical care,
few people die of bacterial pneumonia, so a better comparison in humans would be the
prevalence of respiratory illness in the community, as discussed in Chapter 14 of this
document (epidemiological studies) Such a comparison is proper because both mortality
(animals) and morbidity (humans) result from a loss of pulmonary defenses However,
different exposure levels, patterns, and durations may be required to produce alterations to
human host defenses Table 13-4 summarizes the effects of exposure to NO2 and infectious
agents.
An enhancement in mortality following exposure to NO2 and a pathogenic
microorganism could be due to several factors Studies by Goldstein et al (1973) showed
decreases in pulmonary bactericidal activity following NO2 exposure In then first
experiments, mice breathed aerosols of Staphylococcus aureus (S aureus) labeled with
radioactive phosphorus and then were exposed to NO2 for 4 h Physical removal of the
bacteria was not affected by any of the NO2 concentrations used up to 27,800 jwg/m
(14.8 ppm). Concentrations of 13,200, 17,300, and 27,800 j^g/m3 (7 0, 9 2, and 14 8 ppm)
NO2 lowered bactericidal activity by 7, 14, and 50%, respectively, when compared to
controls Lower NO2 concentrations (3,570 and 7,140 /*g/m3, 1 9 and 3 8 ppm) had no
significant effect The cause of the alteration was an impairment of the AM's bactericidal
activity and not an impairment of the host's mechanical clearance system In another
experiment (Goldstem et al , 1973), mice breathed 1,880, 4,320, and 12,400 (1 0, 2 3, and
6.6 ppm) NO2 for 17 h and then were exposed to an aerosol of radiolabeled S aureus Four
hours later, the animals were examined No difference in the number of bacteria inhaled
was found in the NO2-exposed animals Concentrations of 4,320 and 12,400 jttg/m3 NO2
decreased pulmonary bactericidal activity by 6 and 35 %, respectively, compared to controls
Exposure to 1,880 jig/m3 NO2 had no significant effect Goldstem et al (1973)
hypothesized that the decreased bactericidal activity was due to defects in AM function
Jakab (1987, 1988) confirmed these findings and reported that the concentration of NO2
required to suppress normal pulmonary bactericidal activity in mice depends on the specific
13-38
-------
TABLE 13-4. INTERACTION OF NITROGEN DIOXIDE WITH INFECTIOUS AGENTS3
UJ
NO2 Concentration
/tg/m
100 base
+ 188
peak
940 base
1,880 peak
2,256 base
+ 4,700
peak
376 base
+ 1,504
peak
564-
940
ppm
0 05 base
+ 0 1
peak
0 5 base
1 Opeak
1 2 base
+
2 5 peak
0 2 base
+
08 peak
03-
05
Exposure Gender Age
Continuous, F NS
base +
twice/day 1-h
peaks,
5 days/week
for 15 days
23 h/day, F 6-8
7 days/week weeks
base + twice
daily 1-h
peaks,
5 days/week
for 1 year
Continuous, F 4
3 mo weeks
Continuous,
6 mo
Species Infective
(Strain) Agent Effects
Mouse Streptococcus No effect
(CD-I) sp
Increased mortality
Increased mortality
Mouse Streptococcus Peak plus baseline caused significantly greater
(CD-I) sp mortality than baseline
Mouse A/PR/8 High incidence of adenomatous proliferation of
(ICR JCL) virus peripheral and bronchial epithelial cells, NO2
alone and virus alone caused less severe
alterations
No enhancement of effect of NO2 and virus
Reference
Gardner (1980)
Gardner et al
(1982)
Graham et al
(1987)
Miller etal (1987)
Motomiya et al
(1973)
-------
TABLE 13-4 (cont'd). INTERACTION OF NITROGEN DIOXIDE WITH INFECTIOUS AGENTS2
NO2 Concentration
/tg/m
940
940-
1,880
18,800
940-
52,700
940
940
1,880
2,820
9,400
940
1,880
3,760
9,400
ppm
05
05-10
10
05-28
05
05
10
15
50
05
10
20
50
Exposure Gender
Intermittent, F
6 or 18 h/day,
up to 12 mo
Continuous,
90 days
Continuous, F
39 days
2 h/day,
1, 3, and
5 days
Varied F
3 h/day, F
3 mo
24 h/day, F
7 days/week,
3 mo
3 days
4h M/F
Species
Age (Strain)
NS Mouse
(Swiss)
NS Mouse
(ICR, dd)
NS Mouse
(CD-I)
6-8 Mouse
weeks (CD2FJ,
CD-I)
NS Mouse
(CF-1)
8-10 Mouse
weeks (C57BL/6N)
Infective
Agent
K. pneumoniae
A/PR/8
virus
Streptococcus
sp
Streptococcus
sp
K pneumoniae
Mycoplasma
pulmonis
Effects
Increased mortality after 6 mo
intermittent exposure or after
3, 6, 9, or 12 mo continuous
exposure, following 12 mo
exposure, increased mortality
was significant only in
continuously exposed mice
Increased susceptibility to
infection
Increased mortality with
increased time and
concentration, concentration is
more important than time
Increase in mortality with
reduction in mean survival
time
Significant increase in
mortality after 3-day exposure
to 5 0 ppm, no effect at other
concentrations, but control
mortality very high
Decrease in intrapulmonary
killing only at 5 0 ppm
Reference
Ehrlich and
Henry (1968)
Ito (1971)
Gardner et al (1977a,b)
Ehrlich etal (1979)
McGrath and Oyervides
(1985)
Davis etal (1992)
-------
TABLE 13-4 (cont'd). INTERACTION OF NITROGEN DIOXIDE WITH INFECTIOUS AGENTS3
NO2 Concentration
pg/m
1,880
4,324
12,408
1,880
4,700
9,400
18,800
1,880
1,880
5,640
1,880
4,700
9,400
2,820-
94,000
ppm
10
23
66
10
25
50
100
10
10
30
10
25
50
15-
50
Species
Exposure Gender Age (Strain)
17 h M NS Mouse
(Swiss)
4h F NS Mouse
(Swiss)
48 h M NS Mouse
(Swiss
Webster)
3h F 5-6 Mouse
weeks (CD-I)
6 h/day, 6 days NS 4-6 Mouse
weeks (CD-I)
2h NS NS Mouse
(NS)
Hamster
(NS)
Monkey
(Squirrel)
Infective
Agent
S aureus
after exposure
S aureus
Streptococcus
sp
S aureus
Streptococcus
sp
Cytomegalo-
virus
K pneumoniae
Effects Reference
No difference in number of Goldstein et al
bacteria deposited, but at the two (1973)
highest concentrations, there was a
decrease in pulmonary bactericidal
activity of 6 and 35%,
respectively, no effect at 1 0 ppm
Injection with corticosteroids Jakab (1988)
increased NO2-induced inpairment
of bactericidal activity at
>2 5 ppm
Increased proliferation of Sherwood et al
Streptococcus in lung of exposed (1981)
mice but no effect with
Staphylococcus
Exercise on continuously moving Illing et al (1980)
wheels during exposure increased
mortality at 3 0 ppm
Increase in virus susceptibility at Rose et al (1988,
5 0 ppm only 1989)
Significant increased mortality in Ehrhch (1975)
mice, hamsters, and monkeys at
NO2 concentrations of >3 5,
^35, and 50 ppm, respectively
-------
TABLE 13-4 (cont'd). INTERACTION OF NITROGEN DIOXIDE WITH INFECTIOUS AGENTS8
NO2 Concentration
. 3
/ig/m ppm
2,820 1 5
6,580 3 5
2,820 base 1 5 base
+ 8,100 +
peak 4 5 peak
8,100 45
Species
Exposure Gender Age (Strain)
Continuous F NS Mouse
or (CD-I)
intermittent,
7 h/day,
7 days/week,
up to 15 days
Continuous F NS Mouse
64 h, then (CD-I)
peak for 1,
3 5, or 7 h,
then
continuous
18 h base
1, 3 5, or 7 h
Infective
Agent Effects
Streptococcus After 1 week, mortality with
sp continuous exposure was greater
than that for intermittent, after
2 weeks, no significant difference
between continuous and
intermittent exposure
Increased mortality with increased
duration of exposure, no significant
difference between continuous and
intermittent exposure, with data
adjusted for total difference in
C X T, mortality essentially the
same
Streptococcus Mortality increased with 3 5- and
sp 7-h single peak when bacterial
challenge was after an 18-h
baseline exposure
Mortality proportional to duration
Reference
Gardner etal (1979)
Coffin etal (1977)
Gardner (1980)
Gardner etal (1982)
Graham etal (1987)
2,820
15
when bacterial challenge was
immediate, but not 18 h
postexposure
7 h/day, 4, NS
5, and 7 days
NS Mouse
Streptococcus Elevated temperature (32 °C) Gardner et al (1982)
sp increased mortality after 7 days
3,570
7,140
13,200
17,200
27,800
1 9
38
70
92
148
4 h M NS Mouse
(NS)
5 aureus Physical removal of bacteria
unchanged by exposure
Bactericidal activity decreased by
7, 14, and 50%, respectively, in
three highest NC>2-exposed groups
Goldstein et al (1973)
-------
TABLE 13-4 (cont'd). INTERACTION OF NITROGEN DIOXIDE WITH INFECTIOUS AGENTS8
NC>2 Concentration
/ig/m
2,820-
9,400
2,820
4,700
6,580
9,400
18,800
28,200
3,760
4,700
7,500
9,400
18,800
28,200
9,400
18,800
ppm
1 5-
50
15
25
35
50
10
15
20
25
40
50
10
15
50
10
Exposure Gender
3h F
2h NS
1 5 h/day, NS
5 days/week
for 1, 2, and
3 weeks
4h F
Continuous, M
2 mo
Continuous,
1 mo
Species
Age (Strain)
6-10 Mouse
weeks (CF-1,
CD2Fj)
6-8 Mouse
weeks (Swiss
Webster)
2 Hamster
weeks (Golden
Syrian)
(in vitro)
NS Mouse
(Swiss)
NS Monkey
(Squirrel)
Infective
Agent
Streptococcus
sp
K pneumomae
A/PR/8/34
influenza
virus
S aureus,
Proteus
mirabihs,
Pasteurella
pneumotropica
K pneumomae
or A/PR/8
influenza virus
Effects
Increased mortality in mice exposed to
>2 0 ppm
No effect at 1 5 or 2 5 ppm, increased
mortality at 3 5 ppm and above
Increase in mortality when
K pneumomae challenge 1 and 6 h
after 5 or 10 ppm NC^ exposure, when
K pneumomae challenge 27 h
following NO2 exposure, effect only at
15 ppm
Peak virus production in trachea!
explants occurred earlier
Concentration-related decrease in
bactericidal activity at > 4 0 ppm with
S aureus wnen NC>2 exposure after
bacterial challenge, when NO2 exposure
was before challenge, effect at 10 ppm,
NC>2 concentrations > 5 0 ppm required
to affect bactericidal activity for other
tested microorganisms
Increased viral-induced mortality (1/3)
Increase in Klebsiella-wduced mortality
(2/7), no control deaths
Increased virus-induced mortality (6/6)
within 2-3 days after infection, no
control deaths Increase rn Klebsiella-
induced mortality (1/4), no control
deaths
Reference
Ehrhchetal (1977)
Ehrlich (1980)
Purvis and Ehrlich
(1963)
Ehrhch (1980)
Schiff (1977)
Jakab (1987, 1988)
Henry et al (1970)
-------
TABLE 13-4 (cont'd). INTERACTION OF NITROGEN DIOXIDE WITH INFECTIOUS AGENTS3
N02
2
9,400
18,880
18,800
28,200
65,800
94,000
Concentration
ppm
50
10
10
15
35
50
Species
Exposure Gender Age (Strain)
4h M/F 6-10 Mouse
weeks (C57B1/6N,
C3H/HeN)
2h M/F NS Monkey
(Squirrel)
Infective
Agent
Mycoplasma
pulmonis
K
pneumoniae
Effects
NC>2 increased incidence and seventy
of pneumonia lesions and decreased the
number of organisms needed to induce
pneumonia, no effect on physical
clearance, decreased mycoplasmal
killing and increased growth, no effect
on specific IgM in serum, C57B1/6N
mice generally more sensitive than
C3H/HeN mice At 10 ppm, one
strain (C57B1/6N) of mice had
increased mortality
Clearance of bactena from lungs of
10-, 15-, and 35-ppm groups delayed
or prevented All three animals in
highest exposed group died
Reference
Parker et al (1989)
Henry et al (1969)
TF = Female
NS = Not stated
K pneumoniae = Klebsiella pneumoniae
M = Male
S aureus = Staphylococcus aureus
C X T = The product of concentration and time
-------
invading organism For example, exposure to > 7,520 jwg/m (4 0 ppm) NO2 for 4 h after
bacterial challenge depressed lung bactericidal activity in mice against deposited S aureus,
but it required a concentration of 18,800 to 37,600 /t*g/m3 (10 to 20 ppm) before the lung's
ability to kill Pasteurella and Proteus was unpaired These effects became more significant
with increasing concentrations
The combination of corticosteroid (subcutaneous injection) and NO2 exposure (4 h)
significantly impaired the intrapuhnonary killing of staphylococci at concentrations of
> 4,700 /i*g/m3 (>2 5 ppm) (Jakab, 1988) Without corticosteroids, bactericidal activity was
•5
only decreased at >9,400 jwg/m No NO2- and corticosteroid-related effects on
o
intrapuhnonary killing were noted in mice exposed to 1,880 /jg/m (1 0 ppm) NO2 These
results would demonstrate that such a pretreated host is more susceptible to the effects of
NO2 The implication of this finding is the probable existence of a high-risk population
(because of immunosuppression, chronic lung disease, or old age) whose altered host status
makes them more susceptible to infection following NO2 exposure (Green, 1970)
Parker et al (1989) made similar observations in mice exposed for 4 h to 9,400 or
<1
18,800 /ig/m (5 or 10 ppm) NO2 and infected with Mycoplasma pulmonis The higher
concentration of NO2 increase mortality Both concentrations reduced lung bactericidal
activity and increased the number of bacteria within the lung, producing an increase in the
incidence and seventy of munne respiratory mycoplasmosis lesions There was no effect on
physical clearance When NO2 exposure was decreased (940, 1,880, or 3,760 jug/m , 0 5,
1.0, or 2 0 ppm), using the same exposure model, the bactericidal activity of the lungs was
not affected, although 9,400 /-cg/m3 (5 0 ppm) decreased bactericidal activity (Davis et al,
1992)
Differences in species susceptibility to NO2 or to a pathogen may play a role in the
enhancement in mortality seen in experimental animals An enhancement in mortality was
noted in mice, hamsters, and monkeys exposed to NO2 for 2 h followed by a challenge of
K pneumomae (Ehrlich, 1975) However, differences in susceptibility were noted between
the species Squirrel monkeys exposed continuously to NO2 levels of 9,400 and
18,800 /*g/m3 (5 0 and 10 ppm) for 2 and 1 mo, respectively, showed increased mortality
following a challenge with K pneumomae and reduced lung clearance of viable bacteria
(Henry et al , 1970). Two of seven monkeys exposed 1o 9,400 /tg/m3 for 2 mo died, and the
13-45
-------
rest had bacteria in the lungs on autopsy Ehrlich (1975) found that hamsters exhibited
enhanced mortality after exposure for 2 h to NO2 concentrations of > 65, 800
ppm), but not at 9,400 to 47,000 jwg/m3 (5 to 25 ppm) The mouse was the most
sensitive to NO2 exposure, as evidenced by enhanced mortality following a 2-h exposure to
6,580 /ig/m3 (3 5 ppm), but not to 2,820 to 4,700 ^g/m3 (1 5 to 2 5 ppm) (Ehrlich, 1975)
Purvis and Ehrlich (1963) also reported no effect on mortality in mice exposed for 2 h to
2,820 or 4,700 /ig/m3 (1 5 or 2 5 ppm), increase mortality occurred at 6,580 jug/m3
(3.5 ppm) and higher However, when Streptococcus sp was the infectious agent, a 3-h
exposure to 3,760 jug/m (2 0 ppm) NO2 caused an increased in mortality in mice (Ehrlich
et al., 1977)
Squirrel monkeys exposed to 9,400 or 18,800 /xg/m3 (5 or 10 ppm) NO2 for 2 or 1 mo,
respectively, also showed increased susceptibility to a laboratory induced viral influenza
infection (Henry et al , 1970) All six animals exposed to the highest concentration died
within 2 to 3 days of infection with the influenza virus At the lower concentration, one of
three monkeys died Susceptibility to viral infection was enhanced when the NO2 exposure
occurred 24 h after the infectious challenge
McGrath and Smith (1984) investigated whether changes in susceptibility to bacterial
infections in mice were related causally to the effects of NO2 on respiration Because no
spontaneous changes in respiratory patterns were produced following a 3-day exposure to
<3
9,400 /xg/m (5 0 ppm) NO2, the authors concluded that any such relationship was unlikely
Refer to Section 13 2 2 3 on pulmonary function for details of the study
The importance of the test microorganism used with the infectivity models was also
demonstrated by Sherwood et al (1981) These researchers illustrated that exposure to
q
1,880 [Ag/m (1.0 ppm) NO2 for 48 h increased the propensity of virulent group C
streptococci, but not S aureus, to proliferate within the lungs of mice and cause earlier
mortality
The relationships of concentration and time to susceptibility to respiratory infection and
to subsequent mortality in infections with Streptococcus sp were examined by Gardner et al
(1977a,b) When NO2 concentrations were varied from 2,820 to 52,600 ^g/m3 (1 5 to
28 ppm) and the duration of exposure varied from 0 25 to 4 7 h so that C X T equaled a
value of 7 ppm-h, exposure to high concentrations of NC^ for brief penods of time resulted
13-46
-------
in greater mortality than did prolonged exposures to lower concentrations (Gardner et al ,
1977b) This indicated that susceptibility to infection v/as influenced more by the
concentration of NO2 than by the duration of the exposure
In the same study (Gardner et al , 1977a,b), a linear exposure-duration response was
'•j
observed in mice exposed to from 940 to 52,640 /wg/nT (0 5 to 28 ppm), indicating that
mortality increases with increasing length of exposure to a given concentration of NO^
(Figure 13-1) Mortality also increased with increasing concentration of NO2, as indicated
by the steeper slopes with higher concentrations When C x T was held constant, the
relationship between concentration and time produced significantly different mortality
responses At a constant C x T of approximately 21 ppm-h, a 14-h exposure to
2,820 /ig/m (1 5 ppm) NO2 increased mortality by 12 5%, whereas a 1 5-h exposure to
26,300 jtig/m3 (14 ppm) NO2 enhanced mortality by 58 5% These results confirmed that
concentration is more important than tune in determining the degree of injury induced by
NO2 in this model According to Larsen et al (1979), NO2 modeling studies have shown
that the concentration (c) of NO2 expected to cause a certain mortality level (z) as a function
of the hours of exposure (t) can be expressed as c = 9 55 (2 42)zt"° 33
Gardner et al (1979) and Coffin et al (1977) also compared the effect of continuous
versus intermittent exposure to NO2 followed by bacterial challenge with Streptococcus sp
Mice were exposed either continuously or intermittently (7 h/day, 7 days/week) to 2,820 or
6,580 /ig/m (1 5 or 3 5 ppm) NO2 The results of continuous and intermittent exposure to
6,580 /xg/m (3 5 ppm) for periods up to 15 days indicated that there was a significant
increase in mortality for each of the experimental groups with increasing duration of
exposure When the data were adjusted for the difference in C X T, the mortality was
essentially the same for the continuous and intermittent groups The continuous exposure of
o
mice to 2,820 fug/m NO2 increased mortality after 24 h of exposure During the first week
of exposure, the mortality was significantly higher in mice exposed continuously to NO2 than
in those exposed intermittently By the 14th day of exposure, the difference between
intermittent and continuous exposure became indistinguishable This suggests that fluctuating
levels of NO2 may ultimately be as toxic as sustained high levels (Gardner et al , 1979)
3
When mice were exposed continuously or intermittently (6 or 18 h/day) to 940 ^g/m
(0 5 ppm) NO2 for up to 12 mo, munne resistance to K pneumomae infection was not
13-47
-------
SYMBOL ppmNO2
280
140
• 70
V 35
A 15
• 05
TIME
Figure 13-1. Mortality enhancement for mice exposed to nitrogen dioxide at various
concentrations and for various durations prior to challenge with
streptococci. At all concentrations, prolonged exposure results in
enhanced mortality, but the severity of resistance reduction is more
directly related to concentration.
Source Gardner et al (1977b)
affected during the first month of exposure (Ehrkch and Henry, 1968) Those exposed
continuously exhibited decreased resistance to the infectious agent, as demonstrated by a
significant enhancement in mortality at 3, 6, 9, and 12 mo In another experiment, a
significant enhancement did not occur at 3 mo, but was observed after 6 mo of exposure
After 6 mo, mice exposed intermittently (6 or 18 h/day) to NO2 showed significant increases
in mortality over controls (18%) Only the continuously exposed animals showed increased
mortality (23 %) over controls following 12 mo of exposure After 12 mo of exposure, mice
in the three experimental groups showed a reduced capacity to clear viable bacteria from
their lung This was first observed after 6 mo in the continuously exposed groups and after
9 mo in the intermittently exposed groups These changes, however, were not statistically
tested for significance Therefore, although it is not possible to directly compare the results
of the studies using Streptococcus sp to those using K pneumomae, the data suggest that as
the concentration of NO2 is decreased, a longer exposure time is necessary for the
13-48
-------
intermittent exposure regimen to produce a level of effect equivalent to that of a continuous
exposure
A significant increase in percent mortality and a decrease m relative mean survival tune
was noted in mice exposed continuously to 9,400 /*g/m" (5 0 ppm) for 3 days (McGrath and
Oyervides, 1985) However, similar continuous exposure for 24 h/day, 7 days/week to 940,
3
1,880, and 2,820 ftg/m (0 5, 1 0, and 1 5 ppm) NO2 for 3 mo did not produce a difference
in either of these parameters These subchromc exposure results do not agree with Ehrlich
and Henry (1968), who found excess mortality m mice -after continuous exposure to
^
940 fjig/m (0 5 ppm) for 3 mo, but the short-term exposure results do agree with those of
Gardner et al (1979) and Ehrlich (1980) The inconsistency may also be attributed to the
fact that the McGrath and Oyervides (1985) study had 95 % mortality in the control groups,
making it virtually impossible to detect a NO2-induced enhancement in mortality
Purvis and Ehrlich (1963) investigated the persistence of the effect of NO2 using the
infectivity model They found a significant increase in excess mortality m mice exposed for
3
2 h to 9,400 jig/m (5 0 ppm) NO2, followed by a challenge with K pneumomae They
reported that the increased mortality occurred when the infectious challenge was given 1 or
6 h postexposure, but was no longer present if the infeclious challenge was given 27 h after
the animals were removed from the inhalation chambers
Gardner (1980), Gardner et al (1982), and Graham et al (1987) reported further
investigations on the response of mice to airborne infections during or following intermittent
exposure to NO2 These studies investigated the toxicity of NO2 peak exposures
superimposed on a lower continuous background level of NO2 Such a regimen
approximates the pattern of exposure that humans receive in the urban environment Mice
were exposed to NO2 peaks of 8,460 /*g/m3 (4 5 ppm) for 1, 3 5, or 7 h and then were
challenged with Streptococcus sp either immediately or 18 h postexposure Mortality was
proportional to the duration of the peak when the mice were exposed to bacteria immediately
following NO2 exposure, but mice had recovered from the exposure by 18 h When a peak
3 ^
exposure of 8,460 /-cg/m was superimposed on a continuous background of 2,820 jwg/m
(1 5 ppm) for 64 h preceding and 18 h following the peak, mortality was significantly
enhanced by a peak exposure lasting 3 5 or 7 h when the infectious agent was administered
18 h after the peak exposure Possible explanations for these differences due to the presence
13-49
-------
or absence of a background exposure are that mice continuously exposed are not capable of
recovery or that new AMs or PMNs recruited to the site of infection are unpaired by the
continuous exposure to NO2 The effect of multiple peaks was examined by exposing mice
for 2 weeks to two daily 1-h peaks (morning and afternoon) (5 days/week) of 8,460
superimposed on a continuous background (7 days/ week) of 2,820 /*g/m NO2 Mice were
exposed to the infectious agent either immediately before or after the morning peak exposure
When the infectious agent was given before the morning peak exposure, the increase in
SJ
mortality did not closely approach that of a continuous exposure to 2,820 jwg/m NO2
However, in mice exposed after the morning peak, by the second week of exposure, the
increased mortality over controls approached that equivalent to continuous exposure to
2,820 /ig/m NO2 These findings demonstrate that the pattern of exposure determines the
response and that the response is not predictable based on a simple C x T relationship
Further investigations into the effects of NO2 on munne antibacterial lung defenses
have been conducted using a peak to baseline ratio of 4 1, which is not uncommon in the
urban environment (Miller et al , 1987) For 1 year, mice were continuously exposed
23 h/day, 7 days/week to a baseline of 376 /*g/m (0 2 ppm) or to this baseline level on
which a 1-h peak of 1,500 /*g/m (0 8 ppm) NO2 was superimposed two times a day,
5 days/week The animals exposed to the baseline level did not reveal any significant
treatment-related effects, however, the infectivity mortality of the mice exposed to the
baseline plus peak regimen was significantly greater than that of either the NO2 background
exposed mice or the control mice This chronic study indicates that short-term peaks of NO2
can cause detectable effects on antibacterial lung defenses This baseline and peak exposure
also affected pulmonary function (see Section 13 2 2 3) (Miller et al , 1987)
Mice exposed continuously for 3 mo to 564 to 940 /^g/m3 (0 3 to 0 5 ppm) NO2
followed by a challenge with A/PR/8 influenza virus showed significant pulmonary
pathological responses (Motomiya et al , 1973) A greater incidence of adenomatous
proliferation of bronchial epithelial cells resulted from the combined exposures of virus plus
NO2 than with either the viral or NO2 exposures alone Continuous NO2 exposure for an
additional 3 mo did not enhance the effect of NO2 or the subsequent virus challenge
Ito (1971) challenged mice with influenza A/PR/8 virus after continuous exposure to
940 to 1,880 /ig/m3 (0 5 to 1 0 ppm) NO2 for 39 days and to 18,800 /ig/m3 (10 ppm) NO2,
13-50
-------
2 h daily for 1, 3, and 5 days Acute and intermittent exposure to 18,880 jug/m NO2, as
3
well as continuous exposure to 940 to 1,880 jwg/m NO2, increased the susceptibility of mice
to influenza virus, as demonstrated by increased mortality Further, when isolated hamster
tracheal organ explants were exposed in vitro for 1, 2, and 3 weeks to 3,760 /tg/m3
(2 0 ppm), 1 5 h/day for 5 days/week and then immediately infected with influenza virus
(A/PR/8/34), the maximum virus titer reached was the same for both the exposed and
unexposed explants However, NO2 exposure caused the peak virus production to occur
earlier (Schiff, 1977)
The lower respiratory tract of mice became significantly more susceptible to munne
cytomegalovirus infection after 6-h exposures for 6 days to 9,400 jttg/m3 (5 0 ppm), but not
<4,700 /*g/m3 (<2 5 ppm) NO2 (Rose et al, 1988, 1989) Viral replication was routinely
"3
increased in the lungs of mice exposed to 9,400 /xg/m NO2 over that of controls There was
also an increase in the incidence of lung lesions in the NO2-exposed mice The NO2-exposed
animals could be infected with an inoculum 100 tunes less than that required to infect air-
exposed animals. Further, the NO2-exposed animals were more susceptible to viral
reinfection, whereas the air-exposed controls appeared to resistant to reinfection However,
AM antiviral capacity did not appear to be altered Humoral virus-specific antibody titers
were not affected by NO2, nor were splenic lymphocyte proliferative responses to viral
antigens
Exposure to a NO2 concentration of 9,400 jwg/m (5 0 ppm) did not significantly alter
the course of a parainfluenza (munne sendai virus) infection in mice as measured by the
infectious pulmonary virus titers in the lungs However, this concentration of NO2, when
combined with the virus exposure, did increase the seventy of the pulmonary disease process
(viral pneumomtis) (Jakab, 1988)
Only one human clinical study has examined the effect of NO2 on infectivity rate
(Goings et al, 1989) Subjects were exposed to 1,880, 3,760, or 5,640 /*g/m3 (1.0, 2.0, or
3 0 ppm) NO2 for 3 consecutive days (2 h/day) and inoculated uitranasally with attenuated
influenza A/Korea/reassortment virus on the second of these days No statistically significant
effects were observed, although there was a trend towards an increase in infectivity rates
However, this study had a low statistical power to detect small changes See Section 15 6
for an expanded discussion of this study
13-51
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Stress, in addition to influencing the lethality of a particular exposure concentration, has
been shown to enhance the toxic effect of NO2 Mice placed on continuously moving
O -2
exercise wheels during exposure to 5,640 jug/m (3 0 ppm) NO2, but not 1,880 /wg/m
(1 0 ppm), for 3 h showed enhanced mortality over nonexercised NO2-exposed mice using
the streptococcal infectivity model (Tiling et al, 1980) The presence of other environmental
factors, such as O3 (Ehrlich et al, 1977, Gardner, 1980, Gardner et al , 1982, Graham
et al, 1987), elevated temperatures (Gardner et al , 1982), or tobacco smoke (Henry et al,
1971), also augments the effect of NO2 on host resistance to infection When mice were
stressed by elevated temperature (32 °C) and exposed to 2,820 jwg/rn3 (1 5 ppm) NO2, there
was a significant enhancement in mortality rate after 7 days of exposure (Gardner et al,
1982)
Summary
The host defense system is one of the many potential targets whose function has been
shown to be altered significantly by exposure to NO2 Evidence would indicate that any
breach in these defenses should be considered as a possible indicator of an increased nsk of
infectious pulmonary and/or systemic disease
As discussed in the section on lung morphology, NO2 causes structural alterations in
the ciliated cells of the airways, however, significant impairment of tracheobronchial
clearance rates generally were not seen at levels <, 18,800 /*g/m3 (10 ppm) NO2 (Schlesinger
et al, 1987a,b) This would indicate that even a severely damaged airway epithelium (i e ,
loss of cilia) still has the ability to maintain mucus transport at a normal rate, and that the
exposure of experimental animals to NO2 would have to be to concentrations
> 18,800 jtg/m to induce any significant alterations that would have detrimental health
effects.
Within the pulmonary region of the lung, the primary cellular defense affected by both
acute and long-term exposure to NO2 is the AM Nitrogen dioxide causes a depression of
phagocytic activity, reduces cell viability, disrupts membrane integrity, reduces the total
number of available cells, produces morphological changes, and decreases bactericidal
activity Although a few of these effects were seen following exposure to concentrations
< 1,880 ftg/m3 (1 0 ppm) (Schlesinger, 1987b, Rose et al , 1989), most of the studies
13-52
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showed effects at concentrations between 1,880 and 9,400 jug/m3 (1 0 and 5 0 ppm) NO2
(Goldstein et al , 1973, Greene and Schneider, 1978, Hooftman et al, 1988, Dowell et al,
1971, Suzuki et al, 1986, Acton and Myrvik, 1972) Evidence indicates that these cells are
no longer capable of isolating, transporting, detoxifying, or clearing inhaled substances
The systemic cell-mediated and humoral immune system is also a target for NO2, as
evidenced by experimental animal studies The immunological effects reported seem to be
variable Some studies show effects and others do not It has been suggested that long-term
exposure may result in a suppression of the various humoral and cell-mediated functions,
whereas shorter exposures may cause an enhancement of immunological activity The
response seems to be dependent not only on concentration and duration of exposure, but also
on animal species and the specific immunological end point measured Research on the
systemic immune system in mice, guinea pigs, and monkeys mdicates that subchronic and
chronic exposure at or below 1,880 jwg/m3 (1 0 ppm) can suppress T- and B-cell
responsiveness to mitogens and can decrease the number of T cells (Richters and Damji,
1988, 1990, Maigetter et al, 1978) Nitrogen dioxide influences the production of serum-
neutralizing antibodies to viruses and humoral primary antibody response to SRBCs
(Fujimaki et al, 1982, Ehrlich et al, 1975, Fenters et al , 1973) Other immunological
effects attributed to NO2 include an increase in IgM and IgG, and a decrease in IgA serum
levels (Ehrlich et al, 1975) The significance of many of these changes is uncertain, and
studies conducted at lower levels of exposure and for longer periods of tune are needed to
improve our understanding of these immunological responses In the absence of adequate
data, one can only speculate that if NO2 affects the systemic immune system, it is likely that
it also would affect the pulmonary immune system
The consequence of suppression of the various host defense mechanisms would
ultimately lead to increased microbial proliferation within the lung, resulting in increased
incidence and seventy of pulmonary infections Experimental animal studies have
demonstrated that both acute and chronic exposures to NO2 can significantly increase
susceptibility to viral and bacterial infections The exact exposures producing such effects
are dependent upon the animal species, the microbial species/strain, and the model used
The infectivity model, in which air- and NO2-exposed mice are challenged with a viable
microbial aerosol and mortality is measured, is the most sensitive For example, a 39-day
13-53
-------
a
exposure to 940 pg/m (0 5 ppm) NO2 increased influenza-induced mortality (Ito, 1971), and
«a
a 6-mo exposure to 940 /ig/m (0 5 ppm) NO2 uicreased bactenal-mduced mortahty (Ehrhch
and Henry, 1968) After an acute exposure, 3,760 /*g/m3 (2 0 ppm) is the lowest level
tested to produce significant mortality for the bacterial model (Ehrhch et al, 1977, Ehrhch,
1980)
The mouse streptococcal infectivity model has been applied extensively to elucidate
C X T relationships (Gardner et al, 1977a,b) Exposure concentration has a predominant
influence over that of exposure duration In the urban air, the typical pattern of NO2 is a
low-level baseline exposure on which peaks are superimposed corresponding to peaks of NOX
mobile source emissions When the relationship of the peak to baseline exposure and of
enhanced susceptibility to bacterial infection was investigated, the results indicated that no
simplistic C X T relationship was present, and that peaks had a major influence on the
outcome (Gardner, 1980, Gardner et al, 1982, Graham et al, 1987) When one compares
the effect of a subchromc contmuous one-level exposure to an exposure consisting of baseline
and peaks having a lower C x T, the effect was roughly equivalent In a 1-year chronic
3
study with the infectivity model, the effect of a 376 /*g/m (0 2 ppm) NO2 baseline exposure
-^
(21 h/day, 7 days/week) was compared to baseline plus two daily 1-h peaks of 1,504 jwg/m
(0.8 ppm) NO2 for 5 days/week Only the basehne-plus-peak group exhibited significant
increased susceptibility to bacterial infection (Miller et al, 1987)
The effects associated with NO2 exposure on the host defense system are dependent on
the concentration of the gas, the duration of the exposure, the animal species tested, and the
specific end point of toxicity measured As stated earlier, basic defense mechanisms are
common across mammalian species Thus, the summation of the effects on a number of host
defense systems may make the mammalian host more vulnerable to infectious disease
Although the outcome measured in animals is mortality, morbidity would be expected to
occur first or occur at exposures too low to induce mortahty In humans, especially those
under medical treatment, such a loss in pulmonary defenses would be expected to result in an
increased incidence of morbidity, especially in that segment of the population that may be
more susceptible, such as young children or the elderly In assessing or predicting such
human risk from experimental animal data, it is understood that in humans, different levels
of exposure to NO2 may be required to produce effects similar to those seen in animals
13-54
-------
13.2.2.2 Lung Biochemistry
Studies of lung biochemistry in animals exposed to NO2 have focused on either the
putative mechanisms of toxic action of NO2 or on detection of indicators of NO2-induced
tissue and cell damage One theory to explain NO2 toxicity is that NO2 initiates kpid
peroxidation in unsaturated fatty acids in membranes of target cells (Menzel, 1976) These
changes are thought to cause cell injury or death, and the symptoms associated with NO2
inhalation An alternate theory is that NO2 oxidizes water-soluble, low molecular weight
reducing substances and proteins, resulting in a metabolic dysfunction that evidences itself as
the toxic symptom (Freeman and Mudd, 1981) Nitrogen dioxide may, in fact, act by both
means and, as a consequence, may affect the intermediary metabolism of animals and their
growth and maturation Further, given that NO2 dissolves in water to produce HONO and
HNO3, the possibility of an acid or a pH effect as a primary or secondary mechanism of
injury should also be considered
Lipid Metabolism
Table 13-5 summarizes the effects of NO2 on lipids
Various investigators have performed in vitro experiments on isolated lung cells or
subcellular components to examine the effects of NO2 on oxidation of unsaturated fatty acids
In a series of studies, Patel and Block (1986a,b) and Patel et al (1988) examined the effects
of NO2 exposure on cultured endothelial cells from either pulmonary arteries or aortae of
o
pigs After exposing the cells to 9,400 jttg/m (5 0 ppm) NO2 for 24 h, they observed
changes in membrane fluidity, lipid peroxide formation, 5-hydroxytryptamine uptake, release
of LDH (a marker for cell membrane abnormalities) and increases in GSH reductase and
G-6-P dehydrogenase activities The authors concluded that injury of endothelial cells by
NO2 can be ascribed to decreased membrane fluidity secondary to lipid peroxidation, and
that the altered physical state of the cell membrane causes impaired functionality of the
membrane, leading to cellular abnormalities of metabolism and biochemistry
Sekharam et al (1991) found that oxidative damage in endothelial cells (from the
pulmonary artery of pigs) exposed in vitro to 9,400 /ig/m (5 0 ppm) for 48 h
Phospholipase A^ activity was increased in the cell membrane, but not in the mitochondnal
13-55
-------
TABLE 13-5. EFFECTS OF NITROGEN DIOXIDE ON LIPID METABOLISM3
ON
NO2 Concentration
/tg/m3
75
752
7,520
75
225
752
752
2,256
7,520
752
1,880
5,640
9,400
9,400
752
1,000
10,000
1,880
1,880
5,640
18,800
ppm
00«l
04
40
004
0 12
04
04
12
40
04
10
30
50
50
04
053
53
10
1
30
10
Exposure
Continuous,
9, 18, or 27 mo
Continuous,
6, 9, and 18 mo
Continuous,
1, 2, 4, 8, 12,
16 weeks
72 h
3h
72 h or 1 week
5 h/day, 21 days
Continuous,
2 weeks
2h
Species
Gender Age (Strain)
M 8 Rat
weeks (Wistar)
M 13 Rat
weeks (Wistar)
M NS Guinea pig
(Hartley)
F NS Rat
(NS)
Rabbit
M 15-16 Rabbit
weeks (New Zealand)
Effects
Increased lipid peroxidation (TEA method) at 4 0 ppm after
9 mo and at 0 4 and 4 0 ppm after 18 mo, increased ethane
exhalation at all levels at 9 and 18 mo, ethane exhalation
returned to normal levels in highest group by 27 mo, no
changes in total lipid, phosphokpid, total cholesterol, or
tnglycende contents TBA reactants increased at 4 ppm
(9 mo) and <0 4 ppm (18 mo)
Increased ethane exhalation after 9 and 18 mo
Increased ethane exhalation and TBA-reactive substances
during first week of exposure, returned to normal levels by
fourth week, tendency towards increase thereafter
No effect at 0 4 ppm, increase in lung lipid content in vitamin
C-depleted, but not normal, animals at 1 0 ppm and above
Increased lung lipid content in vitamin C-depleted guinea pigs
No effect on lung lavage fluid composition in normal or
vitamin C-depleted animals
Increase in lipid peroxidation products
Decrease in lecithin synthesis after 1 week, less marked
depression after 2 weeks
1 ppm elevated thromboxane 62 3 ppm depressed
thromboxane 62 10 ppm depressed 6-keto-PGFj,.,, and
thromboxane 82, no changes noted in PGE2, PGE2a, or
LTB4
Reference
Sagaietal (1984)
Ichinoseetal (1983)
Ichinose and Sagai
(1982)
Ichinose etal (1983)
Belgrade et al (1981)
Balabaeva and
Tabakova (1985)
Setoetal (1975)
Schlesinger et al
(1990)
-------
TABLE 13-5 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON LIPID METABOLISM3
NC>2 Concentration
jttg/m
5,450
5,640
18,800
5,640-
30,080
h- 7,520
•^ 9,400
9,400
10,300
18,800
18,800
ppm
29
30
10
3-
16
40
50
50
55
10
10
Exposure
Continuous,
5 days/week,
9 mo
Continuous,
17 days
Continuous,
4 weeks
Ih
3h
24 h
48 h
3 h/day, 7 and
14 days
12 h
1, 3, 5, 7,
14 days
Gender Age
M NS
M NS
NS NS
M/F 21-33
years
NS 6-7 mo
NS 6-7 mo
M 8
weeks
M NS
M NS
Species
(Strain)
Rat
(Long Evans)
Rat
(Sprague-
Dawley)
Dog
(Beagle)
Human
Pig
(in vitro)
Pig
(in vitro)
Rat
(Wistar)
Rat
(Wistar)
Rat
(NS)
Effects
Increase in lung wet weight (12 7%) and decrease in total
hpids (8 7%), decrease in saturated fatty acid content of
BAL fluid and tissue, increase in surface tension of BAL
fluid
Decrease in linoleic and linolemc acid content of BAL
fluid
Decrease in polyunsaturated fatty acids in BAL and lung
tissue
Decrease in phospholipid content of BAL fluid from
animals with NC^-induced intraalveolar edema, increase
in unsaturated fatty content of BAL lecithin at S5 0 ppm
Increased lipid peroxidation products in BAL fluid
In endothehal cells of pulmonary artery and aorta,
changes in membrane fluidity, hpid peroxide formation,
release of LDH, and 5-hydroxytryptamine uptake
Lipid alterations in endothehal cells from pulmonary
artery
Decrease in lysolecithin acyltransferase in lung
homogenate microsomes
Changes in fatty acids of BAL phosphohpids
Changes in prostaglandins and thromboxane 62 in BAL
fluid
Reference
Arner and Rhoades (1973)
Menzeletal (1972)
Dowelletal (1971)
Mohsenin (1991)
Patel and Block (1986a,b)
Pateletal (1988)
Sekharametal (1991)
Yokoyamaetal (1980)
Kobayashi et al (1984)
Kobayashi (1986)
"M = Male
TEA = Thiobatbitunc acid
NS = Not stated
F = Female
6-keto-PGFiQ, = 6-keto-prostaglandmFjQ,
PGEj = Prostaglandm E2
PGE^ = Prostaglandm ~B^a
LTB4 = LeukotneneB4
LDH = Lactate dehydrogenase
BAL = Bronchoalveolar lavage
-------
or microsomal membranes There was a significant increase in lyso-phosphatidyl-
ethanolamine and a significant decrease in phosphatidylethanolamine as a result of the
increase in phospholipase A± activity Phosphatidylsenne content was also increased
However, the total phosphokpid content of the plasma membrane was decreased compared to
that of controls The authors suggested a possible association between the increased activity
of phospholipase Aj and the increase in phosphatidylsenne seen in the cell membrane of the
NO2-exposed endothekal cells after treating endothehal cells with exogenous
phosphatidylsenne and observing an 87% increase in phospholipase Aj activity in the cell
membrane
Rietjens and co-workers (1986, 1987) exposed cultured rat AMs to NO2 or O3 by gas
diffusion through a Teflon® film Based on various experiments using different radical
scavengers, the authors concluded that NO2 and O3 acted by different mechanisms, whereby
NO2 exerted its toxicity via a free radical-mediated peroxidative pathway and O3 via a
pathway involving the formation of lipid ozomdes Both NO2 and O3 appear to act at the
level of hpid oxidation in causing AM toxicity However, is should be noted that the
chemical reactions of NO2 or O3 with organic compounds in aqueous solutions can be very
complex (Glaze, 1986), and prediction of which pathway(s) may predominate in complex
biological systems is by no means straightforward For example, NO2 or O3 may react with
unsaturated fatty acids, a component of lung phosphohpids, but also with water-soluble
reducing substances of low molecular weight or reducible groups on proteins A discussion
of the functional and structural effects of NO2 on AMs appears in Sections 13221 and
13.2.2 4 on host defense mechanisms and morphological effects, respectively
Roehm et al (1971) studied the in vitro oxidation of unsaturated fatty acids by NO2 and
O3. Both NO2 and O3 initiated the oxidation of unsaturated fatty acids through free radicals
Typically, an induction period was noted with either anhydrous thin films or aqueous
emulsions of linolenic acid exposed to 2,820 /*g/m3 (1 5 ppm) NO2 The addition of free
radical-scavenging agents such as vitamin E, butylated hydroxytoluene, or butylated
hydroxyanisol delayed the onset of oxidation in vitro The rate of oxidation of linolenic acid
in thin films was proportional to concentrations of NO2 from 940 to 10,152 jttg/m3 (0 5 to
5.4 ppm) Thin-layer chromatography of the oxidation products of linolenic acid showed a
conversion to polar rntrogen-containing compounds and to peroxides A suggested
13-58
-------
mechanism of formation of these products (Menzel, 1976) involves addition of NO2 across a
double bond between two carbon atoms in an unsaturated fatty acid to form a mtro compound
and a carbon-centered free radical Such a radical can extract an electron from various
potential electron donors, thereby initiating the chain reaction Nitrohydroperoxides and fatty
acid hydroperoxides are produced in vitro from the oxidation of unsaturated fatty acids by
NO2 Phenolic antioxidants can prevent the autoxidatiori of unsaturated fatty acids by NO2
by reacting with both fatty acid hydroperoxyl free radicals and nitrohydroperoxyl free
radicals generated by the addition of NO2 to unsaturated fatty acids It is not known whether
this sequence of reactions is important in the lung in vivo
Sagai et al (1984) and Ichinose et al (1983) reported an increase in thiobarbitunc acid
(TEA) reactants (an indication of hpid peroxides) in lung homogenates of rats exposed to
3
7,520 jttg/m (4 0 ppm) NO2 continuously for 9 mo When exposure was increased to
18 mo, a concentration-related increase in TBA reactants occurred in rats exposed to 75,
752, and 7,520 jwg/m3 (0 04, 0 4, and 4 0 ppm) NO2, but the increase was only significant in
animals exposed to the two highest concentrations
Mohsenin (1991) reported the effects of NO2 exposure on healthy human subjects
Subjects received either a placebo or vitamins C (1,500 itng/day) and E (1,200 ITJ/day) for
3
4 weeks and were then exposed to 7,520 ftg/m (4 0 ppm) for 3 h In BAL fluid, NO2
exposure decreased the elastase inhibitory capacity of o^-protease inhibitor, the major plasma
and lung protease inhibitor of elastase There was also an NO2-induced increase in lipid
peroxidation products (primarily conjugated dienes, but some malondialdehydes) in the BAL
fluid However, when subjects were supplemented with dietary vitamin C and E, these
effects were prevented
The exhalation of ethane in the breath was measured in assays of in vivo lipid
peroxidation (Sagai et al , 1984, Ichinose et al, 1983) In the first series of studies, excess
mortality in chamber control rats forced the use of room control rats in the statistical
analyses, however, there was no major difference between room and chamber control values
At 75, 752, and 7,520 jwg/m3 (0 04, 0 4, and 4 0 ppm) NO2, 9 and 18 mo of exposure
increased the exhalation of ethane The two lower NO2 concentrations also increased ethane
exhalation after 27 mo of exposure, but ethane was within the control range in the
7,520 /*g/m -exposed group Pentane exhalation was measured to determine if the lipid
13-59
-------
peroxidation was bacterial in origin Pentane was only increased after 18 mo of exposure to
75 and 752 /*g/m NO2, supporting the interpretation of the ethane results In the second
series of experiments, chamber control rats were used and rats were exposed to 75, 225, and
752 /ig/m3 (0 04, 0 12, and 0 4 ppm) NO2 for 6, 9, and 18 mo After 6 mo, ethane
•2
exhalation was only increased in the 752 /xg/m group All NO2 concentrations increased
ethane exhalation after 9 and 18 mo of exposure These studies showed that NO2 increased
lipid peroxidation in a concentration- and exposure duration-related manner An inverse
relationship with lung antioxidant metabolism was also found (see later subsection on
antioxidant metabolism) Shorter duration exposures also influence lipid peroxidation in rats,
as measured by ethane exhalation For example, exposure to ^2,256 jtg/m (> 1 2 ppm)
NO2 increases ethane exhalation after 1 week of exposure Levels of ethane had returned to
control values after 4 weeks of NO2 exposure, at which tune ethane levels began a slow rise
again over the remainder of the 16-week exposure period (Ichinose and Sagai, 1982, Ichinose
et al, 1983) Based on their body of work and other related studies, Sagai et al (1984)
suggested that the increased lipid peroxidation may be related to NO2-induced thickening of
alveolar walls, as reported in some lung morphology studies, and decreased O2 tension in
arterial blood
An increase in lung lipid peroxidation products has also been reported in pregnant rats
exposed to 1,000 /tg/m3 or 10,000 jug/m3 (0 53 or 5 3 ppm) NO2, 5 h/day for 21 days
(Balabaeva and Tabakova, 1985) When the pregnant progeny of this group were exposed to
the same NO2 exposure regimen, there was an exposure-related increase in the lung lipid
peroxides Nitrogen dioxide exposure was also reported to have an effect on lipid
peroxidation in the kver in both pregnant and nonpregnant rats and in the placenta The
findings are discussed later in Section 13 2 3 7
Amer and Rhoades (1973) exposed rats for 9 mo to 5,450 mg/m3 (2 9 ppm) NO2 for
24 h/day, 5 days/week % The lung wet weight increased by 13 % compared to that of
controls. The lipid content of the lung was significantly depressed by about 9 % The total
saturated fatty acid content of the lungs was decreased The largest decrease was seen in the
phosphatidylethanolamine Smaller decreases were seen in lecithin (phosphatidylcholine),
phosphatidylinositol, and phosphatidylsenne Values for specific unsaturated fatty acids of
biological importance were not reported The lung surface tension extracts were reported as
13-60
-------
increased The authors suggested that the increased surface tension corresponded to a
decrease in the lung surfactant concentration
Total lecithin was reduced in lung lavage fluid from beagle dogs with NO2-induced
intraalveolar edema (Dowell et al, 1971) Individual dogs were exposed for 1 h to 13,160
to 30,080 /ig/m3 (7 to 16 ppm) NO2 There was a decrease in total phospholipids, as
compared to neutral lipids, in animals with intraalveolar edema In animals without
intraalveolar edema, exposed to 5,640 to 22,560 jwg/m3 (3 to 12 ppm) NO2 for 1 h, the
phospholipid content was slightly greater than in control animals There was also an increase
in the amount of unsaturated fatty acids in the phospholipids from the lungs of animals
>y
exposed to 9,400 to 30,080 jt-cg/m (5 to 16 ppm) NO2 whether or not mtraalveolar edema
was present These changes were not noted in the animals exposed to 5,640 jwg/m NO2
The physiological effects of NO2 exposure in these animals is discussed in Section 13224
on lung morphology
Lecithin synthesis has also been reported depressed in the lungs of rabbits exposed to
1,880 jwg/m3 (1 0 ppm) NO2 for 2 weeks (Seto et al, 1975) The most marked effect was
observed after 1 week of exposure and appeared to decline after the second week of
exposure Yokoyama et al (1980) found few changes m lipid metabolism of rats exposed
O
for 3 h/day for 7 and 14 days to 10,300 //ig/m (5 5 ppm) NO2 Lysolecithin acyltransferase
activity in the microsomal fraction decreased when an unsaturated acid (linoleic) was used,
but not when a saturated acid (palmitic) was the substrate The supernatant fraction of this
enzyme was unchanged, phospholipase Aj and A2 activities were not affected either
Products of arachidonic acid metabolism are also affected by NO2 The concentration
<>
of thromboxane B2 was elevated in the BAL fluid from rabbits exposed to 1,880 /ig/m
(1 0 ppm) NO2 for 2 h (Schlesinger et al, 1990) When exposure was increased to
3
5,640 /jg/m (3 0 ppm), the concentration of thromboxane B2 was depressed Thromboxane
B2 levels were significantly below those of controls 24 h postexposure m rabbits exposed to
18,880 jwg/m3 (10 ppm) NO2 6-Keto-prostaglandin Fltt was also depressed in rabbits
exposed to 18,880 jwg/m3 NO2 Prostaglandins E2 and F2 and LTB4 were not affected
No effects on BAL lipid and protein content were observed in guinea pigs exposed to
752, 1,880, 5,460, or 9,400 jug/rn3 (0 4, 1 0, 3 0, or 5 0 ppm) NO2 for 72 h (Selgrade
et al, 1981) However, vitamin C-depleted guinea pigs, having an average of 25% of the
13-61
-------
normal blood vitamin C content, had greater BAL protein and lipid content, except for those
« A
guinea pigs exposed to 752 /tg/m NO2 In annuals exposed to 9,400 jiig/m NO2, the
changes in BAL fluid composition were correlated with mortality (50%) and alveolar edema
as determined by conventional light microscopy
Effects on Lung Amino Acids, Proteins, and Enzymes
Table 13-6 summarizes the effects of NO2 on proteins and selected enzymes
Nitrogen dioxide can oxidize various reducible ammo acids or side chains of proteins in
aqueous solution (Freeman and Mudd, 1981) Suzuki et al (1988) reported increased
'j
amounts of trytophan metabolites in the urine of rats exposed for 2 weeks to 9,400 ---'—
3
(5 0 ppm) NC«2 Concentrations of NO2 above 9,400 jwg/m produce lung edema with
concomitant infiltration of serum protein and enzymes Also, an influx of inflammatory cells
(predominantly leukocytes) from blood and alterations in the epithelial cell types of the lung
may occur Thus, some reports of changes in lung enzyme activity and protein content may
reflect either edema, altered inflammatory cell populations, and/or changes in cell types,
rather than direct effects of NO2 on lung cell enzymes
As indicated earlier in this section, Sagai et al (1984) reported a concentration-related
increase in TEA reactants in the lungs of rats exposed to 75, 752, and 7,520 /xg/m
(0.04, 0 4, and 4 0 ppm) NO2 continuously for 18 mo However, total lung protein content
was not affected by the NO2 exposure Gelzleichter et al (1992a) investigated the effect of
C X T on total BAL protein, PMNs, and epithelial cells in rats Experimental animals were
exposed to 6,770, 13,500, 20,300, or 27,100 jug/m3 (3 6, 7 2, 10 8, or 14 4 ppm) NO2 for
24, 12, 8, or 6 h, respectively, for 3 consecutive days The cumulative C x T was
o
259 2 ppm-h Concentrations ^ 13,500 /xg/m increased BAL protein to a roughly
equivalent extent The 24-h exposure to 6,770 /xg/m3 caused no effects Epithelial cell
increases followed a pattern similar to that of protein, but PMNs were only increased at the
highest concentration
Nitrogen dioxide has also been reported to increase the protein content of lung lavage in
vitamin C-depleted guinea pigs (Selgrade et al, 1981, Sherwin and Carlson, 1973, Hatch
et al., 1986, Slade et al, 1989) Selgrade et al (1981) found effects as low as 1,880 /*g/m3
13-62
-------
TABLE 13-6. EFFECTS OF NITROGEN DIOXIDE ON LUNG AMMO ACIDS, PROTEINS, AND ENZYMES8
NC>2 Concentration
/tg/m
752
1,880
5,640
9,400
9,400
752
752
752
2,256
7,520
752
2,260
7,520
845
935
ppm
04
10
30
50
50
04
04
04
1 2
40
04
12
40
045
047
Exposure
72 h
3h
Continuous,
1 week
Continuous,
1 week
1 to 14 weeks
7 days
7 h/day,
4 weeks
Continuous,
10, 12, 14 days
A Species
Gender Age ,0f .
6 (Strain)
M NS Guinea pig
(Hartley)
M NS Guinea pig
(NS)
M 22-24 Rat
weeks (Wistar)
M 10 Rat
weeks (Wistar)
M NS Mouse
(Swiss
Webster)
M NS Mouse
(NS)
Effects
No effect at 0 4 ppm, increase in BAL
protein in vitamin C-depleted, but not
normal, animals at 1 0 ppm and above
Increased BAL protein in vitamin C-
depleted guinea pigs 15 h postexposure
No effect on BAL protein
Increased lung protein content of guinea
pigs with an unquantified vitamin C
deficiency
Complex concentration and duration
dependence of effects Example at
0 4 ppm, cytochrome P-450 levels
decreased at 2 weeks, returned to control
level by 5 weeks At 1 2 ppm,
cyiochrome P-450 levels decreased
initially, increased at 5 weeks, and
decreased at 10 weeks Effects on
succinate-cytochrome c reductase also
Decrease in cytochrome P-450 levels at
1 2 ppm
No changes in lung serotonin levels,
increase in brain serotonin and
5-hydroxyrndoleacetic acid content
Increased content of serum protems in
homogenized whole lung tissue
Reference
Selgradeetal (1981)
Sherwin and Carlson
(1973)
Takahashi et al (1986)
Mochitate et al (1984)
Sherwin et al (1986)
Sherwin and Layfield
(1976)
-------
TABLE 13-6 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON LUNG AMMO ACIDS, PROTEINS, AND ENZYMES8
NO2 Concentration
ftg/m3
940
1,880
1,880
14,100
28,200
47,000
56,400
1,880
9,400
1,880 base +
9,400 peak
3,760
3,760
7,500
18,800
5,640
ppm
05
10
10
75
15
25
30
10
50
1 0 base +
50 peak
20
20
40
10
30
Exposure Gender Age
6 h/day, M NS
5 days/week,
4 weeks
6 h/day,
2 days
7 h/day, M/F 14-16
5 days/week, weeks
up to 15 weeks
Base 7 h/day,
5 days/week,
2 1 5-h peaks/day
up to 15 weeks
Continuous, M NS
1-3 weeks
14 days M 12-24
10 days weeks
7 days
7 days M/F 8 weeks
Species
(Strain)
Rat
(Fischer 344)
Rat
(Fischer 344)
Guinea pig
(NS)
Rat
(Wistar)
Rat
(Sprague-
Dawley)
Effects
0 5 ppm increase in urinary hydroxylysine output starting
during Week 1, BAL hydroxylysine level, angiotensin-
converting enzyme level, and BAL protein content
unchanged
1 0 ppm gradual increase in urinary hydroxylysine output,
becoming significant the week after exposure ended, BAL
hydroxylysine level lower following exposure and 4 weeks
postexposure, angiotensin-converting enzyme level increased
Concentration-dependent increase in urinary hydroxylysine
output and BAL hydroxylysine content, but only significant
at S7 5 ppm and 15 ppm, respectively, angiotensin-
converting enzyme levels and BAL protein increased in
highest-exposed groups
Changes in BAL and tissue levels of enzymes early in
exposure, resolved by 15 weeks
Increase in number of LDH-positive cells with time of
exposure Suggests Type 1 cells decrease as Type 2 cells
increase
Increased activity of lung glycolytic enzymes
Various changes in lung homogenate protein and DNA
content and enzyme activities, changes more severe in
vitamin E-deficient rats
Reference
Evans et al
(1989)
Gregory et al
(1983)
Sherwin et al
(1973)
Modulate et
al (1985)
Elsayed and
Mustafa (1982)
-------
TABLE 13-6 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON LUNG AMEVO ACIDS, PROTEINS, AND ENZYMES3
ON
NO2 Concentration
fig/m
6,770
13,500
20,300
27,100
7,520
18,800
7,520
18,800
47,000
8,100
9,020
9,020
9,400
9,400
9,400
ppm
36
72
108
144
40
10
40
10
25
45
48
48
50
50
50
Exposure
24 h
12 h
8h
6h
10 days
7 days
6h/day,
5 days/week,
7, 14, and
21 days
16 h
3h
8h/da>,
7 days
14-72 h
2 weeks
6 h/day,
6 days
Gender Age
M 10-12
weeks
M 21-24
weeks
M NS
M/F NS
M
M 8 weeks
F NS
M 5 weeks
NS 4-6
weeks
Species
(Strain)
Rat
(Sprague-
Dawley)
Rat
(Wistar)
Rat
(Wistar)
Guinea pig
(Hartley)
Mouse
(Swiss
Webster)
Mouse
(NS)
Rat
(Fischer 344)
Mouse
(CD-I)
Effects
Increased BAL protein at > 7 2 ppm
Initial decrease in lung protein content
followed by an increase, changes in
nucrosomal enzyme activities
Increased gamma-glutamyl transferase on
Days 14 and 21, no consistent effect on
alkaline phosphatase, LDH, or total
protein
Increased lung wet weight, alterations in
lung antioxidant levels in vitamin C-
deficient animals
Increased lung lavage fluid protein content
in vitamin C-deficient animals
No significant changes in mng homogenate
parameters
Increase in lung protein (14 to 58 h) by
radioactive label incorporation
Increased amounts of the trytophan
metabolites and xanthuremc and kynuremc
acids excreted m urine during Week 2 of
exposure, but had returned to normal
levels by Week 4
Modest increase in albumin in BAL no
effect on LDH or lysosomal enzyme
peroxidase
Reference
GeMeichter et al (1992a)
Mochitate et al (1984)
Hooftmanetal (1988)
Hatch etal (1986)
Mustafa et al (1984)
Csallany (1975)
Suzuki etal (1988)
Rose etal (1989)
-------
TABLE 13-6 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON LUNG AMINO ACIDS, PROTEINS, AND ENZYMES8
ON
NC>2 Concentration
/tg/m
9,400-47,000
9,400
37,600
94,000
9,400
15,000
17,900
17,900
18,800
18,800
37,600
56,400
75,200
ppm
50-25
50
20
50
50
80
95
95
10
10
20
30
40
Exposure
Continuous,
7 days
3h
Continuous,
1, 3, or 7 days
Continuous,
14 days
7h/day,
5 days/week,
6 mo
7h/day
5 days/week,
24 mo
Continuous,
14 days
4h
Gender Age
M 10-11
weeks
NS NS
M NS
F (NS)
M in utero
and 6
mo
M 18
weeks
M 8 weeks
M NS
Species
(Strain)
Rat
(Sprague-
Dawley)
Rabbit
(New Zealand)
Rat
(Sprague-
Dawley)
Mouse
(NS)
Rat
(Fischer 344)
Rat
(Fischer 344)
Rat
(Wistar)
Rat
(Long Evans)
Effects
Concentration-related increase in rate of
collagen synthesis, 125% increase in rats
exposed to 5 0 ppm
Benzo[a]pyrene hydroxylase activity of
tracheal mucosa not affected
Increased BAL protein at 3 days (Day 7
not measured), increased (120%) collagen
synthesis at 7 days (not measured other
days)
Increase in lung protein
Increase in BAL alkaline phosphatase,
acid phosphatase, and LDH in older rats
only
Increase in BAL levels of LDH and
alkaline phosphatase activities and in
collagenous peptides
Changes in several enzymes in whole lung
homogenates
Increased activities of various enzymes,
sialic acid, and BAL protein, attenuation
by high dietary levels of vitamin E
Reference
Last etal (1983)
Palmer etal (1972)
Last and Warren (1987)
Csallany (1975)
Mauderly etal (1987)
Mauderly etal (1990)
Sagaietal (1982)
Guth and Mavis (1985,
1986)
"NS = Not stated
LTB4 = LeukotaeneB4
LDH = Lactate dehydrogenase
M = Male
BAL = Bronchoalveolar lavage
F = Female
-------
q
(1 0 ppm) after a 72-h exposure, but not after a 1-week exposure to 752 jwg/m (0 4 ppm)
The results of the 1-week exposure apparently conflict with those of Sherwin and Carlson
(1973), who found increased protein content of lavage fluid from vitamin C-deficient guinea
pigs exposed to 752 /4g/m3 NO2 for 1 week Differences in exposure techniques, protein
measurement methods, and/or degree of vitamin C deficiency may explain the difference
between the two studies However, Sherwin and Carlson (1973) also reported increases in
lavage fluid protein from normal guinea pigs exposed to 752 jwg/m (0 4 ppm) NO2
continuously over a 1-week period
Hatch et al (1986) found that the NO2-induced increase in protein in lung lavage fluid
in vitamin C-deficient guinea pigs was accompanied by an increase in lung content of
nonprotein sulfhydryls and vitamin C and a decrease in vitamin E content The increased
susceptibility to NO2 was observed when lung vitamin C was reduced (by diet) to levels
below 50% of normal values A depletion of nonprotein sulfhydryls also enhances
susceptibility to a high level of NO2 exposure (18,800 ^g/m3, 10 ppm) (Slade et al , 1989)
Selgrade et al (1981) expanded earlier studies of Sherwin and Carlson (1973) on the effects
of vitamin C deficiency on NO2 toxicity Taken together, these investigations support a role
for dietary vitamin C in influencing the susceptibility of NO2-exposed animals to increased
protein and lipids in lung lavage Because vitamin C is readily oxidized and reduced, it
could serve to detoxify oxidative products formed by NO2 or to maintain the intracellular
redox potential
Utell et al (1991) and Frampton et al (1989) reported no significant changes in the
content of total protein, albumin, or a2-macroglobuhn, a glycoprotein that may play a role in
the local control of lung protease activity, of BAL fluid from healthy, nonsmoking volunteers
exposed to 94 jKg/m3 (0 05 ppm) NO2 with three 15-min peak exposures to 3,760 /tg/m3
3 3
(2 0 ppm), to 1,128 (jLg/m (0 6 ppm) continuously, or to 2,820 /xg/m (1 5 ppm)
continuously All NO2 exposures were for 3 h, and BAL fluid was obtained at 3 5 h
(94-jwg/m3 + 3,760 jwg/m3 peaks and 2,820-jwg/m3 groups) or 18 h (1,128 /ig/m3-group)
postexposure When BAL was performed 3 5 h after exposure to 1,128 j«g/m NO2, there
was an increase in a2-macroglobului that was not seen under the other exposure regimes
Whether this increase in ce2-macroglobuhn was indicative of an NO2-induced change in the
13-67
-------
protease-antiprotease balance or was a chance observation is not known (Frampton et al ,
1989). (See Section 15 6 on clinical studies for details )
A major concern has been the effect of NO2 on the structural proteins of the lungs
because elastic recoil is lost after exposure Kosmider et al (1973a) reported that the
urinary hydroxyproline and acid mucopolysacchande content of guinea pigs exposed to
1,880 jttg/m (1 0 ppm) NO2 for 6 mo were significantly increased Because the remodeling
of bone is the major source (>90%) of urinary hydroxyproline in normal animals and
dietary ascorbate status would affect hydroxyproline homeostasis, the significance of these
observations to lung structure and function remains to be shown
Because hydroxylysine, a modified ammo acid, is unique to collagen and proteins
containing collagen-like sequences, Evans et al (1989) selected this compound for study as a
biomarker of lung injury During a series of experiments, rats were exposed to 1,880 to
g
56,400 £ig/m (1 to 30 ppm) NO2, 6 h/day for 2 days A concentration-dependent
relationship was noted in the amount of hydroxylysine in the BAL fluid and the urinary
hydroxylysine output The increases in hydroxylysine were, however, only significant at
NO2 exposure levels of > 14,100 /xg/m3 (7 5 ppm) for BAL fluid and 2:28,200 jug/m3
(15 ppm) for urinary output The angiotensin-converting enzyme level and the total protein
concentration of BAL fluid were significantly increased in the highest NO2-concentration
groups
When rats were exposed to 940 or 1,880 jwg/m (0 5 or 1 0 ppm) NO2, 6 h/day,
5 days/week for 4 weeks, there was a gradual increase in urinary hydroxylysme output that
/5
became significant the week after exposure ended in the 1,880 /*g/m exposed group, but was
•3
significant in the 940 /*g/m exposed group starting during the first week of exposure (Evans
_____
et al , 1989). The amount of hydroxylysme in the BAL fluid of rats in the 1,880
group was significantly lower than that of controls immediately following exposure and
remained significantly lower after a 4-week recovery period, whereas the angiotensin-
converting enzyme level was significantly increased, returning to normal values 4 weeks after
exposure ended The hydroxylysme content of lavage fluid and the angiotensin-converting
enzyme level were unchanged in the 940-jwg/m3 exposed group The total protein content of
the BAL fluid was not significantly altered at either exposure level
13-68
-------
Last and co-workers have examined the effects of NO2 on collagen synthesis rates in
lung minces from animals exposed in vivo for 7 days In one study (Last et al, 1983), rats
were continuously exposed to 9,400 to 47,000 j»g/m3 (5 to 25 ppm) NO2 for 7 days The
authors found a linear concentration-response curve for plots of collagen synthesis rate, with
a correlation coefficient (least squares analysis) for fit of the data to a straight line of 0 92
Linear extrapolation of the line to an estimated no-observable-effect level gave a value of
about 1,880 to 5,640 /ttg/m3 (1 0 to 3 0 ppm) NO2 Last and Warren (1987) confirmed that
exposure to 9,400 jt*g/m3 NO2 increased collagen synthesis It was assumed by these
workers, although not proven, that the increases in lung collagen synthesis rate observed
after acute exposure regimens are predictive of increases in total lung collagen (pulmonary
fibrosis) after longer periods of exposure
Only a modest increase in albumin, indicating a mild degree of injury to the pulmonary
capillary membrane, was noted in mice exposed to 9,400 /tg/m3 (5 0 ppm) NO2, 6 h/day for
6 days or 9,400 jwg/m3, 6 h/day for 2 days prior to viral inoculation and 6 h/day for 4 days
immediate following inoculation (Rose et al, 1989) However, only minimal
histopathological changes were noted in the NO2-exposed, viral-inoculated animals
Lysosomal enzyme peroxidase and lactate dehydrogenase (LDH) activities were not affected
See also Section 13 2 2 1 on host defense mechanisms
Sherwin et al (1972) exposed guinea pigs to 3,760 jug/m3 (2 0 ppm) NO2 for 1, 2, or
3 weeks They examined lung sections histochemically for LDH With this technique, LDH
is thought to be primarily an indicator of Type 2 cells rather than Type 1 cells The number
of Type 2 cells per alveolus was determined In control lung sections, a mean value of
1 9 Type 2 cells per alveolus was found, with a range of 1 5 to 3 4 Exposure to NO2
significantly increased the LDH content of the lower lobes of the lung by increasing the
number of Type 2 cells per alveolus The increase was progressive over the 3-week
exposure period The authors suggested that the increase in lung LDH content was due to
the replacement of Type 1 cells by Type 2 cells, as shown in some of the morphological
studies
An increase in LDH in BAL fluid was reported in rats exposed to 1,880 to
9,400 jig/m3 (1 0 to 5 0 ppm) NO2, 7 h/day, 5 days/week for 2 7 weeks (Gregory et
1983) By 15 weeks of exposure, LDH had returned to control values, even though
13-69
-------
'J
histological changes persisted A baseline (1,880 |wg/m [1 0 ppm]) plus peak (two 1 5-h
peaks to 9,400 jttg/m3 [5 0 ppm]) exposure had no effects Alkaline phosphatase and LDH
activities, as well as collagenous peptides were increased in BAL fluid of chronically exposed
rats (17,900 jig/m3, 9 5 ppm, 7 h/day, 5 days/week, 24 mo) (Mauderly et al , 1990)
Glycolytic pathways are also increased by NO2 exposure, apparently due to an increase
in Type 2 cells (Mochitate et al , 1985) The most sensitive enzyme was pyruvate kinase
^
After a 14-day exposure to 3,760 jwg/m (2 0 ppm) NO2, the activity of this enzyme was
increased When the exposure concentration was increased to 7,520 and 18,800
(4 and 10 ppm), the pyruvate kinase activity was increased by Day 4 and 7, respectively
Alterations in lung xenobiotic metabolism follow a complex pattern based on exposure
duration and concentration in rats exposed to 752, 2,260, or 7,520 pg/m3 (0 4, 1 2, or
4.0 ppm) NO2 (Takahashi et al , 1986) At 752 |Kg/m3, cytochrome P-450 levels had
decreased by the second week of exposure, but returned to normal levels by the fifth week of
exposure, where they remained at Week 10 An initial decrease in cytochrome P-450 was
also seen in animals exposed to 2,260 /ig/m3 NO2, cytochrome P-450 levels returned to
control level by Week 5 and decreased below control levels by Week 10 A similar pattern
of response occurred in the highest concentration tested Only 7,520 jug/m affected other
microsomal electron-transport systems The activity of succinate-cytochrome c reductase was
<>
decreased by the fourteenth week of exposure to 752 j^g/m and even sooner at higher levels
of exposure Mochitate et al (1984) also found a decrease in cytochrome P-450 levels after
a 7-day exposure of rats to ^2,260 /*g/m3 NO2
The activity of benzo[a]pyrene hydroxylase in the tracheobronchial region of the lungs
of rabbits exposed to 9,400, 37,600, or 94,000 /ig/m3 (5, 20, or 50 ppm) NO2 for 3 h was
studied by Palmer et al (1972) No effect was observed Law et al (1975) studied the
effect of NO2 on benzo[a]pyrene hydroxylase, microsomal O-methyl transferase, catechol
Omethyl transferase, and supernatant catechol Omethyl transferase activity in rat lungs
Exposure to 75,200 or 132,000 jtig/m3 (40 or 70 ppm) NO2 for 2 h had no effects Thus,
the studies of Palmer et al (1972) and Law et al (1975) agree that NO2 exposure does not
effect total benzo[a]pyrene hydroxylase activity of the lung The Omethyl transferase
activity studied by Law et al (1975) relates to the ability of the lung to metabolize
catecholamine hormones
13-70
-------
Effects on Antioxidant Metabolism and Influence of Antioxidants
Table 13-7 summarizes the effects of NO2 on antioxidant metabolism and antioxidants
Menzel (1970) proposed that antioxidants might protect the lung from NO2 damage by
inhibiting hpid peroxidation Data related to this hypothesis have been reported by Ayaz and
Csallany (1978), Csallany (1975), Fletcher and Tappel (1973), Menzel et al (1972),
Mohsenin (1991), Slade et al (1989), and Thomas et al (1968) Many laboratories have
observed changes in the activity of enzymes in the lungs of NO^-exposed animals that
regulate levels of glutathione (GSH), the major water-soluble reductant in the lung's
armamentarium (Tyson et al , 1982), or in lung content of GSH in rodents exposed to NO2
Buthiomne sulfoxime, an inhibitor of GSH synthesis, has also been shown to cause increased
•7
lung damage in mice exposed to 1,960 ji*g/m (1 0 ppm) O3, suggesting a role for GSH as a
protective agent against oxidant gases in vivo (Sun et al, 1988) Chow and Tappel (1972)
proposed an enzymatic mechanism for the protection of the lung against lipid peroxidation
damage by O3, involving coupled reactions of glucose-6-phosphate dehydrogenase (G-6-P
dehydrogenase) (to produce reduced nicotinamide-adenine dinucleotide phosphate [NADPH]),
GSH reductase (to regenerate nicotinamide-adenine dinucleotide phosphate [NADP]), and
GSH peroxidase (to regenerate GSH) Chow et al (1974) exposed rats to 1,880, 4,330, or
2
11,600 jwg/m (1 0, 2.3, or 62 ppm) NO2 continuously for 4 days to examine the effect on
the GSH peroxidase system by measuring the activities of GSH reductase, G-6-P
dehydrogenase, and GSH peroxidase in the soluble fraction of exposed rat lungs Linear
regression analysis of the correlation between NO2 concentrations and enzymatic activities
showed a significant positive correlation coefficient of 0 63 for GSH reductase and of
0 84 for G-6-P dehydrogenase No correlation was found between the GSH peroxidase
activity and the NO2 exposure concentration The activities of GSH reductase and G-6-P
dehydrogenase were significantly increased during exposure to 11,600 /-cg/m NO2 The
possible role of edema and cellular inflammation in these findings was not examined These
researchers concluded that because exposure of rats to NO2 had an insignificant effect on
lung GSH peroxidase activity, but did significantly increase the activities of GSH reductase
and G-6-P dehydrogenase, it appears that this oxidant attacks mainly GSH and NADPH,
13-71
-------
TABLE 13-7. EFFECTS OF NITROGEN DIOXIDE ON ANTIOXTOANT METABOLISM AND
INFLUENCE OF ANTIOXTOANTS3
NO2 Concentration
/ 3
US/to.
75
752
7,520
752
2,260
7,520
752-
940
940
1,880
1,880
1,880
4,330
11,600
ppm
004
04
40
04
12
40
04-
05
05
10
10
10
23
62
Exposure Gender
Continuous, M
9 and 18 mo
Continuous, M
4 mo
Continuous, F
1 5 years
Continuous, F
17 mo
4 h/day, NS
6 days
Continuous, M
4 days
Species
Age (Strain)
8 weeks Rat
(Wistar)
13 weeks Rat
(Wistar)
NS Mouse
(NS)
4 weeks Mouse
(C57B1/6J)
NS Rat
(Sprague-
Dawley)
8 weeks Rat
(Sprague-
Dawley)
Effects
NPSHs increased at >0 4 ppm after 9 or 18 mo,
GSH peroxidase activity decreased at 0 4 ppm
after 18 mo and at 4 0 ppm after 9 and 18 mo,
GSH reductase activity increased after a 9 mo
exposure to 4 0 ppm, G-6-P dehydrogenase was
increased after a 9- and 18-mo exposure to
4 0 ppm, no effects on 6-P-G dehydrogenase,
SOD, or disulfide reductase, some GSH
S-transferase had decreased activities after 18-mo
exposure to >0 4 ppm
Duration-dependent pattern for mcrease in
activities of antioxidant enzymes, increase,
peaking at Week 4 and then decreasing
Concentration-dependent effects
Growth reduced, vitamin E (30 or 300 mg/kg
diet) improved growth
At 1 ppm, GSH-peroxidase activity decreased in
vitamin E-deficient mice, and increased in
vitamin E-supplemented mice
Vitamin E-supplement reduced lipid peroxidation
Activities of GSH reductase and G-6-P
dehydrogenase mcreased at 6 2 ppm proportional
to duration of exposure, plasma lysozyme and
GSH peroxidase not affected at 6 2 ppm, no
effects at 1 0 or 2 3 ppm
Reference
Sagai et
Ichinose
Ichinose
(1982)
Csallany
al (1984)
etal (1983)
and Sagai
(1975)
Ayaz and Csallany
(1978)
Thomas
Chow et
etal (1967)
al (1974)
-------
TABLE 13-7 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON ANTIOXTOANT METABOLISM AND
INFLUENCE OF ANTIOXTOANTS3
u>
NO2 Concentration
•$
2,260
3,380
3,760
18,800
3,760
7,500
18,800
5,600
5,600
13,200
18,800
28,200
ppm
12
1 8
20
100
20
40
100
30
30
70
10
15
Exposure
Continuous,
3 days
3 days
14 days
10 days
7 days
7 days
4 days
4 days
4 days
1-7 days
Species
Gender Age (Strain)
M 12 weeks Rat
(Sprague-Dawley)
M/F 5->60 Rat
days (Wistar)
Guinea pig
(Dunkin Hartley)
M 12-24 Rat
weeks (Wistar)
M/F 1 day to Rat
> 8 weeks (Sprague-Dawley)
M NS Rat
(Sprague-Dawley)
Effects
Increases in G-6-P dehydrogenase, isocitrate
dehydrogenase, disulfide reductase, and
NADPH cytochrome c reductase activities at
1 8 ppm only
Decreased SOD activity in 21-day-old animals
G-6-P dehydrogenase increased at >2 ppm, at
2 ppm, 14 days of exposure needed
Increased lipid peroxidation (TBA-reactive
substances) with vitamin E deficiency
No effects on parameters tested
Increase in lung weight, G-6-P dehydrogenase,
GSH reductase, and GSH peroxidase
activities
Increase in lung weight, G-6-P dehydrogenase,
6-P-G dehydrogenase, and GSH reductase
activities
Increase in lung weight, DNA content, G-6-P
dehydrogenase, 6-P-G dehydrogenase, GSH
reductase, disulfide reductase, GSH
peroxidase, succrnate oxidase, and cytochrome
oxidase activities, no effect on lung protein
Reference
Leeetal (1989,
1990)
Azoulay-Dupuis
etal (1983)
Mochitake et
(1985)
al
Sevanian et al
(1982)
Mustafa et al
(1979)
-------
TABLE 13-7 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON ANTIOXIDANT METABOLISM AND
INFLUENCE OF ANTIOXTOANTS3
NO2 Concentration
3
/tg/m ppm
7,520 4 0
11,000 6 0
28,000 15
53,000 28
17,900 9 5
17,900 9 5
Exposure
3h
4 h/day,
30 days
7 days
7 h/day,
5 days/week,
6 mo
7 h/day
5 days/week,
24 mo
Gender Age
M/F 21-33
years
F NS
M in
utero
and
6 mo
M 18
weeks
Species
(Strain)
Human
Mouse
(NS)
Rat
(Fischer 344)
Rat
(Fischer 344)
Effects
Decreased elastase inhibitory capacity
and increased lipid peroxidation
products in BAL of subjects not
administered supplement of vitamin C
and E prior to NO2 exposure
Increase in GSH reductase and G-6-P
dehydrogenase activities
Increase in GSH levels, and GSH
reductase, G-6-P dehydrogenase, and
GSH peroxidase activities
Increase in GSH reductase activity in
younger rats and GSH peroxidase
activity in older rats
Increase in GSH reductase activity in
BAL
Reference
Mohsenin (1991)
Csallany (1975)
Mauderly et al (1987)
Mauderlyetal (1990)
M = Male
NPSHs = Nonprotem sulfhydryls
GSH = Glutathione
G-6-P dehydrogenase = Glucose-6-phosphate dehydrogenase
6-P-G dehydrogenase = 6-phosphosgluconate dehydrogenase
SOD = Superoxide dismutase
F = Female
NS = Not stated
NADP = Nicotinamide-adeninedmucleotide phosphate
ICD = Isocitiate dehydrogenase
NADPH = Nicotinamide-ademnedinucleotide phosphate (reduced form)
TEA = Thiobarbitunc acid
-------
whereas O3 not only initiates lipid peroxidation, but also directly attacks these reducing
substances
Nitrogen dioxide effects on antioxidant metabolism appears to follow a concentration-
and exposure-duration response Lee et al (1990) reported that G-6-P dehydrogenase,
6-phosphogluconate dehydrogenase (6-P-G dehydrogenase), and NADP-specific isocitrate
dehydrogenase were not affected after rats were exposed to 2,260 jwg/m (1 2 ppm) NO2
continuously for 3 days When NO2 exposure was increased from 3 days to 16 weeks, there
was an increase in GSH peroxidase, GSH reductase, G-6-P dehydrogenase, 6-P-G
dehydrogenase, SOD, and disulfide reductase activities in rats from the first week of
exposure that reached a maximum by the fourth week of exposure and thereafter gradually
declined over the 16-week exposure period (Ichinose et al, 1983) The increase in
antioxidant activity was exposure-dependent over the exposure range of 75 to 7,520 j«g/m3
(0 04 to 4 0 ppm) NO2
Sagai et al (1984) and Ichinose et al (1983) studied the effects of prolonged (9 and
18 mo) exposures to 75, 752, and 7,520 jwg/m3 (0 04, 0 4, and 4 0 ppm) NO2 on rats
•3
Nonprotein sulfhydryl levels were increased in the 752- and 7,520-/*g/m -exposed groups
after both exposure durations Nine- and 18-mo exposures to 7,520 ftg/m caused a decrease
in the activity of GSH peroxidase and an increase in G-6-P dehydrogenase activity
^
Glutathione peroxidase activity was also decreased in rats exposed to 752 ju,g/m NO2 for
18 mo Three GSH S-transferases were also studied, two of which (aryl S-transferase and
aralkyl S-transferase) exhibited decreased activities after 18 mo of exposure to 752 and
7,520 jug/m NO2 No effects were observed on the activities of 6-P-G dehydrogenase,
SOD, or disulfide reductase The decreases in antioxidant metabolism were inversely related
to the formation of lipid peroxides (see previous subsection on lipid metabolism) Shorter
exposures (4 mo) to NO2 between 752 and 7,520 jwg/m also cause concentration- and
duration-dependent effects on antioxidant enzyme activities (Ichinose and Sagai, 1982) For
example, G-6-P dehydrogenase activity increased, reaching a peak at 1 mo, and then
decreased towards control Briefer (2-week) exposures to 752 pg/m NO2 caused no such
effects in rats or guinea pigs (Ichinose and Sagai, 1989)
2
Age susceptibility to the effects of NO2 (17,900 jug/m , 9 5 ppm, 7 h/day,
5 days/week) was examined by Mauderly et al (1987) Rats were exposed for 6 mo,
13-75
-------
beginning in utero or at 6 mo of age In the older rats, only GSH peroxidase was increased,
whereas in the younger rats only GSH reductase was increased
Malnutrition of animals can drastically affect their response to toxicants, including
NO2 Experimental interest in this area has mainly focussed on dietary kpids, vitamin E and
other lipid-soluble antioxidants, and vitamin C and other water-soluble antioxidants For
example, Sevaman et al. (1982) reported an increase in the amount of TEA reactants in lung
homogenate of vitamin E-deficient rats after 7 days of exposure to 5,640 jwg/m3 (3 0 ppm)
NO2 Ayaz and Csallany (1978) exposed weanling mice continuously for 17 mo to 940 or
•2
1,880 /tg/m (0 5 or 1 0 ppm) NO2 and fed the animals a basal diet that was either deficient
in vitamin E or supplemented with 30 or 300 mg/kg of diet Blood, lung, and liver tissues
were assayed for GSH peroxidase activity Exposure to 1,880 /*g/m NO2 suppressed GSH
peroxidase activity in the blood and lungs A combination of vitamin E deficiency and
1,880 /^g/m NO2 exposure resulted in the lowest GSH peroxidase activity in blood and lung
Liver GSH peroxidase activity was unaffected by either vitamin E deficiency or NO2
exposure Other studies (Hatch et al, 1986, Selgrade et al, 1981, Slade et al, 1989,
Sherwin and Carlson, 1973) have also shown that vitamin C deficiency increases
susceptibility to NO2-induced increases in BAL protein
Summary
Studies on the biochemical effects of NO2 on the lung have focused on the mechanisms
of the toxic action or indicators of tissue and cell damage One theory describing the toxic
action of NO2 is that of lipid peroxidation of unsaturated fatty acids in target cell membranes
(Menzel, 1976). An alternate theory is that NO2 oxidizes water-soluble low molecular
weight reducing substances and proteins (Freeman and Mudd, 1981) Studies show that
regardless of the toxic action, many of the effects are concentration- or exposure duration-
dependent
Exposure to 75 /xg/m3 (0 04 ppm) NO2 increased lipid peroxidation (as indicated by
increased ethane exhalation) in the lungs of rats exposed for 9 mo or longer Ethane
exhalation was also increased in rats exposed to 752 ^g/m3 for 6 mo, but not in rats exposed
o
to 75 or 225 jttg/m (0 04 or 0 12 ppm) over the same time period (Sagai et al, 1984,
Ichinose et al, 1983) Increases in lipid peroxidation products have also been reported in
13-76
-------
healthy, nonsmoking humans exposed to 7,520 ji*g/m3 (4 0 ppm) NO2 for 3 h (Mohsemn,
1991)
Increases in lavage fluid and urinary levels of hydroxylysine were found in rats exposed
to 1,880 to 56,400 jwg/m3 (1 to 30 ppm) NO2, 6 h/day for 2 days The increases were,
however, only significant at NO2 levels > 14,100 /tg/m (7 5 ppm) for lavage fluid and
28,200 /*g/m (15 ppm) for urinary output (Evans et al, 1989) Urinary secretion of
hydroxyprohne and acid mucopolysacchandes have been reported in guinea pigs exposed to
1,880 jitg/m3 (1 0 ppm) NO2 for 6 mo (Kosmider et al 1973a) The significance of these
observations to lung structure and function is unknown
No changes in blood and lung GSH peroxidase activity were reported in mice exposed
•3
to 940 /ig/m (0 5 ppm) NO2 continuously for up to 17 mo, however, when the exposure
concentration was increased to 1,880 /*g/m3 (1 0 ppm), a suppression of GSH peroxidase
activity was noted (Ayaz and Csallany, 1978) This enzyme activity was not affected in rats
exposed continuously to up to 11,600 /*g/m3 (6 2 ppm) NO2 for 3 or 4 days (Chow et al,
-3
1974, Lee et al., 1990), but was significantly decreased in rats exposed to 752 jug/m for
18 mo (Sagai et al, 1984, Ichinose et al , 1983)
Nitrogen dioxide effects on antioxidant metabolism appears to be both concentration-
and exposure duration-dependent No effect on G-6-P dehydrogenase, 6-P-G dehydrogenase,
and NADP-specific isocitrate dehydrogenase activities was noted in rats exposed to
2,256 jwg/m3 (12 ppm) NO2 continuously for 3 days (Lee et al , 1990) However, an
increase in GSH peroxidase, GSH reductase, G-6-P dehydrogenase, 6-P-G dehydrogenase,
SOD, and disulfide reductase activities has been reported in rats exposed to 75 to
7,520 /*g/m3 (0 04 to 4 0 ppm) NO2 from the first week of exposure that reached a maximum
by the fourth week of exposure and thereafter gradually declined over the 16 week exposure
period (Ichinose et al , 1983) When exposure was incieased to 9 to 18 mo, there was an
increase in G-6-P dehydrogenase activity, but only in rats exposed to 7,520 /*g/m3
No effects were observed on the activities of 6-P-G dehydrogenase, SOD, or disulfide
reductase (Sagai et al , 1984, Ichinose et al ,1983)
13-77
-------
13.2.2.3 Pulmonary Function
The key issues addressed by investigators evaluating the effects of NO2 on pulmonary
function in experimental animals were (1) the effects of low-level, long-term exposures to an
urban pattern of NO2, the lowest concentrations that stimulated respiratory reflexes and
impaired gas exchange in the lung, and (2) differences in responses between very young and
mature animals Compared with humans, rats and hamsters used in experimental studies of
NO2 have very immature lungs at birth Humans have approximately 50 million alveoli at
birth, which multiply rapidly until age 3 years and slowly until about age 8 years, when
alveolar development is complete Growth continues until maturity, 16 to 18 years, through
alveolar enlargement Rats and hamsters are born with no true alveoli Alveolar
proliferation is most rapid between 4 and 30 days of age and is essentially complete by
40 days of age Although hamster lungs have reached adult volumes and elasticity at 40 days
of age, lung growth through alveolar enlargement continues in rats to 5 mo of age
(Mauderly, 1989) Changes in pulmonary function parameters in the experimental animals
from exposure NO2 are shown in Table 13-8
Nitrogen dioxide concentrations in urban areas are not constant, but consist of peak
exposures superimposed on a relatively constant background level Miller et al (1987)
evaluated this urban pattern of NO2 exposure in mice using continuous 7 day/week, 23 h/day
o
exposures to 376 jttg/m (0 2 ppm) NO2 with two daily (5 days/week) 1-h peak exposures to
"2
1,500 /ig/m (0 8 ppm) NO2 for 32 and 52 weeks Mice exposed to clean air and to the
o
constant background concentration of 376 [Ag/m served as controls Data from animals
examined immediately and 30 days following both exposure regimens were combined for
analysis because there was no statistical difference between the groups (i e , immediately
and 30 days postexposure) Most of the differences in pulmonary function were measured
between groups exposed to background concentrations with diurnal peaks and those exposed
to constant background NO2 levels, although the same pattern of effects was found when
comparing peak- and air-exposed animals Both end-expiratory volume and vital capacity,
the difference in lung volume between maximum inflation and deflation, were significantly
lower in mice exposed to NO2 with diurnal peaks than in mice exposed to the constant level
of NO2. Lung distensibility, measured as respiratory system compliance, also tended to be
lower (p = 0 072) in mice exposed to diurnal peak exposures of NO2 compared with
13-78
-------
TABLE 13-8. EFFECTS OF NITROGEN DIOXIDE ON PULMONARY FUNCTION3
NC>2 Concentration
/ 3
Ag/rn
376
376 base +
1,504 peak
750
2,250
7,520
940 base +
2,820 peak
1,880 base +
5,640 peak
3,760 base +
11, 280 peak
940
1,880
ppm
02
0 2 base +
0 Speak
04
12
40
0 5 base +
1 Speak
1 0 base +
3 Opeak
2 0 base +
6 0 peak
05
1 0
Species
Exposure Gender Age (Strain)
23 h/day, F 6-8 Mouse
7 days/week weeks (CD-I)
base +
two 1-h
peaks/day,
5 days/week,
32 and
52 weeks
1, 2, and Rat
3 mo
Continuous, M 1 day Rat
with two and (Fischer 344)
daily 1-h 7 weeks
peaks for 1,
3, or 6 weeks
6 h/day, M NS Rat
5 days/week, (Fischer 344)
4 weeks
Effects
Decreased vital capacity and respiratory
system compliance following exposure to
0 2 ppm + 0 8-ppm peak compared with
air-exposed and 0 2-ppm exposed rats
Decreased heart rate following 1-mo
exposure to 1 2 and 4 0 ppm, decreased
body weight and PaO2 following 3-mo
exposure to 4 0 ppm NO2
Increased lung volume (at Weeks 3 and 6)
and compliance (at Week 3) in neonates
exposed to the two highest exposure
levels Decreased body weight and lung
compliance in older rats following
6 weeks exposure to the highest
concentration, older rats recovered by
3 weeks postexposure (younger rats not
tested)
No effects 1 0 ppm, increase in vital
capacity immediately following exposure
and an increase in compliance 4 weeks
postexposure at 0 5 ppm Functional
changes associated with changes in mean
linear intercept
Reference
Miller etal (1987)
Suzuki etal (1981)
Stevens et al (1988)
Evans etal (1989)
-------
TABLE 13-8 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON PULMONARY FUNCTION3
u>
00
o
NO2 Concentration
/tg/m3
940 (base) +
2,820 (peak)
1,880
3,760
9,400
9,400
9,400
9,400
18,800
37,600
75,200
ppm
0 5 (base) +
1 5 (peak)
10
20
50
50
50
50
10
20
40
Exposure
22 h/day,
7 days/week
base + 6-h
peak/day,
5 days/week,
-1, 3, 12, 52,
and 78 weeks
Continuous,
493 days
8 h/day,
5 days/week,
8 weeks
30 nun/day,
twice/week
for 7 weeks
2 h, resting
or exercising
Continuous,
2 mo
24 h
Gender Age
M 60 days
M NS
M 8
weeks
M NS
F 2-9
years
M NS
M 15-16
weeks
Species
(Strain)
Rat
(Fischer 344)
Monkey
(Squirrel)
Hamster
(Golden
Syrian)
Guinea pig
(NS)
Dog
(Beagle)
Monkey
(Squirrel)
Mouse
(JCL ICR)
Effects
Decreased AFEF^ following 78 weeks of NC>2
exposure, frequency of breathing decreased
throughout, with greatest decrease observed at
78 weeks
Monkeys challenged with monkey-adapted
influenza virus Minor NO2-induced changes in
tidal volume, minute volume, and respiration rate
Increase in fixed lung volume, but no change in
vital capacity or lung compliance following NO2
exposures in both normal and elastase-treated
animals
Last 5 weeks of NO2 exposure followed by 10-min
exposure to aerosolized albumin Increased
dyspneic breathing during fourth through seventh
week of NO2-albumin exposure
Statistically significant decrease in the ventilation
equivalent for O2 in exercising dogs
Monkeys challenged with monkey-adapted
influenza virus Decreased tidal volume and
increased respiratory rate
Concentration-related decrease in forced swimming
time immediately following exposure, elevated
blood lactic acid immediately and 24 h following
4-min forced swim at 5 0 ppm
Reference
Tepperetal (1992)
Fentersetal (1973)
Lafumaetal (1987)
Yoshidaetal (1980b)
Kleinman and Mautz
(1991)
Henry et al (1970)
Suzuki etal (1982a)
-------
TABLE 13-8 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON PULMONARY FUNCTION3
NO2 Concentration
[ig/m ppm
9,400 50
18,800 10
37,600 20
9,400 50
188,000 100
10,200 5 4
^ 13,200- 7 0-146
w 275,000
oo
i— i
14,000 7 5
28,000 15 0
17,900 9 5
Exposure Gender
24 h M
Continuous, F
3-day to
5 Oppm
followed by
8 nun to
100 ppm
3 h/day for M
7, 14, or
30 days
In M/F
2h NS
2 and 4 h
7 h/day, M
5 days/week,
24 mo
Species
Age (Strain)
15-16 Mouse
weeks (JCL ICR)
NS Mouse
(CF-1)
7-9 Rat
weeks (Wistar)
5-10 Guinea pig
weeks (CRL COBS)
NS Sheep
(NS)
18 weeks Rat
(Fischer 344)
Effects
Concentration-related increase in
respiration rate, decrease in PaCO2 at
5 ppm and in PaC>2 at > 10 ppm
The irritant response to 8-min, 100-ppm
NO2 exposure, typified by increased
respiratory rate and decreased tidal volume
and minute volume, was lessened by
preexposure to 5 0 ppm NO2 for 3 days
Nonsignificant tendency toward increased
lung volume at low inflation pressures at
30 days
Concentration-related increase in
sensitivity to inhaled histarmne aerosols
10 mm following NO2 exposure, but not
2 and 19 h following exposure,
concentration-related increase in
respiratory rate 10 mm following exposure
and decrease in tidal volume 10 mm and
2 and 19 h following exposure
Increased pulmonary resistance
immediately following 4-h exposure to
15 ppm NC«2, no consistent effects of
exposure on airway reactivity to inhaled
carbachol, arterial blood gases, and
pulmonary and systematic hemodynanucs
Increased lung volumes and lung
compliance and decreased rate of forced
exhalation in NO2-exposed rats, no
physiologically significant interaction
between NO2 and elastase-treatment
Reference
Suzuki etal (1982b)
McGrath and Smith (1984)
•t
Yokoyamaetal (1980)
Silbaughetal (1981)
Abraham etal (1980)
Mauderlyetal (1990)
-------
TABLE 13-8 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON PULMONARY FUNCTION3
N02 Concentration
/tg/m ppm
17,900 9 5
18,800 10
28,200 15
Exposure Gender
7 h/day, M
5 days/week,
6 mo
2h M/F
Age
in utero
and
6 mo
NS
Species
(Strain)
Rat
(Fischer 344)
Monkey
(Squirrel)
Effects
No substantive effects of NO2
Monkeys challenged with Klebsietta
pneumonia Decreased tidal volume that
returned to normal or elevated levels
within 24 h in most animals
Reference
Mauderlyetal (1987)
Henry et al (1969)
P = Female
PaC>2 = Arterial oxygen tension
M = Male
NS = Not stated
AFEF25 = Change in forced expiratory flow at 25% of forced vital capacity
C>2 = Oxygen
PaCC>2 = Arterial carbon dioxide tension
00
to
-------
constant NO2 exposure These changes suggest that up to 52 weeks of low-level NO2
exposure with diurnal peaks produces some decrease in lung distensibility, resulting in
decreased respiratory system compliance and vital capacity Vital capacity was still
decreased 30 days postexposure Lung morphology (by light microscopy) in these mice
showed no exposure-related lesions, however, the absence of observable morphologic lesions
does not preclude the presence of subtle morphologic changes as discussed in
Section 13 22 4 The decrease in lung distensibility measured in this study is consistent
with the increase in vitro lung collagen synthesis rates measured in lung minces from rats
exposed in vivo for 7 days to 9,400 to 47,000 jwg/m3 (5 to 25 ppm) NO2 (Last et al , 1983)
Tepper et al (1992) exposed rats to 940 jwg/m3 (0 5 ppm) NO2, 22 h/day, 7 days/week,
•2
with a 6-h peak slowly rising to 2,820 /tg/m (1 5 ppm) NO2, 5 days/week for up to
78 weeks Evaluations of pulmonary function conducted at 1, 3, 13, 52, and 78 weeks
revealed a small, but statistically significant decrease in the frequency of breathing that was
paralleled by a trend toward increased tidal volume, expiratory resistance, inspiratory and
expiratory tune In general, these variables were most affected following 78 weeks of
exposure Also, at the 78-week evaluation, delta flow at 25 % forced vital capacity was
decreased Taken together, these results might indicate arrway obstruction, however, a more
prudent conclusion would be that few, if any, significant functional effects were observed
that might suggest degenerative lung disease
Effects of exposure to diurnal peaks of NO2 were also studied by Stevens et al (1988)
These investigators exposed 1-day- and 7-week-old rats 1o 940, 1,880, or 3,760 /ig/m3
(0 5, 1 0, or 2 0 ppm) NO2 with two daily 1-h peak exposures at three tunes the baseline
concentration for 1, 3, and 7 weeks The effects on rats beginning exposure at 1 day and
7 weeks of age were substantially different In rats begmning exposure at 1 day of age,
respiratory system compliance was increased following 3 weeks, but not 6 weeks of exposure
to the 1,880 to 3,760 jwg/m3 NO2 baselines with peak exposures In rats beginning exposure
at 7 weeks of age, respiratory system compliance was decreased following 6 weeks of
exposure to the two highest levels, and body weight was decreased following 3 and 6 weeks
q
of exposure to the 3,760 jwg/m baseline with peak exposures The decreased compliance in
the older rats similarly exposed to NO2 was associated with morphological changes,
particularly in the centnacinar alveolar region (Chang et al , 1986, Section 13 2 2 4) In the
13-83
-------
older rats (younger rats not tested), pulmonary function changes returned to normal values by
3 weeks after exposure ceased
Mauderly et al (1987) did not measure substantive differences in effects on pulmonary
function parameters between rats exposed to 17,900 /ig/m (9 5 ppm) NO2, 7 h/day,
5 days/week either beginning exposure at 6 mo of age for 6 mo or beginning from the tune
of conception until 6 mo after birth There was, however, a statistically significant increase
in minute volume in the NO2-exposed younger animals compared to air-exposed animals
The increase in minute volume was attributed to an increase in tidal volume Because there
were no changes in lung volumes, mechanics, or gas exchange, the increased minute volume
apparently does not reflect a compensation for unpaired lung function No morphologic or
morphometnc differences were observed either Differences in the observed effects of NO2
between this study and that of Stevens et al (1988) may possibly be attributed to differences
in exposure schedules (7 h/day, 5 days/week versus 23 h/day with diurnal peaks), to
differences in the stage of lung development at the tune of sacrifice (alveolar expansion
essentially complete at 6 mo, but still continuing at 13 weeks), or to some other factor
In adult rats receiving a longer (24-mo) intermittent exposure to 17,900 /*g/m (9 5 ppm)
NO2, lung volumes and compliance were increased (Mauderly et al, 1990) The percentage
of forced vital capacity exhaled in 1 s was decreased There were no structural correlates
(light microscopy) to these changes
Lung volumes and capacities were evaluated in anesthetized rats after exposure to
O
940 or 1,880 jug/m (0 5 or 1 0 ppm) NO2, 6 h/day, 5 days/week for 4 weeks (Evans et al ,
1989). No pulmonary function effects were noted in rats exposed to 1,880 /ig/m NO2
immediately after exposure ended or 4 weeks postexposure There was, however, a
significant increase in vital capacity at the end of the 4-week exposure penod, and an
<>
increase in compliance following the 4-week recovery penod in the 940-/*g/m -exposed
group. The changes in the pulmonary function parameters were associated with changes in
the mean linear intercept in the NO2-exposed animals Except for a nonsignificant trend for
an increased lung volume at low inflation pressure, Yokoyama et al (1980) observed no
changes in ventilatory mechanics of rats exposed for 14 or 30 days (3 h/day) to
o
10,200 jug/m (5 4 ppm) NO2 Histological changes were minor A discussion of the
13-84
-------
biochemical changes and morphological findings in the lungs of these exposed animals
appears in Sections 13 2 2 2 and 13 2 2 4
Only minor changes in tidal volume, minute volume, and respiration rate were reported
in squirrel monkeys exposed to 1,880 /tg/m3 (1 0 ppm) NO2 continuously for 493 days
(16 mo) following viral challenge with monkey adapted influenza A/PR/8/34 virus (Fenters
et al , 1973) These changes were not affected by the viral challenge, although the authors
stated that slight emphysema and thickening bronchial and bronchiolar epithelium were noted
in monkeys exposed to NO2 and the influenza virus When influenza challenge followed
•3
exposure to 9,400 jug/m (5 0 ppm) for 2 mo, there was a decrease in tidal volume and an
increase in respiratory rate that had returned to normal values after 4 weeks (Henry et al ,
1970)
Kleinman and Mautz (1991) conducted pulmonary function studies on a group of beagle
3
dogs exposed to 9,400 /-cg/m (5 0 ppm) NO2 for 2 h while standing at rest or exercising
Resting dogs developed a shorter breathing time and a trend towards an increase in VE and
VO2 compared to air-exposed animals, however, neithei of these responses was significant
The only statistically significant effect in NO2- and air-exposed exercising dogs was a
decrease in the ventilation equivalent for oxygen (O2) Other pulmonary function parameters
examined included tidal volume, minute ventilation, ventilation equivalent for CO2 ,
pulmonary resistance, and dynamic compliance
Lafuma et al (1987) exposed 12-week-old hamsteis to 3,760 /ig/m3 (2 0 ppm) NO2,
8 h/day, 5 days/week for 8 weeks Half the animals had been pretreated intratracheally with
elastase to produce a condition of experimental emphysema Fixed lung volumes (20 cm
water with 25% glutaraldehyde) were significantly higher in NO2-exposed animals than in
air-exposed controls, independent of elastase treatment Vital capacity and pulmonary
compliance were not affected by NO2 exposure, however, morphometncally, the
emphysematous lesion produced by elastase appeared to be aggravated by the NO2 exposure
o
Mauderly et al (1990) exposed rats to higher concentrations of NO2 (17,900 jttg/m ,
9 5 ppm) for longer tunes (7 h/day, 5 days/ week for 24 mo) and also found no increased
susceptibility, related to pulmonary function or morphological changes, to NO2 in elastase-
treated animals Although there was an interaction of NO2 and elastase treatment on forced
expiratory flow at 10% of forced vital capacity, the authors attributed this response to either
13-85
-------
a statistical fluke or a change of little physiological significance See Section 13 2 2 4 for a
further discussion of structural changes
Suzuki and Tsubone, along with their colleagues, have conducted extensive studies on
the effects of NO2 on respiratory and cardiac function in mice and rats Because many
cardiovascular effects observed following exposure to NO2 are most likely secondary to
pulmonary edema and/or stimulation of sensory receptors in the respiratory tract, these
changes will be discussed together
Suzuki et al (1984) reported that the heart rate in unanesthetized mice was lower
•^t
following 1-mo exposure to 2,260 and 7,520 /*g/m (1 2 and 4 0 ppm) NO2, but not
following 2- and 3-mo exposures Arterial O2 tension (PaO2) was decreased following a
o
3-mo exposure to 7,520 /tg/m NO2 Respiratory rate was not affected by NO2 exposures
Suzuki et al. (1981) also exposed rats for up to 3 mo to between 752 and 7,520
2|
(0.4 and 4 0 ppm) NO2 After 3 mo of exposure to 7,520 jttg/m NO2, anesthetized rats,
artificially ventilated at high frequencies, had a significant reduction in PaO2
Effects of 24-h exposures to 9,400, 18,800, 37,600, and 75,200 /*g/m3 (5, 10, 20, and
40 ppm) NO2 on swimming performance in mice were evaluated by Suzuki et al (1982a)
Blood lactic acid levels measured in mice after a 4-min forced swim were approximately
o
50% higher following exposure to 9,400 jttg/m NO2 compared with controls Exposures to
higher concentrations of NO2 (>18,800 jug/m ) resulted in a concentration-related decrease
in maximum forced swimming time Changes in blood lactic acid levels in the Suzuki et al
(1982a) study indicate that although mice exposed to 9,400 jug/m3 NO2 were able to swim as
long as control mice, the cardiorespiratory system was not able to supply sufficient O2 to
meet the metabolic demands of swimming, and anaerobic pathways were activated producing
lactic acid
Suzuki et al (1982b) evaluated breathing pattern and gas exchange in mice following
exposure to 9,400, 18,800, or 37,600 /tg/m3 (5, 10, or 20 ppm) NO2 for 24 h The irritant
effect of exposure to 9,400 |wg/m3 NO2 resulted in increased respiratory rate and an
associated decrease in arterial CO2 tension (PaCO2), but no effect on PaO2 Respiratory
rates were increased at the two highest NO2 exposure concentrations, but because of
impaired gas exchange associated with increased lung wet weight and lung water content,
PaCO2 was unchanged and PaO2 was decreased following exposure The studies of Suzuki
13-86
-------
and Tsubone together have shown that 30-min to 3-mo exposures of mice and rats to NO2
concentrations of 9,400 /ig/m NO2 and greater stimulated respiratory reflexes that slow the
heart rate and produce concentration-related pulmonary edema, decreasing blood oxygenation
and impairing maximum exercise performance
McGrath and Smith (1984) found that the irritant response in mice to an 8-min
exposure to 188,000 jwg/m3 (100 ppm) NO2 was lessened by a 3-day continuous preexposure
to 9,400 /ig/m3 (5 0 ppm) NO2 Considering both 188,000 fcg/m3 NO2 exposures and
phenyl diguamde injections as irritant challenges, 3 to 7 days of exposure to 7,520 to
o -3
9,400 jwg/m (4 0 to 5 0 ppm) NO2 lessened the response to 188,000 /*g/m NO2 exposure,
suggesting the development of a tolerance or attenuated response to NO2 (McGrath and
Smith, 1984), but heightening of the response to phenyl diguamde injections (Tsubone and
Suzuki, 1984)
Yoshida et al (1980b) exposed guinea pigs to 9,400 j«g/m3 NO2 (5.0 ppm) After four
preliminary 30-min NO2 exposures, spread over 2 weeks, animals were exposed twice a
week for 10 weeks to 30 mm of NO2, followed 20 mm later by a 10-min exposure to
aerosolized albumin After 5 weeks of NO2 plus albumin exposures (two 30-min
exposures/week), animals were exposed to aerosolized acetylcholine for 10 mm Results
were evaluated by grading the animals breathing pattern on a scale of 1 to 7, with
1 representing normal breathing and 7 almost total apnea with only rare respiratory efforts
Using this relatively subjective measure, the authors state that dyspneic breathing patterns
were more severe in animals exposed to NO2 followed by exposure to aerosolized albumin
compared to animals exposed to albumin alone, with the greatest differences occurring
between the fourth and seventh week of exposure to albumin Animals previously exposed to
NO2 plus albumin were also more affected by the final exposure to acetylcholine Although
the authors state that the effects they observed are statistically significant, quantitative
measures of pulmonary function would allow for much more thorough statistical evaluation
and better definition of the functional changes occurring in the lungs
Summary
Changes in pulmonary function parameters following NO2 exposure in experimental
animals have shown consistent patterns among different treatment conditions and animals
13-87
-------
species A significant increase in vital capacity has been reported immediately following the
-3
exposure of rats to 940 /tg/m (0 5 ppm) NO2, 6 h/day, 5 days/week for 4 weeks (Evans
et al., 1989) When the animals were examined 4 weeks later, there was an increase in
compliance in the NO2-exposed animals The changes in these pulmonary function
parameters were associated with changes in the mean linear intercept Exposures to diurnal
peaks of NO2 superimposed on a constant background level, simulating NO2 patterns in the
urban environment, produced a decrease in lung distensibility m both mice and rats (Miller
et al, 1987, Stevens et al, 1988) These changes were very subtle, and in mice occurred at
concentrations of 376 /tg/m3 (0 2 ppm) NO2 with peak exposures of 1,504 jwg/m3 (0 8 ppm)
after 52 weeks of continuous exposure (Miller et al, 1987) Impaired gas exchange was a
predominant feature in mice following several months of exposure 7,520 /xg/m (4 0 ppm)
NO2 and was reflected in decreased PaO2 (Suzuki et al, 1984) When NO2 exposure was
increased to 9,400 j«g/m (5 0 ppm) for 24 h, increased anaerobic metabolism, manifested by
increased production of lactic acid, occurred (Suzuki et al, 1982)
Newborn and older animals are affected differently by NO2 exposures, particularly rats
exposed subchromcally to continuous background concentrations with diurnal peaks
(1,880 and 3,760 jwg/m3 [1 0 and 2 0 ppm] with two daily 1-h peaks at three times the
baseline concentration [Stevens et al, 1988]) Lung distensibility was increased transiently
in 1-day-old rats exposed to NO2 for 3 weeks, but was decreased in rats that were 7 weeks
old at the beginning of exposure When pulmonary function parameters were compared
between 6 mo old rats exposed to NO2 concentrations of 17,900 jug/m3 (9 5 ppm) for 6 mo
and rats exposed to the same NO2 regimen beginning at conception and continuing until 6 mo
of age, there were no substantive differences noted between the two exposed groups
(Mauderly et al, 1987)
All these studies taken together demonstrate that NO2 produces subtle to major changes
in pulmonary function, depending on the concentration and duration of exposure Lung
distensibility and gas exchange are the parameters most consistently affected by NO2
exposure
13-88
-------
13.2.2.4 Morphologic Studies
Inhalation of NO2 produces morphological alterations in the respiratory tract
Tables 13-9, 13-10, and 13-11 provide an overview of results of studies of respiratory system
morphology following acute, subchronic, and chronic exposures to a wide range of NO2
concentrations Examination of the tables shows variability in responses at similar exposure
levels in different studies There are several possible explanations for these differences in
response Species, strain, and age of the experimental animals and the diet they were fed
appear to be major factors Although in most recent studies, the authors specify that specific
pathogen-free animals were ordered from the supplier, the possibility of intercurrent disease
acquired after they were shipped or during the experimental protocol can only be excluded by
serology, microbial culture, and a complete necropsy at the end of the experiment
Other major factors that influence the results reported are the methods and instruments
used for morphological evaluation Because NO2 does riot affect the cells and tissues of the
lungs in a uniform manner (Stephens et al, 1972, Crapo et al , 1984), sampling procedures
are important to the detection of lesions Sampling procedures can also influence the
validity of the morphometnc data derived using stereological procedures The type of
microscopy used for morphological observations is also critical Light microscopy (LM)
permits detection of many NO2 lesions and the examination of much larger areas of lungs
than does transmission electron microscopy (TEM) However, LM provides neither the
magnification nor the resolution required to detect some very significant morphological
lesions that follow acute or low-concentration exposures Conversely, inspection of small
areas using TEM can easily miss the limited sites affected by NO2 Scanning electron
microscopy (SEM) is especially useful for examining the many surfaces of lungs at both low
and high magnification Scanning electron microscopy is the method of choice for inspecting
airways for some subtle NO2-induced changes in cell surfaces Although qualitative
judgments of the extent and seventy of lesions can be made using all of the above
instruments, quantitative evaluation requires morphometnc methods employing appropnate
sampling and stereological procedures Morphometnc techniques, especially those using
TEM (Chang et al 1986, 1988, Kubota et al 1987), are essential for evaluating subtle
changes resulting from exposure to low concentrations or evaluating the extent of recovery of
either the epithelium or the underlying connective tissue interstitium
13-89
-------
TABLE 13-9. EFFECTS OF ACUTE EXPOSURE TO NITROGEN DIOXIDE ON LUNG MORPHOLOGY3
N02 Concentration
/tg/m
940
1,880
940
3,760
18,800
3,760
18,800
5,640-
30,100
9,400
20,000
ppm
05
10
05
20
10
20
10
3-16
50
106
*M = Male
F = Female
NS = Not stated
Exposure
4h
Continuous,
up to 6 days
3 days
3 days
Ih
24 h/day,
3 days
6h
Procedures Gender Age
LM M NS
LM M NS
LM M/F 5, 10, 21,
TEM 45, 55,
>60 days
LM
TEM
SEM
LM NS NS
TEM
LM M 60-75 days
LM M 6 weeks
TEM
SEM
Species
(Strain)
Rat
(Sprague-Dawley)
Rat
(Wistar)
Rat
(Wistar)
Guinea pig
(Dunkin Hartley)
Dog
(Beagle)
Rat
(Sprague-Dawley)
Rat
(Wistar)
Effects
Loss of cytoplasic granules in and
rupture of mast cells at 0 5 ppm;
degranulation and decreased number of
mast cells at 1 ppm
Increased number of mast cells in
trachea as exposure duration increased
At 2 ppm. no lesions observed
At 10 ppm fibrin deposition in alveoli,
some bronchiolar cilia loss
At 2 ppm thickening of alveolar walls,
edema, increase in macrophage number,
loss of bronchiolar cilia, inflammation
At 10 ppm severe loss of cilia in
trachea and bronchioles, edema,
hemorrhage, inflammation, focal
emphysema (enlarged airspaces),
increased number of Type 2 cells
Edema at >7 0 ppm, some damage to
alveolar cell mitochondria and cell
membrane at S3 0 ppm
No major morphological changes
observed
Cilia loss, shortening of cilia, focal
hypertrophy of bronchiolar epithelium
Reference
Thomas et al (1967)
Hayasmetal (1987)
Azoulay-Dupuis et al
(1983)
Dowelletal (1971)
Messihaetal (1983)
Romboutetal (1986)
LM = Light microscopy
TEM = Transmission electron microscopy
SEM = Scanning electron microscopy
-------
TABLE 13-10. EFFECTS OF SUBCHRONIC EXPOSURE TO NITROGEN DIOXIDE ON LUNG MORPHOLOGY3
NC>2 Concentration
a
jttg/m
207
865
5,260
16,500
640
940
base +
2,820
peak
3,760
^ base +
V3 11,300
H-* peak
ppm
0 11
046
28
88
034
05
base +
15
peak
20
base +
60
peak
Exposure Procedures
Continuous, LM
1 mo TEM
6 h/day, LM
5 days/week,
6 weeks
23 h/day, LM
7 days/week TEM
base, 1-h peaks
twice/day,
5 days/week
3X base
concentration,
6 weeks
Species
Gender Age (Strain) Effects Reference
F 1,3, Rat Various morphometnc changes, depending on age Kyono and Kawai
12, 21 mo (JCL-SD) and exposure level Multiphasic pattern (e g , (1982)
decrease in air-blood barrier thickness from
1-12 mo of age, and increase at 21 mo old)
M NS Mouse Type 2 cell hypertrophy and hyperplasia, increase Sherwin and Richters
(Swiss in mean linear intercept and amount of alveolar (1982)
Webster) wall area
M 1 day and Rat In proximal alveolar region 0 5 ppm base + Crapo et al (1984)
6 weeks (Fischer 344) peak caused Type 2 cells to become spread over Chang et al (1986,
more surface area in neonates and adults, Type 2 1988)
cell hypertrophy and increase in number of AMs
in adults, Type 2 cells thinner in neonates,
2 0 ppm base 4- peak (only adults studied) caused
similar changes plus an increase in numbers of
Type 1 cells, which were smaller than normal
Type 1 cells 0 5 ppm base + peak had increased
interstitial matrix and fibroblast volume In
terminal bronchiolar region 0 5 ppm base + peak
caused no effects on percentage distribution of
ciliated cells and Clara cells in neonates or adults,
but neonates (only) had an increase in ciliated cell
surface area and mean luminal surface area of
Clara cells 2 0 ppm base + peak (only adults
studied) resulted in fewer ciliated cells with a
reduced surface area and alterations in the shape of
Clara cells
-------
TABLE 13-10 (cont'd). EFFECTS OF SUBCHRONIC EXPOSURE TO NITROGEN DIOXIDE ON
LUNG MORPHOLOGY3
NC>2 Concentration
a
/ig/m ppm Exposure Procedures Gender Age (Strain)
1,000 053 Continuous, LM NS NS Guinea pig
90 days (NS)
Rabbit
(New
Zealand)
Dog
(Beagle)
Monkey
(Squirrel)
Rat
(Sprague-
Dawley)
H- 1,000 0 53 Continuous, LM M 6 weeks Rat
£ 2,500 1 33 up to 28 days TEM (Wistar)
N> 5,000 27 SEM
20,000 10 6
20,000 10 6 6 h/day,
up to 28 days
Effects
No lesions were visible by the technique used
At 0 53 and 1 33 ppm no pathology
At 2 7 ppm focal thickening of centnacinar septa by
2 days, progressive loss of cilia and abnormal cilia m
trachea and main bronchi at >4 days, hypertrophy of
bronchiolar epithelium at >8 days At Days 16 and 28,
all epithelial cells hypertrophied At 10 6 ppm
hypertrophy of ciliated and Clara cells in terminal
bronchioles by 2 days, hyperplasia of bronchioli by
4 days, extensive shortening and loss of cilia in trachea
through bronchioles by 4 days, necrosis of Type 1 cells,
increase m number of Type 2 cells, thickening of
proximal alveolar septa, increased number of
macrophages and accumulation in bronchioles, alveolar
dilatation
Delayed onset of changes and changes less severe
Reference
Steadman et al
(1966)
Rombout et al
(1986)
-------
TABLE 13-10 (cont'd). EFFECTS OF SUBCHRONIC EXPOSURE TO NITROGEN DIOXIDE ON
LUNG MORPHOLOGY3
u>
NC«2 Concentration
3
Ltg/m PP^-
1,320- 07-08
1,500
1,880- 1 0-1 5
2,820
3,760 2 0
3,760 2 0
3,760 2 0
7,520 4 0
18,800 10
47,000 25
Exposure Procedures Gender Age
Continuous, LM F 3 weeks
1 mo TEM
8h/day, LM M 8 weeks
5 days/week,
8 weeks
Continuous, LM M NS
7-21 days
Continuous, LM M 8 weeks
6 weeks TEM
SEM
6h/day, LM M NS
5 days/week,
up to 21 days
Species
(Strain)
Mouse
(JCL ICR)
Hamster
(Golden
Syrian)
Guinea pig
(NS)
Rat
(Wistar)
Rat
(Wistar)
Effects Reference
At exposure end mucous hypersecretion, focal Nakajima
degeneration and desquamation of mucous membrane, et al
terminal bronchiolar epithelial hyperplasia, some alveolar (1980)
enlargement, shortening of cilia, Type 1 cell edema After
1 mo postexposure minimal lesions persisted in some
bronchioles, lymphocytic infiltration of tracheal and
bronchial mucosa
Moderate alveolar enlargement, primarily at bronchiolar- Lafuma
alveolar duct junction, increase in mean linear intercept, et al
decrease internal surface area of lung, no lesions m (1987)
bronchial, bronchiolar, alveolar duct, or alveolar
epithelium, no change in macrophage number
Type 2 cell hypertrophy at 7 or 21 days Sherwin
etal
(1973)
Evidence of intercurrent disease, which may mask changes Azoulay
due to NO2 exposure Some cilia loss ui terminal et al
bronchioles, some distended or disrupted alveolar walls (1978)
At 4 ppm no lesions in nasal cavity or lungs At Hooftman
10 ppm no lesions m nasal cavity, increased cellulanty of et al
walls of bronchioles, alveolar duct, and adjacent alveoli by (1988)
21 days, hypertrophy or hyperplasia of small bronchi and
bronchiolar epithelium by 7 days At 25 ppm no lesions
in nasal cavity, hypertrophy or hyperplasia of small
bronchi or bronchiolar epithelium by 7 days, increase in
cellulanty of walls of respiratory bronchioles, alveolar
ducts and adjacent alveoli by 7 days, some mononuclear
infiltration of penbronchial areas
-------
TABLE 13-10 (cont'd). EFFECTS OF STJBCHRONIC EXPOSURE TO NITROGEN DIOXIDE ON
LUNG MORPHOLOGY3
NO2 Concentration
3
Hg/m ppm
18,880 10
18,800 10
18,800 10
Exposure
Continuous,
14 days
Continuous,
1 mo
Continuous,
6 weeks
Species
Procedures Gender Age (Strain)
IM M NS Rat
TEM (Wistar)
LM F 1, 3, Rat
TEM 12, 21 mo (JCL SD)
TEM M NS Guinea pig
(NS)
Effects Reference
3 days swelling and vacuolar degeneration of Type 1 Hayashi
cells, swelling of Type 2 cells, desquamation of alveolar et al
cells, interstitial edema, goblet cell hyperplasia in trachea (1987)
and bronchi 7-14 days desquamation of Type 1 cells,
hypertrophy and hyperplasia of Type 2 cells, slight
thickening of alveolar wall, interstitial edema,
desquamation of endothelial cells, swelling and vacuolar
degeneration of noncihated bronchiolar cells, loss of cilia
and desquamation of bronchial epithelium
Increased arithmetic mean thickness of air-blood barrier, Kyono and
increased thickness of both interstitial matrix and of cells, Kawai
hyperplastic foci in middle and terminal bronchi (1982)
Type 2 cell hyperplasia, increase in hpid bodies and Yuen and
lamellae in cells Sherwin
(1971)
= Light microscopy
TEM = Transmission electron microscopy
F = Female
M = Male
NS = Not stated
AMs = Alveolar macrophages
SEM = Scanning electron microscopy
-------
TABLE 13-11. EFFECTS OF CHRONIC EXPOSURE TO NITROGEN DIOXIDE ON LUNG MORPHOLOGY3
NO2 Concentration
/wg/m
80
750
7,520
,_, 380 base
& 1,470
peak
940
1,880
7,520
ppm
004
04
40
0 2 base
078
peak
05
10
40
Exposure
Continuous,
9-27 mo
23 h/day,
7 days/week
base + 2 1-h
peaks,
5 days/week,
52 weeks.
Continuous,
7 mo
Species
Procedures Gender Age (Strain)
LM M 8 weeks Rat
TEM (JCL,
Wistar)
LM F 6-8 Mouse
weeks (CD-I)
LM M/F 4 weeks Rat
TEM (JCL-
SD)
Effects
At 0 04 ppm no significant change, but some
tendency towards increase in arithmetic mean thickness
of air-blood barrier At 0 4 ppm slight increase m
arithmetic mean thickness of air-blood barrier by
18 mo, becoming significant by 27 mo, some
interstitial edema and slight change in bronchiolar and
alveolar epithelium by 27 mo At 4 ppm
hypertrophy and hyperplasia of bronchiolar epithelium
and mcrease in arithmetic mean thickness of air-blood
barrier at 9 mo, Clara cell hyperplasia, interstitial
fibrosis, hypertrophy of Type 1 and Type 2 cells, and
some decline in arithmetic mean thickness of air-blood
barrier at 27 mo
Slight to moderate interstitial pneumonia considered to
be due to intercurrent disease rather than NO2
exposure Intercurrent disease may have masked
effects of NO2
At 0 5 ppm swelling of terminal bronchiolar cilia,
hyperplasia of Type 2 cells At 1 ppm cilia loss m
terminal bronchioles, hyperplasia of Type 2 cells,
interstitial edema At 4 ppm cilia loss in terminal
bronchioles, hyperplasia of Type 2 cells, interstitial
edema, decrease in number lamellar bodies in Type 2
cells, lysosomes with osmiophilic lamellar structure in
ciliated cells of terminal bronchioles
Reference
Kubotaetal (1987)
Miller etal (1987)
Yamamoto and
Takahashi (1984)
-------
TABLE 13-11 (cont'd). EFFECTS OF CHRONIC EXPOSURE TO NO2 ON LUNG MORPHOLOGY
NO2 Concentration
jj.gfm ppm Exposure Procedures Gender Age
940 05 6-24h/day, LM NS NS
3-12 mo
940 05 Continuous, LM M NS
up to 19 mo^ TEM
1,500 08 Continuous, LM NS 4
lifetime weeks
(up to 33 mo)
1,880 1 0 Continuous, LM M NS
16 mo TEM
£ 1,880 10 6h/day, LM M NS
vb 9,400 50 5 days/week,
up to 18 mo
1,880 1 0 6 h/day, LM M NS
5 days/week,
18 mo
Species
(Strain) Effects Reference
Mouse 3 mo pneumomtis and alveolar size increase, loss of cilia Blair et al (1969)
(NS) in respiratory bronchioles and bronchiolar inflammation with
24 h/day, 6-12 mo pneumomtis, cilia loss, bronchial and
bronchiolar inflammation, alveolar size increase
Rat Type 2 cell hypertrophy and interstitial edema by 4 mo, Hayashi et al
(Wistar) increased thickness of alveolar septa by 6 mo, fibrous (1987)
pleural thickening by 19 mo
Rat Minimal changes slight enlargement of alveoli and alveolar Freeman et al
(Sprague- ducts, some rounding of bronchial and broncmolar epithelial (1966)
Dawley) cells, increase in elastic fibers around alveolar ducts
Monkey Slight to moderate interstitial pneumonia considered to be Fenters et al (1973)
(Squirrel) result of intercurrent disease rather than NC^ exposure
Intercurrent disease may have masked effects of NC>2
Dog At 1 ppm 6 mo no lesions observed, no pathology, Wagner et al
(Mongrel) 12 mo dilated alveoli and alveolar ducts, 18 mo dilated (1965)
alveoli, edema, thickening alveolar septa by chrome
inflammatory cells No differences between exposed and
control dogs, so intercurrent disease may have been present
Intercurrent disease may have masked effects of NC>2 At
5 ppm 6 mo no pathology, 12 mo dilated alveoli and
alveolar ducts, 18 mo edema, congestion, and thickened
alveolar septa due to inflammatory cells
Guinea Evidence of intercurrent disease that may have masked N(>2 Wagner et al
pig morphological effects (1965)
(English)
-------
TABLE 13-11 (cont'd). EFFECTS OF CHRONIC EXPOSURE TO NITROGEN DIOXIDE ON LUNG MORPHOLOGY
N02 Concentration
/ig/m3
1,880
9,400
1,880
ppm Exposure
1 0 7 h/day,
50 5 days/week,
15 weeks
1 0 Base 7 h/day,
Procedures Gender Age
LM M/F 14-16
weeks
(
Species
(Strain)
Rat
(Fischer 344)
Response
No lesions observed Subpleural
accumulations of macrophages and focal
areas of hyperinflation at 5 ppm or base +
peak exposures No change in mean linear
intercepts
Reference
Gregory et al (1983)
base + base + 5 days/week,
9,400 50 2 1 5-h peaks/day
peak peak 15 weeks
3,760 20 Continuous, LM
2 years TEM
NS 4 weeks Rat
(NS)
Loss of cilia in terminal bronchioles,
abnormal cihogenesis, crystalloid
inclusions in bronchiolar epithelial cells,
increased thickness of collagen fibrils and
basement membrane in terminal
bronchioles
Stephens et al
(1971a,b)
3,760 2 0 Continuous, up to LM
12 mo TEM
M 4 weeks Rat
(Wistar)
Loss of cilia by 72 h, decreased number of Stephens et al
ciliated cells by 7 days, hypertrophy and
hyperplasia of bronchiolar epithelium and
an "apparent" return to normal after
21 days exposure
(1972)
3,760 2 0 Continuous, up to LM
360 days
M 4 weeks Rat
(Wistar)
No change in turnover of terminal
bronchiolar epithelial cells, increase in
turnover of Type 2 cells in peripheral
alveoli by 1 day, but normal by 7 days
Evans et al (1972)
3,760 20 Continuous, LM
14 mo
M/F NS Monkey (Macaco.
speciosd)
M NS Rat
(Sprague-Dawley)
Bronchiolar epithelial hypertrophy,
especially adjacent to alveolar ducts,
change to cuboidal cells in proximal
bronchiolar epithelium
Minimal effect some terminal bronchiolar
epithelial hypertrophy
Funosietal (1973)
-------
TABLE 13-11 (cont'd). EFFECTS OF CHRONIC EXPOSURE TO NITROGEN DIOXIDE ON LUNG MORPHOLOGY2
NC>2 Concentration
/&g/m3 ppm Exposure
3,760 2 0 Continuous,
lifetime (up
to 763 days),
0 8 ppm for
69 days, then
2 Oppm
7,520 4 0 Continuous,
16 weeks
9,400 50 6 h/day,
5 days/week,
14 mo
£ 9,400 50 4-7 5 h/day,
\b 5 days/week,
00 5 5 mo
17,900 95 7 h/day,
5 days/week,
6 mo
17,900 95 7 h/day,
5 days/week,
24 mo
Species
Procedures Gender Age (Strain)
LM M NS Rat
(Sprague-
Dawley)
LM M 4 weeks Rat
(Sprague-
Dawley)
LM M NS Mouse
(C57BL/6,
Webster)
LM NS NS Guinea pig
(New
England)
LM M inutero Rat
and 6 mo (Fischer
344)
LM M 18 weeks Rat
(Fischer
344)
Effects
Alveolar distension, especially near alveolar duct
level, increased variability in alveolar size,
hypertrophy in terminal bronchiolar cells, no
inflammation
Bronchial epithelial hyperplasia
No lesions observed
Some dilation of terminal bronchioles, trachea!
inflammation, desquamative pneumomtis
No histological changes, no effects on mean linear
intercepts
Minimal inflammatory response, mild hyperplasia of
bronchiolar epithelium extending into proximal
alveoli Slight thickening of terminal bronchiolar
walls No change in mean linear intercept
Emphysemic rats had similar response
Reference
Freeman
etal (1968b)
Haydon et al
(1965)
Wagner et al
(1965)
Balchum
etal (1965)
Mauderly
etal (1987)
Mauderly
(1989)
Mauderly
etal (1989,
1990)
aLM = Light microscopy
TEM = Transmission electron microscopy
M = Male
F = Female
NS = Not stated
-------
The large degree of interspecies variability in responsiveness to NO2 is clearly evident
from those few studies (Table 13-9 and 13-10) where different species were exposed under
identical conditions and examined using the same methods (Wagner et al, 1965; Funosi
et al , 1973, Azoulay-Dupuis et al, 1983) Such differences in response may be due to
inherent species differences in sensitivity of cells at target sites, to differences in effective
dose of NO2 reaching target sites due to anatomical and ventilatory differences, or to a
combination of these factors Thus, the choice of experimental animal affects the magnitude
of changes observed following exposure Morphological lesions may not be detectable in
some species by less sensitive methods of evaluation However, in most cases, the sites and
types of morphological lesions produced by NO2 inhalation are similar in all species when
effective concentrations are used and appropnate tissues are examined using sensitive
methods In direct comparisons, the guinea pig, hamstei, and monkey all appear to be more
severely affected by equivalent exposure to NO2 than is the rat, which is the most commonly
used experimental animal
Sites Affected
The extent of injury to an individual cell is related to both the sensitivity of that cell
type and the dose of NO2 delivered to the site occupied by that cell Among cells of a
specific type, several factors (e g , the maturity of the cell and its antioxidant capacity) may
influence its sensitivity The dose of NO2 to which an individual cell is exposed is
determined by the concentration of NO2 at the site in the respiratory system occupied by that
individual cell and the surface area of that cell that is exposed to that concentration Thus,
sensitivity of cells and the magnitude of morphologically detectable injury are not the same
In lungs, morphological evidence of injury is first noted, and is usually most severe, in
the epithelium of the centnacinar region (i e , at the junction of the conducting airways with
the gas exchange area) The centnacinar region mcludes the terminal or respiratory
bronchioles and the immediately adjacent alveoli, often called proximal alveoli Within this
region, those cells that are most sensitive to NO2-induced injury are the ciliated cells of the
bronchiolar epithelium and the Type 1 cells of the alveolar epithelium
Ciliated cells located in terminal or respiratory bronchioles lose some or all of their
cilia or become necrotic and are shed from the epithelium Nonsecretory bronchiolar cells
13-99
-------
(Clara cells in rodents) appear less sensitive to NO2, but they do lose secretory granules and
surface projections With continued exposure, nonciliated bronchiolar cells increase m
number (hyperplasia) and in size (hypertrophy) These cells are the progenitor cells for
replacement of the ciliated cells that were sloughed from the airway epithelium Following
chronic exposure, there is also increased loss of cilia both in bronchioles and more proximal
conducting airways Remaining cilia may have abnormal structure and location
In centnacinar (proximal) alveoli, but not in alveoli located at the periphery of acini,
short-term (acute) exposure to NO2 results in necrosis and sloughing of Type 1 alveolar
epithelial cells, which leaves the underlying basal lamina bare This is followed by
proliferation of, and replacement by, Type 2 alveolar epithelial cells, which are progenitor
cells for Type 1 cells Because Type 2 cells have a cuboidal shape, rather than the squamous
shape of Type 1 cells, this may result in a few centriacinar alveoli with a thickened epithelial
component of the air-blood barrier
Bronchiolar epithelium was observed in centriacinar alveoli by Mauderly et al (1990)
o
in rats exposed to 17,900 /*g/m (9 5 ppm) NO2, 7 h/day, 5 days/week for 24 mo
In addition to the usually described mild hyperplasia of the terminal bronchiolar epithelium,
they reported an extension of bronchiolar cell types into centnacinar alveoli, giving the
appearance of respiratory bronchioles which was not observed in controls They also
reported a slight progression of this lesion, but not of the epithelial hyperplasia Nettesheim
et al (1970) described this lesion as "alveolar bronchiolization" and reported it in mice
chronically exposed to a synthetic smog Although it may appear to be only a slight
modification of alveolar epithelial hypertrophy, replacement of one type of epithelium by
another (e g , alveolar by bronchiolar), and the progression of the lesion with increasing
length of exposure, may have quite different consequences
Although the centriacinar epithelial changes have received the most attention, as they
\
are easily seen and quantitated using a variety methods, there have also been reports of
changes in the centriacinar basal lamina and connective tissue interstitium that underlie the
epithelium. In earlier studies, Freeman et al (1969, 1972) reported proliferation of new
connective tissue at the junction of terminal bronchioles and alveolar ducts from rats exposed
o
to 28,200 jtg/m (15 ppm) NO2 These interstitial changes persisted following postexposure
"recovery" penods of up to 52 weeks In more recent studies, Kyono and Kawai (1982)
13-100
-------
reported morphometric increases in the interstitium following continuous exposure of
1-mo-old rats to 940, 5,640, and 18,800 /*g/m3 (0 5, 3 0, and 10 0 ppm) NO2 for 1 mo
Chang et al (1986), in a TEM morphometric study of centriacinar alveoli from rats exposed
3
to an urban pattern consisting of a baseline of 940 or 3,760 /*g/m (0 5 or 2 0 ppm) with 1-h
peaks twice a day to 3 tunes these levels NO2, reported an increase in mean volume, but not
number, of interstitial fibroblasts and in volume of interstitial matrix These interstitial
2
changes were statistically significant in rats exposed to the lower concentration (940 pg/m
with peaks to 2,820 jitg/m ) NO2 for 6 weeks In the same study, they also reported
increased surface density of the basement membrane (basal lamina) in the rats exposed to
3,760 jitg/m3 (2 0 ppm) baseline with peaks to 11,300 jwg/m3 (6 0 ppm) for 6 weeks Kubota
et al (1987) reported morphometric increases in the interstitium of rats exposed to
752 jwg/m3 (0 4 ppm) for 27 mo and to 7,520 /ig/m3 (4 0 ppm) for 18 mo These qualitative
and quantitative morphological studies correlate well with the increased collagen synthesis
rate in NO2-exposed rats discussed in Section 13 2 2 2 on lung biochemistry
Chronic exposures may result in airspace enlargement characteristic of animal models
of emphysema In most, but not all cases, tissue destruction is not present, so the condition
does not meet the 1985 National Heart, Lung and Blood Institute (NHLBI) definition of
human emphysema (National Institutes of Health, 1985) A detailed discussion of
emphysema in relation to NO2 exposure follows the discussion on susceptibility to NO2-
induced morphological changes
Progression and Regression of Morphological Changes
The temporal progression of early events due to NO2 exposure focusing on centriacinar
epithelial cells has best been described in the rat (e g , Evans et al, 1972, 1973a, 1974,
1975, 1977, Freeman et al , 1966, 1968b, Stephens et al , 1971a, 1972) The earliest
2
alterations resulting from exposure to concentrations of >3,760 /*g/m (2 0 ppm) are seen
within 24 to 72 h of continuous exposure and include desquamation of the Type 1 cells and
ciliated bronchiolar cells, resulting in bare basal lamina in the centriacinar region and
accumulation of fibrin in small airways Accumulations of AMs were also reported
Epithelial repair by replacement of destroyed cells begins within 24 to 48 h of continuous
exposure The new cells in the bronchioli are denved from nonciliated cells, whereas in the
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alveoli, the damaged Type 1 cells are replaced with Type 2 cells Cell renewal, as measured
by incorporation of tntiated thymidine ( H-thymidine) by Type 2 cells, is observed within
12 h after the initial NO2 exposure During continued exposure, the number of H-thymidine
labeled cells becomes maximal by about 48 h and decreases to preexposure levels by about
o
6 days (Evans et al, 1975) If exposure levels are very high (> 18,800 /-tg/m , 10 ppm),
however, resolution and return to normal-appearing centnacmar epithelial cells may be
delayed or prevented, and the presence of increased numbers of Type 2 cells may be
prolonged or permanent
The resolution of NO2-induced morphologic changes may be complete after the
exposure ends or some lesions may remain, depending upon details of the exposure regime,
species of animal studied, and the methods of evaluation For example, Rombout et al
O
(1986) exposed rats to 20,000 jitg/m (10 6 ppm) continuously for 28 days By LM and
SEM, bronchiolar epithelial hyperplasia appeared totally resolved beyond 4 days after
cessation of exposure, and hypertrophy was totally resolved after 16 days of postexposure
recovery After a 1-week postexposure period, all changes seen by LM and SEM appeared
completely recovered However, some abnormal cilia were observed by TEM 4 weeks after
the exposure ended and some remnants of the NO2-induced lesions were still seen by TEM
56 days after the exposure ended Kubota et al (1987) examined the tune course of alveolar
epithelial lesions ui small groups of rats exposed to 7,520 jig/m3 (4 0 ppm) NO2, 24 h/day
for up to 27 mo. In the quantitative or morphometnc portions of this study, Kubota et al
(1987) deliberately avoided centnacmar or proximal alveoli, so the changes they describe
represent those hi the overall gas exchange area rather than only those in the most involved
alveoli (i.e , centnacmar or proximal alveoli), which are the only alveoli usually studied
One phase, which lasted for 9 to 18 mo of exposure, consisted of a decrease in number and
an increase in cell volume of Type 1 epithelium, an increase in the number and volume of
Type 2 cells, and an increase in the relative ratio of Type 2 to Type 1 cells A second
phase, which occurred at 18 to 27 mo of exposure, showed some recovery of alveolar
epithelium Thus, epithelial changes tend to resolve, at least partially, during continued
chronic exposure to low concentrations and to resolve rapidly during postexposure penods
Changes in the interstitium, the basal lamina and connective tissues under the
epithelium, develop much more slowly and resolution, if it occurs, is prolonged In the two
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studies of epithelium discussed above, Rombout et al (1986) reported TEM observations of
increased collagen and elastin in the interstitium of rats exposed to 20,000 ^g/m (10 6 ppm)
for 28 days, and Kubota et al (1987) reported morphometnc increases of the mean alveolar
air-blood barrier in rats exposed to 752 jwg/m3 (0 4 ppm) for 27 mo and in rats exposed to
7,520 jug/m (4 0 ppm) for 18 mo These authors considered the interstitial changes
progressive and leading to fibrosis, rather than resolving as do epithelial changes
In summary, epithelial changes have been the focus of most studies Epithelial changes
progress rapidly and regress rapidly and relatively completely both during chronic exposures
and during postexposure periods In contrast, changes in the interstitium have been the
subject of fewer studies, even though significant interstitial changes have been reported
following chronic exposure to low concentrations of NO2 Interstitial changes develop more
slowly, may progress during exposure, and regress slowly, if at all, during postexposure
penods
Effects of Nitrogen Dioxide as a Function of Exposure Pattern
Although the extent and degree of morphologic alterations in the epithelium and
interstitium appear to correspond to NO2 exposure concentrations, little is actually known
about effects of other modifying factors, such as the exposure duration and concentration
relationship, short-term peaks in concentration, or cycles of exposure and postexposure
The relative roles of C and T in response to subchronic exposure were examined by
Rombout et al (1986) These researchers exposed rats to 1,000 to 5,000 /ig/rn3 (0 53 to
2 66 ppm) NO2 for up to 28 days, or to 20,000 /^g/m3 (10 6 ppm) for either 6 h, 6 h/day for
28 days, or 24 h/day for 28 days They concluded that concentration played a more
important role in inducing lesions than did exposure duration, as long as the product of
C X T was constant, and that the effect of concentration was stronger with intermittent
exposure than with continuous exposure They also reported that continuous exposure was
important to the development of strong AM response These findings are similar to those
using the mfectivity model discussed in Section 13 2 2 1
Ambient concentrations of NO2 are often characterized by transient peaks superimposed
upon a lower and relatively constant baseline concentration, unlike experimental exposures,
which are most commonly at a constant concentration The morphological effects of
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exposure patterns involving transient peaks were examined in a number of studies
However, some studies lacked a group exposed to the constant baseline concentration for
comparison with those exposed to the baseline concentration plus transient peaks Thus, the
findings of these studies do not elucidate the relative contributions of the baseline and peaks
to the responses
A study that included a baseline concentration group was that by Gregory et al (1983)
They exposed rats for 7 h/day, 5 days/week for up to 15 weeks to atmospheres consisting of
the following concentrations of NO2 (1) 1,880 /jg/m3 (1 0 ppm), (2) 9,400 ^g/m3
(5.0 ppm), or (3) 1,880 /*g/m3 (1 0 ppm) with two 1 5-h peaks of 9,400 ^g/m3 (5 0 ppm)
per day (i e , animals were exposed to NO2 at 1,880 jwg/m3 for 1 5 h, 9,400 jwg/m3 for
1.5 h, 1,880 Atg/m3 for 3 h, 9,400 jwg/m3 for 1 5 h, and 1,880 /*g/m3 for 0 5 h) No change
in lung weight was found in any exposure group After 15 weeks of exposure, routine LM
histopathology showed minimal effects, with focal hyperinflation and areas of subpleural
accumulation of macrophages found in some of the animals exposed either to a constant level
of 9,400 ftg/m3 or to 1,880 jiig/m3 with the 9,400-/ig/m3 peaks Because the 1,880 /wg/m3
group without peak exposures did not have these changes, the peak exposures appear to have
contributed to increased morphological effects Changes were also reported in the lung
biochemistry of the NO2-exposed animals as discussed in Section 13 2 2 2
Miller et al (1987) exposed mice for 1 year-(23 h/day, 7 days/week) either to a
*>
continuous baseline concentration of 380 /*g/m (0 2 ppm) NO2, or to this baseline onto
which was superimposed, for 5 days/week, two 1-h peaks (given in the morning and
o
afternoon) of 1,470 /ig/m (0 78 ppm) Morphologic examination (LM) performed after
32 and 52 weeks of exposure, and then 1 mo after all exposures had ended, revealed no
treatment-related lesions in either exposure group, although host defense and pulmonary
function changes were noted (Sections 13 22 I and 13 2 2 3)
\
Crapo et al (1984) and Chang et al (1986) reported on the NO2-induced changes in
the centnacinar (proximal) alveolar region of 1-day and 6-week-old rats exposed for 6 weeks
o
to a baseline concentration of 940 or 3,760 j^g/m (0 5 or 2 0 ppm), 23 h/day for
7 days/week onto which were superimposed two daily 1-h peaks of three tunes the baseline
concentration for 5 days/week The rats were studied using TEM morphometnc analyses
In the older rats (6 weeks old at the start of the exposure) at both exposure levels, Type 2
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cells increased in total volume and mean cell volume, but not in number/mm , and they
occupied a larger percentage of the basal lamina (basement membrane) In the older rats
3 3
exposed to 3,760 /tg/m , but not 940 /ig/m , with peaks. Type 1 cells had a larger total
-3
volume and increased numbers/mm , with a smaller mean surface area The percentage of
the basal lamina occupied by Type 1 cells was decreased in older rats of both exposure
groups They also had mcreased total volume and number of AMs, but the mean cell
volume was mcreased only in the 940-jwg/m group The total volume of interstitial matrix
3 3
and of fibroblasts mcreased in the 940-jwg/m older group, but not the 3,760-jttg/m older
group The number of interstitial fibroblasts did not change, but their mean cell volume
mcreased in the older groups Both age groups of rats reacted in a generally similar manner
Pulmonary function changes in sunilianly exposed animals were assessed by Stevens et al
(1988), indicating a correlation between decreased compliance and thickening of the alveolar
interstitium (Section 13 2 2 3)
The terminal bronchiolar region of these rats was also examined morphometncally
(Chang et al., 1988) The lower exposure level caused no effects At the higher level, there
was a 19 % decrease in ciliated cells per unit area of the epithelial basement membrane and a
reduction in the mean surface area of 29 % in the remaining cilia in the high exposure group
In the Clara cells, the siz£ of the dome protrusions were decreased, giving the bronchial
epithelium a flattened appearance
Susceptibility to Nitrogen Dioxide-Induced Morphological Changes
Susceptibility to morphological effects may be influenced by many factors Some
factors that have been studied include age, compromised lung function, and acute infections
Age of the animal at the tune of exposure may be responsible for some of the variability in
morphological response seen in the same species exposed to comparable concentrations
Stephens et al (1978) exposed rats from 1 to 40 days of age to 26,320 /tg/m3 (14 ppm) NO2
for 24, 48, or 72 h Using LM and TEM, they found only minor injury and loss of cilia in
terminal bronchioles of rats exposed before weaning at 20 days After weaning, there was a
progressive increase in cellular response in both terminal bronchioles and alveoli, with a
plateau reached at about 35 days of age Other investigators expanded on these observations
using lower concentrations
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Azoulay-Dupms et al (1983) exposed both rats and guinea pigs aged 5 to > 60 days to
o
3,760 or 18,800 /ig/m (2 or 10 ppm) for 3 days No rats of either age died during the
exposure, but in the guinea pigs exposed to 18,800 jug/m (10 ppm), mortality increased with
increasing age from 4% in the 5-day-old group to 60% in the 55-day-old group and 67% in
the mothers In both species, older animals showed greater effects of exposure than did the
o
newborns. At 3,760 jug/m (2 0 ppm), no histological effects were observed in rats of any
age. Neither did exposure of guinea pigs < 45 days old to this level produce histological
o
effects The 45-day-old guinea pigs exposed to 3,760 jwg/m had thickened alveolar walls,
alveolar edema, and inflammation, whereas animals older than 45 days showed similar, but
more frequent, alterations that seemed to increase with age The mother guinea pigs exposed
*y o
to 3,760 /*g/m had focal loss of cilia in bronchiok At 18,800 jwg/m , only rats ^45 days
old responded, confirming the observations of Stephens et al (1978) Rats in this group had
fibrinous deposits in alveoli and focal loss of cilia in bronchioles Guinea pigs of all ages
were affected by exposure to 18,800 ^g/m3 All guinea pigs in that exposure group had loss
of cilia in bronchioles and the trachea, edema, an increase in number of Type 2 cells, and
alveolar inflammation Guinea pigs greater than 60 days old and mother guinea pigs exposed
o
to 18,800 /ig/m had the most severe lesions, including foci of interstitial pneumonia and
emphysema, presumably enlarged airspaces This study clearly demonstrated the relative
insensitivity of newborn rats and guinea pigs to NO2 and the greater sensitivity of guinea
pigs compared to rats of the same age The investigators noted that the "lungs of newborn
guinea pigs at birth were more mature than newborn rat lungs," which may explain some,
but probably not all, of the species differences in response of similarly aged rats and guinea
pigs (See Section 13 2 2 1 on host defense mechanisms )
An extensive series of exposures designed to relate morphometnc changes in the air-
blood barrier to age was performed by Kyono and Kawai (1982) Rats at 1, 3, 12, and
3
21 mo of age were exposed continuously for 1 mo to 207, 865, 5,260, or 16,500 /tg/m
(0.11, 0.46, 2 8, or 8 8 ppm) NO2 Light and electron microscopic analyses were used to
evaluate the exposure effects Various morphometnc parameters were assessed, including
arithmetic mean thickness of the air-blood barrier (i e , the thickness of the tissue between
the surfaces of the alveolar and capillary lumens) and the volume density of various alveolar
wall components Because these investigators were interested in effects on the overall gas
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exchange area, they deliberately excluded centriacinar alveoli, the site of major NO2 damage,
from the morphometnc analyses The arithmetic mean thickness of the air-blood barrier
tended to increase in an exposure-dependant manner in all age groups The arithmetic mean
thickness of the air-blood barrier was significantly increased in all age groups in rats exposed
3 3
to 16,544 jwg/m or 5,264 jwg/m , with the exception of 12-mo-old rats, and in 3-mo-old rats
exposed to 207 jwg/m Response of arithmetic mean thickness to NO2 was greatest in the
1-mo-old rats, decreased from 1 to 12 mo and increased again at 21 mo The total
interstitmm (matrix and cells) increased with NO2 concentrations over 5,264 /tg/m3 and was
a major component of the increase in volume density of alveolar wall tissue Alveolar
Type 1 and 2 cells showed various degrees of response, depending on both age at onset of
exposure and exposure concentration In general, the response of each lung component did
not always show a simple concentration-dependent increase or decrease, but suggested a
multiphasic reaction pattern The investigators suggested that part of this observation may
have been due to varying stages of impairment and repair Further, they concluded that age-
dependent differences in response to the same concentration of NO2 occurred, and that the
degree of response decreased with aging from 1 to 12 mo, after which it increased at 21 mo
This study by Kyono and Kawaii (1982) is especially important, as it is one of the few
studies that examined the interstitium, and all studies of the interstitium (Chang et al , 1986,
Kubota et al , 1987) have reported increases in one or both components of the interstitium
These morphological studies correlate well with reports of increased collagen synthesis rates
discussed in Section 13 2 2 3 on lung biochemistry
A possible concern in assessing NO2 toxicity is the effect on adults from exposure
during early life, especially during the period of lung development Unfortunately, there are
few data to allow evaluation of this Mauderly et al (1987) compared developing rats
'j
(exposure began in utero) and 6-mo-old animals exposed to 17,900 jwg/m (9 5 ppm) NO2 for
7 h/day, 5 days/ week for 6 mo Lung development, as determined at young adulthood, was
not significantly affected by earlier exposures There was no significant LM evidence of
lung injury in either group of animals, and there were no exposure-related differences in the
morphometnc parameters studied (i e , alveolar mean linear intercept or the internal surface
area of the lungs) Thus, the available data base indicates that alveoli of NO2-exposed rats
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develop normally, but does not provide information concerning other morphological
parameters of lung growth and development
Chang et al (1986, 1988) exposed 1-day- or 6-week-old rats for 6 weeks to a baseline
o
of 940 /ig/m (0 5 ppm) NO2 for 23 h/day, 7 days/week, with two 1-h peaks (given in the
morning and afternoon) of 2,820 /*g/m3 (15 ppm), 5 days/week, and examined the proximal
alveolar and the terminal bronchiolar regions The detailed results are described in the
previous section The older animals had an increase in the surface density of the alveolar
basement membrane that was not found in the younger animals Although both age groups
responded in a generally similar manner, the 6-week-old animals seemed to be generally
more susceptible to injury than were the 1-day-old animals, as they had more variables that
were significantly different from their control groups Although there was no qualitative
evidence of morphological injury in the terminal bronchioles of the younger rats, there was a
19% increase in the average ciliated cell surface that was not evident in the older rats The
authors also reported a 13 % increase in the mean luminal surface area of Clara cells in the
younger animals versus control animals of the same age
Although the investigators concluded that the 6-week-old rats were as sensitive or more
sensitive than 1-day-old rats, an alternative conclusion that the reverse is true cannot be ruled
out by this study The 1-day-old rats were probably not sensitive to the effects of NO2 until
they were 20 days old (Stephens et al, 1978, Azoulay-Dupuis et al, 1983), after which their
sensitivity would increase until they were 35 days old Thus, the effects measured in the
1-day-old rats may have resulted only from the last 22 days of exposure rather than the entire
42 days of effective exposures of the older rats If the measured effects were essentially the
same as the investigators concluded, the 1-day-old rats would be more sensitive than the
older animals as they responded quantitatively similarly to about one half the exposure tune
as the older animals Another alternative conclusion is that the 1-day-old rats, due to the
20 nonreactive exposures, were in a different phase of damage and repair at the time of
examination The exposure method may also have had an influence Prior to weaning,
neonates are exposed with their mother, often on bedding The resulting huddling behavior
and reactivity of NO2 with the bedding could affect exposure of the neonates
In general, it seems that neonates, specifically prior to weaning, are resistant to the
morphological effects of NO2, and that responsiveness increases with age after weaning until
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a plateau is reached Furthermore, the responsiveness of mature animals appears to decline
somewhat with age, until an increase in responsiveness occurs at some point in senescence
However, the morphological response to NO2 in animals of different ages involves
similarities in the cell types affected and in the nature of the damage incurred Age-related
differences occur in the extent of damage and in the time required for repair, the latter taking
longer in older animals The reasons for age differences in sensitivity are not known, but
may involve differences in diet, variable sensitivity of target cells during different growth
phases, or differences in site-specific doses
Preexisting respiratory disease as a susceptibility factor has been studied rarely in
animals, however, an animal model of emphysema (i e , with enlarged airspaces but not
airspace wall destruction) has been used Lafuma et al (1987) exposed both normal and
•3
elastase-induced emphysematous hamsters to 3,760 j«g/m (2 0 ppm) NO2, 8 h/day,
5 days/week for 8 weeks and analyzed lung tissue with tnorphometnc assays The
emphysematous lesions produced by elastase appeared to be aggravated by subsequent
exposure to NO2 When compared to hamsters treated with elastase and exposed to clean
air, elastase-treated hamsters exposed to NO2 had increases in mean linear intercept and
pulmonary volume (volume of fixed lung as measured by saline displacement) and a decrease
in internal alveolar surface area The investigators suggested that these results may imply a
role for NO2 in enhancing preexisting emphysema However, there no changes in pulmonary
mechanics of the animals (Section 13 2 2 3) In a more comprehensive, longer term study,
Mauderly et al (1989, 1990) exposed elastase-treated and control rats to 17,900 jwg/m3
(9 5 ppm) NO2, 7 h/day, 5 days/week for 2 years or to filtered air They evaluated
pulmonary function, clearance, lung collagen, BAL, and histopathology (by LM), including
quantitative methods for alveolar size and internal surface area There were no additive
morphological effects of NO2 on the emphysematous rat lungs In summary, it appears that
elastase-induced emphysema in rats does not increase susceptibility to NO2 exposure, but that
the similar procedures using hamsters may result in enhancement of the emphysema
Acute infectious lung disease before and during NO2 exposure could also affect
morphologic responses to NO2 exposure Fenters et al (1973) challenged squirrel monkeys
<»
with an influenza virus at various tunes during continuous exposure to 1,880 jwg/m
(1 0 ppm) NO2 for 16 mo, and compared the response to that seen in animals not challenged
13-109
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but exposed to NO2 Only the virus-challenged animals showed responses to NO2, which
included thickening of bronchial and bronchiolar epithelium The investigators also reported
"slight emphysema" in viral-challenged exposed monkeys However, the lungs were not
fixed in a manner appropriate for the diagnosis of emphysema using the current definition for
human emphysema (National Institutes of Health, 1985) See the following subsection
addressing NO2-induced emphysema in experimental animals This study suggests that acute
lung disease may increase responses to NO2 The effects of NO2 on infectivity due to
challenges with microorganisms are discussed in Section 13 22 1
Emphysema Following Nitrogen Dioxide Exposure
In evaluating the reports of emphysema following NO2 exposure, it is necessary to
consider both the current and previous definitions of emphysema and to try to determine the
morphological lesions the investigators observed that led them to the diagnosis of
emphysema Several professional groups have presented definitions of emphysema Those
definitions have changed significantly from those proposed by a group of British physicians
meeting under the auspices of Ciba in 1959 (Fletcher et al, 1959) Presumably, both
investigators who wrote manuscripts and reviewers for journals that published manuscripts
concerning emphysema in NO2-exposed animals were aware of, and used, the latest
definition at the lime the manuscripts were written and reviewed Thus, the date of
publication of the reports of effects of NO2 inhalation relative to the dates of publication of
the definitions of emphysema must be considered in the diagnosis of emphysema The most
recent definition, by MHLBI, Division of Lung Diseases Workgroup (National Institutes of
Health, 1985), differentiates between emphysema in human lungs and animal models of
emphysema When reports of emphysema following NO2 exposures of animals are to be
extrapolated to potential hazards for humans, the definition of human emphysema, rather than
that for animal models of emphysema, must be used Thus, the current definitions of
emphysema in human lungs and in animal models are critical to this review
The report from the National Institutes of Health (1985) first defines respiratory
airspace enlargement "Respiratory airspace enlargement is defined as an increase in
airspace size as compared with the airspace size of normal lungs The term applies to all
varieties of airspace enlargement distal to the terminal bronchioles, whether occurring with
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or without fibrosis or destruction " Emphysema is one of several forms of airspace
enlargement In human lungs, "Emphysema is defined as a condition of the lung
characterized by abnormal, permanent enlargement of airspaces distal to the terminal
bronchiole, accompanied by destruction of their walls, and without obvious fibrosis "
Destruction is further defined "Destruction in emphysema is further defined as
nonuniformity in the pattern of respiratory airspace enlargement so that the orderly
appearance of the acinus and its components is disturbed and may be lost" The report also
indicates that "Destruction may be recognized by subgross examination of an inflation-fixed
lung slice " Emphysema in animal models was defined differently The stated reason for
this difference in the definitions of emphysema in humans and in animal models was
"In order to foster the development of new knowledge, animal models of emphysema are
defined as nonrestnctively as possible An animal model of emphysema is defined as an
abnormal state of the lungs in which there is enlargement of the airspaces distal to the
terminal bronchiole Airspace enlargement should be determined qualitatively in appropriate
specimens and quantitatively by stereologic methods " Thus, in animal models of
emphysema, airspace wall destruction need not be present "Appropriate specimens"
presumably refers to lungs fixed in the inflated state and is similar to the 1962 American
Thoracic Society Committee's lequirement for tissue fixation This document states "It is
still not clear whether the airspace enlargement of age is due to age alone or to the
combination of age and environmental history, but the occurrence of these changes in nearly
all subjects suggests that the changes are normal" (Meneely et al, 1962) Control animals of
the same age as the experimental animals appear necessary to avoid potential confusion due
to age This National Institutes of Health committee also noted that, to date, animal models
of emphysema fall into two general classes "The first class centers on testing the
pathogemcity of agents suspected of being relevant to the genesis of emphysema, models
produced by NO2, cadmium, and tobacco smoke are examples of this type The second class
of models is analytical, for testing specific hypotheses of the pathogenesis of emphysema "
Both classes of studies are in this review
These definitions of emphysema in human lungs and of animal models of emphysema
are significantly different from the 1959 Ciba definitions (Fletcher et al, 1959) One of the
lasting benefits of these Ciba definitions was the development of the concept that emphysema
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should be defined in terms of morbid anatomy rather than altered physiology or clinical
observations (Fletcher and Pride, 1984) The Meneely et al (1962) definition continued that
concept and expanded on the most useful instruments for morphological examination of
properly fixed lungs All of the definitions since 1959 have required that lungs be distended
and fixed before they are cut so the airspaces can be examined in the inflated state
Thus, in reviewing reports of emphysema following experimental NO2 exposure,
important considerations include (1) whether the tissue was fixed in an inflated state,
(2) whether airspaces distal to the terminal bronchiole were enlarged beyond normal and
whether that enlargement was determined quantitatively by stereologic methods (control
animals of identical age as exposed animals should be used for stereologic studies to exclude
the possibility that airspace enlargement was due to age), and (3) whether or not airspace
wall destruction, as defined by the NHLBI workgroup (National Institutes of Health, 1985),
was present. The presence of airspace wall destruction, as defined by the NHLBI
workgroup, is critical In published reports of emphysema following NO2 exposure,
evidence of airspace wall destruction can only be obtained by careful review of the authors'
description of the lesions or by examining the micrographs the author selected for
publication In reviewing the research reports, the authors' descriptions of tissue changes
relative to the definition of destruction in the NHLBI workgroup document (National
Institutes of Health, 1985) are quoted, and all published micrographs were examined for
evidence of destruction as defined by this 1985 NHLBI document Because of the changes in
the definitions of emphysema and in the methods used for evaluation of results of NO2
exposures, the studies are reviewed chronologically
o
Freeman et al (1964) reported emphysema in rats exposed to 47,000 jwg/m (25 ppm)
NO2 for 32 to 65 days Control rats of the same age were maintained in an identical
chamber They reported that "alveolar ducts and alveoli in experimental rats are more
\
variable in size and many are much larger than in controls " The methods for fixation of the
lungs are not mentioned, nor are the methods for evaluating size of airspaces Although the
size of the lungs from exposed rats was stated to be increased, lung weights rather than lung
volumes were reported The conclusion that emphysema was present was presumably based
on the large size of the lungs and the observation of enlarged airspaces that were variable in
size, as no evidence of destruction of airspace walls was presented In terms of the 1985
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NHLBI definitions, these lesions appear to represent airspace enlargement rather than
emphysema of the type seen in human lungs
Wagner et al (1965) exposed dogs, rabbits, guinea pigs, rats, hamsters, and mice to
9,400 jug/m3 (5 0 ppm) NO2 for up to 18 mo They did not observe morphological effects
due to the exposure
Haydon et al (1965) reported emphysema in rats exposed for varying periods from
51 to 813 days to 22,600 jig/m3 (12 ppm) NO2, but not in other rats exposed to 7,520 or
O
1,500 /ig/m (4 0 or 0 8 ppm) NO2 The lungs were fixed via the trachea in the inflated
state The alveoli were described as "quite variable in size, being either dilated or
collapsed " The most staking microscopic abnormality was the "persistent variation in size
of alveoli " The diagnosis of emphysema was based on alveolar size and variation in size
and on the "grossly distended, air-filled lungs that fail to collapse when removed from the
thorax " They reported "occasional rupture of alveolar walls " In terms of the 1985 NHLBI
definitions, these lesions appear to be primarily airspace enlargement rather than emphysema
of the type seen in human lungs
Haydon et al (1967) also reported emphysema in rabbits exposed continuously
(presumably 24 h/day) for 3 to 4 mo to 15,000 or 22,600 /*g/m3 (8 or 12 ppm) NO2 The
lungs were fixed via the trachea in an expanded state They reported enlarged lungs that
failed to collapse when the thorax was opened In 100-//m thick sections from formaldehyde-
fixed dried lungs, they reported "dilated" airspaces with "distorted architecture" In those
and other tissue preparations, they reported that the airspaces appeared "grossly enlarged and
irregular, which appears to be due to disrupted alveoli and the absence of adjacent alveolar
collapse " Thus, in appropriately fixed lungs, they reported evidence of enlarged airspaces
with destructive changes in alveolar walls Although no stereology was done, this appears to
be emphysema of the type seen in human lungs Davidson et al (1967) reported physiologic
changes in these rabbits, but no new observations related to the criteria for emphysema
Unlike their previous reports of emphysema in rats exposed to higher concentrations of
NO2, Freeman et al (1968b) reported that rats exposed continuously (24 h/day) to
-3
3,760 jwg/m (2 0 ppm) NO2 for 112 to 763 days had only equivocal increases in lung weight
and distension of airspaces These lungs were fixed in a distended state via the trachea
These NO2 exposures did not result in emphysema
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Freeman et al (1968a) summarized many of their previously pubhshed NO2 exposures
in a manuscript resulting from a meeting (symposium) presentation They again followed the
definition of emphysema proposed by the 1959 Ciba Symposium and did not present new
data relative to enlarged airspaces or destruction of alveolar walls No new information
relevant to emphysema following NO2 exposure was presented
At the same meeting (symposium), Kleinerman and Cowdrey (1968), reported results of
exposures of hamsters to 84,600 to 103,000 /*g/m3 (45 to 55 ppm) NO2, 22 to 23 h daily for
10 continuous weeks. Some of the hamsters were held in room air for a 4-week
postexposure period. The lungs were fixed in a distended state by formaldehyde fumes via
the trachea Fixed-lung volumes were estimated by water displacement Fixed-lung volumes
of exposed and postexposed hamsters were significantly larger than similar-aged controls
Although the size of alveolar spaces appeared enlarged in the exposed animals, as compared
to similarly fixed controls, there was no evidence of destruction The authors concluded that
"emphysema had not been produced in this experiment" These authors also reported
previously unpublished observations that 21 to 23 h/day exposure of guinea pigs, rabbits, and
rats to concentrations of NO2 resulting in a mortality of approximately 35 % does not result
in emphysema due to the nondestructive character of the tissue response They based their
conclusions on the definition of emphysema proposed by the 1962 American Thoracic
Society and discussed the necessity for inflation fixation of lungs using standard techniques
The conclusion that emphysema of the type seen in human lungs was not produced appears
appropriate
Gross et al (1968) studied the effects of NO2 on control and pneumocomotic lungs
using hamsters and guinea pigs Most exposures were for 2 h/day for 5 days/week The
*j
NO2 concentration appears to have been planned for 41,360 jwg/m (22 ppm), but varied with
o
an initial range of 94,000 to 169,000 jwg/m (50 to 90 ppm) during the first 4 weeks and then
was reduced to ranges of 56,400 to 94,000 jwg/m3 (30 to 50 ppm) for a total exposure period
of 12 mo The lungs were fixed in a distended state via the trachea with formaldehyde
fumes No morphometry of airspaces was reported The complex experimental design and
deaths of animals during exposure made interpretation difficult In hamsters, "since more
animals with emphysema were found in the group not exposed to NO2 it can also be
concluded that long-term exposure of hamsters to NO2 did not cause emphysema " In guinea
13-114
-------
pigs, this exposure resulted in "multiple small foci of emphysema with a prevalence of only
15 % More animals (guinea pigs) without pneumocomosis developed this emphysema than
did animals with pneumocomosis " Both enlarged airspaces and destruction of alveolar walls
can be seen in some of the published micrographs Although emphysema with alveolar wall
destruction was present, the relationship of the emphysema to NO2 exposure is not clear
Blair et al (1969) exposed mice to 940 /*g/m3 (0 5 ppm) NO2, 6, 18, or 24 h/day for
1 to 12 mo The method of fixation of the lungs is not entirely clear They were fixed by
immersion, presumably by immersion of slices of lung, which would be collapsed rather than
fixed in a distended state as recommended by the American Thoracic Society in 1962
(Meneely et al, 1962) It is possible that the lungs might have been distended with air, the
trachea tied and then immersed in fixative that would diffuse into the lung through the thin
pleura Control unexposed mice had pneumomtis Although these investigators made an
attempt to measure alveolar size, they properly conclude that their measurements "did not
represent quantitatively whole lung structure " Thus, the data concerning enlarged
airspaces is not reliable due to the types of lung fixation and alveolar morphometry They
also mentioned alveolar "septal breakage", but not destruction as defined by the 1985 NHLBI
definition, and the septal breakage was not a factor in the increased size of alveoli
No evidence of emphysema was presented in this publication
Buckley and Loosli (1969) studied the effects of 75,200 )wg/m3 (40 ppm) NO2 for 6 or
8 weeks on germ-free mice Following exposure, the mice were either sacrificed and the
lungs were examined, or the mice were infected with microorganisms for additional studies
The lungs were fixed by inflation via the trachea They made no mention of lung or alveolar
size or of the presence or absence of alveolar destruction, even though they cited two of the
earlier studies of Freeman and Haydon In the published micrographs of control- and NO2-
exposed mice, the airspaces appear to be the same size and evidence of destruction was not
seen No evidence of emphysema was presented in this publication
•y
Freeman et al (1972) exposed rats to 37,600 /*g/m (20 ppm) NO2, which was reduced
during the exposure to 28,200 jttg/m3 (15 ppm) or to 18,800 j«g/m3 (10 ppm) for varying
periods up to 33 mo Following removal at necropsy, the lungs were fixed via the trachea at
25 cm of fixative pressure Morphometry of lung and alveolar size was performed in a
suitable, although unconventional, manner The morphometry indicated enlargement of
13-115
-------
alveoli and reduction in alveolar surface area They also both reported alveolar destruction
and illustrated alveolar destruction in their figures They correctly conclude that they have
demonstrated emphysema in their NO2-exposed rats However, it is not entirely clear
3
whether both experimental groups or only the group exposed to 28,200 ngfm NO2 had
emphysema
3
Ehrlich and Fenters (1973) exposed squirrel monkeys to 9,400 or 18,800 jwg/m (5 or
10 ppm) NO2 for 3 mo or to 1,880 j^g/m3 (1 0 ppm) NO2 for 16 mo and then challenged the
monkeys with influenza virus Pieces of lung were probably fixed in a collapsed condition
by immersion, as they state "At autopsy representative lung tissues were obtained for
histopathological examination " Neither airspace wall destruction nor morphometry are
mentioned in the article They concluded that "slight emphysema" was present in the
monkeys exposed to the two highest exposure levels and to the virus and "slight to moderate
^t
emphysema" was present in those exposed to 1,880 jug/m NO2 and to the virus They also
o
reported emphysema was not present in monkeys exposed to 1,880 jwg/m NO2 without the
viral challenge The morphological methods used preclude useful information concerning
emphysema following NO2 exposure
Stephens et al (1976), in a long abstract of papers presented at that year's Aspen
O
conference, stated that "Rats exposed for long periods (3 to 5 mo) to 28,200 /*g/m (15 ppm)
NO2 or 0.8 ppm O3 develop a disease which closely resembles emphysema " Because it is
only a long abstract and the emphasis of the paper was cellular injury, there were no data
relative to alveolar size, nor was there information concerning the presence or absence of
alveolar destruction Thus, this paper does not provide new data relative to the presence of
emphysema of the type seen in human lungs
Port et al (1977) studied experimental and spontaneous emphysema in rat, mouse,
hamster, horse, and human lungs and compared them with normal control lungs from the
same species. Only six mice and one rat had been exposed to NO2 The mice were exposed
to 188 /ig/m3 (0 1 ppm) NO2 with a 2-h peak of 1,880 /xg/m3 (1 0 ppm) NO2 daily for
6 mo Control mice of the same age were also examined Only one NO2-exposed and one
o
control rat were studied The exposed rat breathed 28,200 jwg/m (15 ppm) NO2 from
35 days to approximately 5 mo of age, when clinical illness became apparent The NO2 was
then administered intermittently, based on the clinical signs, to permit survival for at least
13-116
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2 years The control rat was approximately the same age as the exposed one The lungs
were fixed in a distended condition via the trachea at a constant pressure The lungs were
examined by LM and SEM The NO2-exposed rat had distended alveoli and evidence of
airspace wall destruction Both dilated airspaces and evidence of alveolar wall destruction
were reported in the NO2-exposed mice lungs Although airspace wall destruction was
demonstrated, the small number of animals studied severely limits the value of this study
Hyde et al (1978) studied dogs that had been exposed 16 h daily for 68 mo to either
filtered air or to 1,210 ^g/m3 (0 6 ppm) NO2 with 310 jwg/m3 (0 16 ppm) NO or to
270 /tg/m3 (0 14 ppm) NO2 with 2,050 /*g/m3 (1 1 ppm) NO The dogs then breathed clean
air during a 32- to 36-mo postexposure period The right lungs were fixed in a distended
state via the trachea at 30 cm fixative pressure Semiautomated image analysis was used for
3 3
morphometry of airspaces The dogs exposed to 1,210 /-tg/m NO2 with 310 jttg/m NO had
statistically significantly larger lungs with enlarged airspaces and evidence of destruction of
-3
alveolar walls These effects were not observed in dogs exposed to 270 //eg/m NO2 with
o
2,050 jitg/m NO, implying a significant role of the NO2 in the production of the lesions
The lesions in dogs exposed to the higher NO2 concentration meet the criteria of the 1985
NHLBI workshop for emphysema of the type seen in human lungs
Lam et al (1983) exposed 3- and 21-day-old hamsters to 56,400 to 65,800 /*g/m3
(30 to 35 ppm) NO2, 23 h/day for 7 days The hamsteij, then breathed room air until they
were 1 year old, when they weie killed and examined The lungs were fixed via the trachea
at 25 cm of fixative pressure and fixed lung volumes were determined by displacement
Appropriate stereologic methods were used to determine the mean linear intercept and
internal surface area The authors did not report emphysema or evidence of alveolar
destruction, nor was emphysema demonstrated in the published micrographs of exposure-
related lesions The group exposed starting at 3 days old, but not those exposed starting at
21 days old, had longer mean linear intercepts, indicating larger alveoli Although they
indicated that these changes were "compatible" with "early emphysema", they did not
specifically conclude that the hamsters had emphysema This conclusion is appropriate as no
evidence of emphysema of the type seen in human lungs was presented
Kleinerman et al (1985) descnbe the effects following exposure of hamsters to
56,400 jttg/m3 (30 ppm) NO2, 22 h/day for 12 mo The authors did not descnbe the
13-117
-------
methodology used in detail A statistically significant longer mean linear intercept was noted
in the exposed hamsters, indicating larger alveoli However, there was no indication that
destruction was or was not present The authors concluded that "a small definable degree of
emphysema developed" using the 1985 National Institutes of Health workgroup's definition
of emphysema in animal models Evidence of emphysema of the type seen in humans was
not presented.
Stavert et al (1986) exposed rats that had received a single intratracheal instillation of
sterile normal saline to either 65,800 jttg/m (35 ppm) NO2 for 6 h/day or to filtered air for
25 days The rats were held an additional 10 weeks and then were examined The lungs
were fixed by intratracheal formalin at 25 cm water pressure and morphometry was
performed according to standard, acceptable techniques They reported that,
microscopically, the lungs from these two groups appeared identical The mean linear
intercepts of these two groups were nearly identical, indicating similar-sized alveoli They
did not report destruction, nor is it evident in their published micrographs They concluded
that this exposure regimen "does not bring about irreversible changes in the lungs of rats
which are consistent with either centnlobular or panlobular emphysema " This conclusion is
appropriate.
Glasgow et al. (1987) exposed rats to 56,400 jwg/m3 (30 ppm) NO2, 24 h/day for up to
140 days. The primary objective of this study was to evaluate neutrophil recruitment and
degranulation from NO2-induced emphysema The lungs were fixed via the trachea in a
appropriate manner and the mean linear intercept was determined by semiautomatic image
analysis, which was compared to manual methods Exposed rats had significantly longer
mean linear intercepts, indicating larger alveoli and alveolar wall destruction The authors
concluded that the exposed rats had emphysema based on the 1985 National Institutes of
Health's workgroup definition of emphysema in animal models However, it appears that
they either pooled data from several ages of control rats or terminated all of the control rats
at or before the day the exposures were started Thus, controls may have been younger than
the exposed rats In addition, there may have been differences m the methods by which the
lung tissues were processed, exposed and control rats may not have been processed at the
same time For example, in their Figure 1 (the example of alveolar wall destruction), the
alveoli in the control lung do not appear to be as fully distended, as the alveolar walls are
13-118
-------
less straight than those of the lungs from the exposed rat The figure legend does not
indicate the ages of either the control or exposed rat Although these problems may not
influence the investigators' main objectives, they are troublesome with respect to the
presence or absence of emphysema The results are inconclusive with respect to the
production of emphysema of the type seen in human lungs
Blank et al (1978) used NO2 to produce an animal model of emphysema based on the
1985 National Institutes of Health's workgroup definition The objective of this study was to
determine the effect of beta-aminopropionitnle, which inhibits cross-linking of collagen and
3
elastin, on that animal model They exposed rats to 56,400 /*g/m (30 ppm) NO2, 24 h/day
for up to 8 weeks The lungs were fixed by appropriate methods, and the mean linear
intercept was determined by semiautomatic image analysis Control and exposed rats fed the
usual rat chow were terminated at the same time and age They reported longer mean linear
mtercepts in the exposed rats, indicating larger alveoli They mentioned that the lungs were
examined for alveolar wall destruction, but do not clearly indicate whether destruction was
present They properly refer to the NO2-induced lesions as "emphysema-like" rather than
emphysema of the type seen in human lungs
Lafuma et al (1987) studied the effect of exposure to 3,760 pig/m (2 0 ppm) NO2,
8 h/day, 5 days/week for 8 weeks on control hamsters and on hamsters with elastase-induced
emphysema The lungs were fixed via the trachea at a constant pressure Relative alveolar
sizes were estimated using standard stereological methods They found statistically
significantly larger lung volumes and mean linear mtercepts and smaller internal surface
areas, indicating larger alveoli in hamsters exposed to NO2 alone They did not state
whether airspace wall destruction was or was not found There is no evidence of
destruction in the published micrographs of lungs from that group of hamsters There is no
evidence from this publication that NO2 alone produced emphysema of the type seen in
human lungs
The study by Mauderly et al (1989, 1990), discussed in the previous section, is also
relevant to emphysema in that the findings were negative They studied the mean linear
q
intercept and internal surface areas of rats exposed to 17j900 /tg/m (9 5 ppm) NO2,
7 h/day, 5 days/week for 2 years There was no significant effect on either of these
parameters
13-119
-------
Table 13-12 provides an overview of the results of the studies discussed in this section
Summary
The anatomic region most sensitive to NO2, and withui which injury is generally first
noted, is the area that encompasses the terminal conducting airways and adjacent alveolar
ducts and alveoli Within this region, those cells most sensitive to NO2-induced injury are
the ciliated cells of the bronchiolar epithelium and the Type 1 cells of the alveolar
epithelium There is, however, a large degree of interspecies variability in responsiveness to
NO2 exposure, with guinea pigs, hamsters, and monkeys being more severely affected than
the rat when exposed under the same conditions and examined using the same techniques
(Azoulay-Dupuis et al, 1983, Wagner et al, 1965, Funosi et al , 1973) When effective
NO2 concentrations are used and appropriate tissues are examined using sensitive methods,
similar morphological lesions are found in all species In alveoli, necrosis and sloughing of
Type 1 cells, leaving bare basal lamina, is followed by proliferation of Type 2 cells that
replace damaged Type 1 cells In bronchioles, ciliated cells lose some or all of their cilia
and nonciliated bronchiolar cells (Clara cells in rodents) lose their dome-like luminal surface
projections Ciliated cells may become necrotic and sloughed from the basal lamina
Nonciliated bronchiolar cells, progenitor cells for ciliated cells, proliferate and become
3
larger. Such effects have been reported following acute exposure to ^3,760 /-cg/m
(2.0 ppm) NO2, depending on the species tested (Azoulay-Dupuis et al , 1983, Rombout
et al., 1986) As the exposure duration increases, similar effects are noted, but hyperplasia
and hypertrophy of Type 2 cells and nonciliated bronchiolar cells are the predominant
epithelial changes (Sherwin and Richters, 1982, Rombout et al , 1986, Nakajima et al ,1980,
Sherwin et al, 1973, Yamamoto and Takahashi, 1984) By LM, these changes may appear
to regress during an exposure to low concentrations, or soon after the exposure ends (Kubota
\
et al, 1987) Increases in the thickness of the basal lamina and interstitium are slower to
develop and probably slower to resolve (Kubota et al , 1987)
Both exposure concentration and duration are important factors affecting response, with
concentration perhaps playmg a more significant role (Rombout et al , 1986) Age may also
be a factor affecting response, with neonates being more resistant to the morphological
effects of NO2 and responsiveness increasing with age until weaning or shortly after weaning
13-120
-------
TABLE 13-12. EFFECTS OF NITROGEN DIOXIDE ON THE DEVELOPMENT OF EMPHYSEMA3
NO2 Concentration
jtig/m3
188 with 2-h peaks
to 1,880
270 plus 2,050 /tg/m3
NO
1,210 plus 3 10 jug/m3
NO
940
1,504
7,520
1,880
3,760
3,760
9,400
18,800
ppm
0 1 with peaks
to 10
0 14
06
05
08
40
10
20
20
50
100
Exposure
Daily, 6 mo
16 h/day, 68 mo
6, 18, or 24 h/day,
1-12 mo
51-813 days
16 mo, with and
without viral
challenge
Continuous,
112-763 days
8 h/day, 5 days/week,
8 weeks
3 mo
Gender
NS
F
NS
M
NS
M
M
NS
Age
4 weeks
6 mo
NS
4 weeks
NS
NS
2 mo
NS
Species
(Strain)
Mouse
(NS)
Dog
(Beagle)
Mouse
(NS)
Rat
(Sprague-
Dawley)
Monkey
(Squirrel)
Rat
(Sprague-
Dawley)
Hamster
(Golden Syrian)
Monkey
(Squirrel)
Emphysema Reference
+ Portetal (1977)
Hydeetal (1978)
Blair etal (1969)
Haydonetal (1965)
± Ehrlich and Fenters (1973)
- Freeman et al (1968b)
- Lafuma et al (1987)
Ehrhch and Fenters (1973)
-------
TABLE 13-12 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON THE DEVELOPMENT OF EMPHYSEMA3
Atg/m3
9,400
15,040
22,600
28,200
28,200
37,600
28,200
17,900
47,000
56,400
56,400
NO2 Concentration
ppm
50
80
120
150
15
reduced to either 20 reduced to
or 18,800 15 or 10
95
25
30
30
Exposure Gender
6 h/day, 5 days/week, M
up to 18 mo
3-4 mo (presumably NS
24 h/day)
3-5 mo NS
Continuously for NS
35 days, then
intermittently for at least
2 years
Up to 33 mo M
7 h/day, 5 days/week, M
2 years
32-65 days NS
22 h/day, 12 mo NS
Continuous, up to M
140 days
Species
Age (Strain)
NS Dog
(Mongrel)
Rabbit
(NS)
Guinea pig
(English)
Rat
(Sherman)
Hamster
(Golden Syrian)
Mouse
(C57BL/6,
CAFj/JAX, HLA)
6-12 mo Rabbit
(NS)
NS Rat
(NS)
16 weeks Rat
(NS)
4 weeks Rat
(Wistar)
18 weeks Rat
(Fischer 344)
NS Rat
(Sprague-Dawley)
NS Hamster
(NS)
NS Rat
(Sprague-Dawley)
Emphysema Reference
Wagner et al (1965)
+ Haydonetal (1967)
- Stephens et al (1976)
± Portetal (1977)
+ Freeman et al (1972)
- Mauderlyetal (1989,
1990)
- Freeman et al (1964)
- Kleinerman et al (1985)
- Glasgow etal (1987)
-------
TABLE 13-12 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON THE DEVELOPMENT OF EMPHYSEMA3
NO2 Concentration
jag/m3
56,400
56,400-65,800
65,800
75,200
94,000-169,000 for
4 weeks, reduced to
56,400-94,000
i— '
£ 84,600-103,000
to
OJ
ppm Exposure Gender
30 Contmuous, up to M
8 weeks
30-35 23 h/day, 7 days M
35 6 h/day, 25 days M
40 6 or 8 weeks F
50-90, reduced 2 h/day, 5 days/week, NS
to 30-50 12 mo
45-55 21-23 h/day, M
10 weeks
Age
6 weeks
3 days
12 weeks
NS
NS
NS
Species
(Strain)
Rat
(Sprague-Dawley)
Hamster
(NS)
Rat
(Fischer 344)
Mouse
(Swiss)
Hamster
(NS)
Guinea pig
(NS)
Hamster
(Syrian)
Emphysema Reference
Blank et al (1978)
Lametal (1983)
Stavertetal (1986)
Buckley and Loosli (1969)
± Gross etal (1968)
— Kleinerman and Cowdrey
(1968)
"M = Male
F = Female
NS = Not stated
NO = Nitric oxide
+ = Emphysema, — = No emphysema, ± = Equivocal
Emphysema is defined according to the 1985 National Heart, Lung and Blood Institute Workshop criteria for human emphysema Although several of the papers reviewed reported finding emphysema,
some of these studies (especially the early studies) were reported according to previous, different criteria, some reports did not folly describe the methods used, and/or the results obtained were not in
sufficient detail to allow independent confirmation of the presence of emphysema Thus, a " —" (i e , no emphysema) should only be interpreted as lack of proof of emphysema, because it is conceivable
that if the study were repeated with current methods and the current criteria were applied, some results might be positive
-------
(Stephens et al, 1978, Azoulay-Duplis et al , 1983) Responsiveness in mature animals
appears to decline with age until an increase occurs at some point in senescence (Kyono and
Kawai, 1982)
Several groups of investigators who experimentally exposed different species of
laboratory animals to NO2 have reported emphysema of the type seen in human lungs as
defined by the NHLBI workgroup (National Institutes of Health, 1985) In human lungs, the
workgroup defined emphysema as "a condition of the lung characterized by abnormal,
permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by
destruction of their walls, and without obvious fibrosis " "Destruction in emphysema is
defined as nonuniformity in the pattern of respiratory airspace enlargement so that the
orderly appearance of the acinus and its components is disturbed and may be lost " Studies
in this group include those by Haydon et al (1967), Freeman et al (1972), and Hyde et al
(1978) Results of studies by several additional groups of investigators are inconclusive
because, although they demonstrated enlargement of airspaces, they did not document or
report whether or not destruction as defined by the NHLBI workgroup (National Institutes of
Health, 1985) occurred The latter group includes several studies testing specific hypotheses
of the pathogenesis of emphysema, which, as appropriate to their research objectives, used
the NHLBI workgroup's definition of animal models of emphysema rather than the definition
of emphysema in human lungs The definition of emphysema in animal models requires
airspace enlargement, but not destruction Several studies were considered inconclusive due
to problems with control animals or to insufficient numbers of animals It is important to
note that a 2-year exposure of rats to 17,900 jwg/m3 (9 5 ppm) NO2 did not result in evidence
of emphysema, even though Mauderly et al (1987, 1990) used appropriate morphologic and
morphometnc methods
13.2.3 Extrapulmonjiry Effects
Exposure to NO2 produces a wide array of health effects beyond the confines of the
lung. Although the aggregate data are inconclusive and do not portray any single issue as
paramount, the evidence suggests that NO2 and/or some of it's reactive products penetrate
the lung epithelial and endothelial layers to enter the blood and produce alterations in blood
13-124
-------
and various other organs Effects on the systemic immune system are included in
Section 13 22 1 (Host Defense Mechanisms)
13.2.3.1 Body Weight
Traditionally, the measurement of body weight in animal toxicology studies has been
considered a primary and sensitive end point However, its biological significance and
extrapolation to humans are still generally uninterpretable The measurement of body weight
may be useful in examining questions related to differences m species sensitivity, age, and
different exposure scenarios A compilation of the effects of NO2 on body weight can be
found in Table 13-13
The most comprehensive study was performed by Wagner et al (1965), who exposed
rabbits, guinea pigs, rats, hamsters, and four strains of mice to 1,880, 9,400, or
47,000 Atg/m3 (1, 5, or 25 ppm) and dogs to 1,880 or 9,400 /tg/m3 NO2 For all species
examined, no significant differences in body weight were observed after 6, 12, and 18 mo of
exposure Similarly, a study by Steadman et al (1966) indicated that 90 days of continuous
exposure to between 900 and 21,600 ftg/m3 (0 48 and 115 ppm) NO2 resulted in scattered
reductions in body weight gain in five species of animals (dogs, rabbits, squirrel monkeys,
guinea pigs, and rats) evaluated at three intervals (30, 60, and 90 days) The authors
concluded that there was no significant weight loss, however, no statistical analysis of the
o
data was presented Also, increased mortality was observed at the 9,200-jWg/m (4 9-ppm)
concentration in guinea pigs and rabbits, and m all species at 21,600 /
-------
TABLE 13-13. EXTRAPULMONARY EFFECTS OF NITROGEN DIOXIDE: BODY WEIGHT3
to
ON
NOj Concentration
/ig/m
94
320-1,504
470-564
658
900-
21,600
940-3,760
base +
peaks to
3 X base
1,300-
1,500
1,504
1,880
ppm
005
0 17-08
0 25-0 30
035
048-
115
05-20
base +
peaks to
3 X base
07-08
08
10
Exposure Gender
90 days NS
8 h/day, M
5 days/week,
1-12 weeks
8 h/day, M
5 days/week,
3-12 weeks
8 h/day, M
5 days/week,
6 weeks
Continuous, NS
90 days
22 h/day base + M
2 1-h peaks/day,
5 days/week,
1, 3, and 6 weeks
30 days F
Continuous, M
lifetime
8 h/day, M
6 mo
Age
NS
Adult
in utero
inutero
and older
mice
NS
1 day and
7 weeks
8 weeks
4 weeks
NS
Species
(Strain)
Rat
Mouse
(Swiss Webster)
Mouse
(Swiss Webster)
Dog
(Beagle)
Rabbit
(New Zealand)
Guinea pig
(NS)
Rat
(Sprague-Dawley)
Monkey
(Squirrel)
Rat
(Fischer 344)
Mouse
(ICR)
Rat
(Sprague-Dawley)
Guinea pig
(NS)
Effects
No effect
No effect
Reduced body weight gain compared to
controls at 3 and 12 weeks, but not at
6 weeks
Reduced body weight gain in newborns,
but not older rats
No effect
Reduced body weight gain in older rats
after 3 and 6 weeks exposure to 1 0 or
2 0 ppm No effects in younger rats
No effect
No effect
Reduced body weight gain
Reference
Shalamberidze (1969)
Richtersetal (1987)
Kuraitisetal (1981)
Steadmanetal (1966)
Stevens et al (1988)
Nakajimaetal (1969)
Freeman et al (1966)
Kosnuder et al (1973b)
-------
TABLE 13-13 (cont'd). EXTRAPULMONARY EFFECTS OF NITROGEN DIOXIDE: BODY WEIGHT2
NO2 Concentration
/tg/m ppm Exposure Gender Age
1,900- 1 0-25 18 mo M NS
47,000
£ 9,400 5 0
i
i— >
t-}
•^ 2,400- 13-30 2 h/day,
5,680 15-17 weeks
3,760 2 0 Continuous, M 8 weeks
up to 6 weeks
3,760 20 Continuous, M NS
lifetime
18,800 10 Up to 62 days M/F inutero
"M = Male
F = Female
NS = Not stated
Species
(Strain) Effects
Rabbit No effect
(NS)
Guinea pig
(English)
Rat
(Sherman)
Hamster
(Golden Syrian)
Mouse
(C57BL/6,
CAiyJAX,
Webster)
Dog No effect
(Mongrel)
Rabbit Reduced body weight gam.
Rat No effect
(Wistar)
Rat No effect
(Sprague-
Dawley)
Rat Reduced body weight gain and
(NS) length of pups
Reference
Wagner et al (1965)
Mitina (1962)
Azoulay et al (1978)
Freeman et al (1968c)
Freeman et al (1974)
-------
Kuraitis et al (1981) also reported that the body weights of newborn mice exposed to
658 jwg/m (0 35 ppm) NO2 for 6 weeks were significantly less than age-matched controls
No decrease in body weight was observed when older mice were exposed Also, Nakajima
et al (1969) found no effects in 8-week-old mice exposed for 30 days to 1,316 or
1,504 /Kg/m3 (0 7 or 0 8 ppm) NO2
Only very high exposure concentrations of NO2 have been found to cause reduced body
weights in rats No exposure-related effects were found in rats exposed for 90 days to
94 /*g/m3 (0 05 ppm) NO2 (Shalambendze, 1969) Freeman et al (1966, 1968b) found no
effects on body weight after lifetime exposure to 1,504 or 3,760 j^g/rn (0 8 or 2 0 ppm)
NO2
Although the data in mice suggest that newborns may be more sensitive to NO2
exposure than older mice, other evidence (see Section 13 2 2 4), including body weight data,
do not support this contention for rats In contrast to the effects on body weight observed in
newborn mice, Stevens et al (1988) reported that 1-day-old rats were less responsive to NO2
-------
13.2.3.2 Hematologic Changes
Alterations of blood constituents as a result of NO2 exposure may be due to a variety of
causes Direct effects of NO2, formation of nitrites and nitrates, or secondary effects
emanating from other organs such as the lung, liver, heart, kidneys, and spleen could all
result in alterations of blood content and chemistry However, the significance of many of
these hematological changes is uncertain
Effects on Blood Cell Counts and Hemoglobin
Several authors have shown effects on the number of red blood cells (RBCs) and
hemoglobin concentration, although the results have been inconsistent. A summary of these
studies can be found in Table 13-14 In some of these studies, leukocytes (white blood cells
[WBCs]) and platelet counts were also examined (Table 13-15)
Shalambendze (1969) exposed rats continuously to 94 /*g/m3 (0 05 ppm) NO2 for
90 days, causing no change in blood hemoglobin or RBC counts Yakunchuk and
-------
TABLE 13-14. EFFECTS OF NITROGEN DIOXIDE ON RED BLOOD CELLS AND HEMOGLOBIN3
NC>2 Concentration
3
|tg/m PPm
94 005
940-1,500 + 05-08 +
1,500 0 8
1,880 1 0
1,880 1 0
^ 9,400 5 0
nL 1,880-56,400 1-30
o
2,400-5,640 1 3-3 0
3,760 2 0
3,760 2 0
Exposure Gender Age
Continuous, NS NS
90 days
Continuous, M/F 4 weeks
1 to 1 5 mo
Continuous, M 7 weeks
5 days
Continuous, M NS
16 mo
Continuous, M NS
18 mo
18 h NS NS
2 h/day,
15 and 17 weeks
Continuous, M/F NS
14 mo
M
Continuous, M 8 weeks
up to 6 weeks
Species
(Strain)
Rat
Mouse
(ICR JCL)
Mouse
(ICR)
Monkey
(Squirrel)
Dog
(Mongrel)
Mouse
(NS)
Rabbit
Monkey
(Macaca
speciosd)
Rat
(Sprague-
Dawley)
Rat
(Wistar)
Effects
No effect on blood hemoglobin
orRBCs
Addition of 50 ppm CO to N02
failed to affect carboxyhemo-
globin
No effect on methemoglobin
No effect on hematocnt or
hemoglobin with NO2 and
influenza exposure
No changes in hemoglobin or
hematocnt
Concentration-related increase in
methemoglobin and
nitrosylhemoglobrn
Decreased RBCs
With or without NaCl
o
(330 jttg/m ) polycythemia with
reduced mean corpuscular
volume and normal mean
corpuscular hemoglobin
No effect on hemoglobin,
hematocnt or RBC count, no
methemoglobin was observed
Reference
Shalambendze (1969)
Nakajima and Kusumoto (1970)
Nakajnna and Kusumoto (1968)
Fentersetal (1973)
Wagner et al (1965)
Caseetal (1979)
Mitina (1962)
Funosietal (1973)
Azoulayetal (1978)
-------
TABLE 13-14 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON RED BLOOD CELLS AND HEMOGLOBIN2
NO2 Concentration
jtig/m ppm
9,400- 5-40
75,200
18,800 10
Exposure Gender Age
1 h F 4 mo
2 h/day, F 6-8 weeks
5 days/week, up
to 30 weeks
Species
(Strain)
Mouse
(JCL ICR)
Mouse
(BALB/c)
Effects
No increase in methemoglobin
Increased nitrite and especially
nitrate
Small decrease in hemoglobin
and mean corpuscular
hemoglobin concentration
Reference
Odaetal (1981)
Holtetal (1979)
aNS = Not stated
RBCs = Red blood cells
M = Male
F = Female
CO = Carbon monoxide
NaCl = Sodium chloride
-------
TABLE 13-15. EFFECTS OF NITROGEN DIOXIDE ON LEUKOCYTES AND PLATELETS3
NO2 Concentration
o
/tg/m ppm
600 032
1,880 1 0
1,880 1 0
9,400 50
2,400-5,640 1 3-3 0
3,760 2 0
18,800 10
18,800 10
Exposure Gender
Continuous, M
3 mo
Continuous, M
16 mo,
fol!6wed by
viral challenge
Continuous, M
18 mo
2h/day,
15 and 17 weeks
Continuous, M/F
14 mo M
Continuous, M
14 days
2 h/day, F
5 days/week, up
to 30 weeks
Species
Age (Strain)
NS Rat
(NS)
NS Monkey
(Squirrel)
NS Dog
(Mongrel)
Rabbit
NS Monkey
(Macaca
speciosa)
Rat
(Sprague-
Dawley)
18 weeks Rat
(Wistar)
6-8 weeks Mouse
(BALB/c)
Effects
Increased leukocytes after
8-10 weeks, no difference after
3 mo of exposure
Increased leukocyte count in
viral-challenged NC^-exposed
animals
No effect on leukocyte count
Increased leukocytes followed by
decreased phagocytic activity
Neutrophil/lymphocyte ratio
tendency to shift upwards in both
animal species tested
Decreased platelets at 1 to
7 days of exposure, but not after
14 days
Increased leukocytes at 5 weeks,
but not at 15 or 30 weeks
Reference
Yakimchuk and Chehkanov
(1972)
Fentersetal (1973)
Wagner et al (1965)
Mitina (1962)
Funosietal (1973)
Kobayashi et al (1983)
Holtetal (1979)
"M = Male
NS = Not stated
F = Female
-------
and magnesium-activated phosphatase activity (Wagner et al , 1965) Rabbits exposed to
2,400 to 5,680 /*g/m3 (1 3 to 3 0 ppm) NO2 for 15 or 17 weeks had a decrease in the
number of RBCs and a significant increase in the number of WBCs (Mitina, 1962)
Funosi et al (1973) exposed rats to 3,760 ± 1,880 jttg/m3 (2 0 ± 1 0 ppm) NO2 for
14 mo and found polycythemia with reduced mean corpuscular volume, but normal mean
corpuscular hemoglobin concentrations Exposure to NO2 also increased the ratio of PMNs
to lymphocytes Because exposures occurred while the rats were in plastic cages inside the
exposure chamber, the actual concentration of NO2 would probably be less than the stated
concentration However, the reported observations are supported by similar findings in
monkeys that were simultaneously exposed in wire cages
Azoulay et al (1978) reported no effects on rat RBC parameters (hemoglobin,
3
hematocnt, and RBC count) after a continuous NO2 exposure of 3,760 /jg/m (2 0 ppm)
lasting between 1 day and 6 weeks Other factors that index or potentially alter oxygen
affinity to hemoglobin (the partial oxygen pressure at which hemoglobin is half-saturated
with O2, n Hill factor [hemoglobin binding affinity], pH, oxygen combining capacity, and
2,3-diphosphoglycerate [a measure of tissue deoxygenation]) were not affected
Although not a direct measure of RBC content, the number of RBCs in the red pulp of
the spleen of mice was increased by a 6-week, 5-day/week, 8-h/day exposure to 658 jwg/m3
(0 35 ppm) NO2 (Kuraitis et al , 1981) This finding could be interpreted as supporting the
polycythemia that was sometimes observed in NO2-exposed animals Spleen weights and the
size of spleen lymphoid nodules were also increased
Three studies examined the production of physiologically inactive hemoglobin
(methemoglobin) that might be produced if nitrites or nitrates reacted with hemoglobin
Nakajima and Kusumoto (1968) found that the amount of methemoglobin was not increased
•3
when mice were exposed to 1,504 pg/m (0 8 ppm) NOZ for 5 days Methemoglobin was
not detected after a 6-week exposure of rats to 3,760 jitg/m3 (0 2 ppm) (Azoulay et al ,
1978) Oda et al (1981) also found no increase in methemoglobin, but nitrites and
especially nitrates were elevated in the blood of mice exposed for 1 h to between 9,400 and
75,200 ng/m3 (5 and 40 ppm) NO2 In contrast, Case et al (1979) showed that mice
exposed to 1,880 to 56,400 jwg/m NO2 (1 to 30 ppm) exhibited a concentration-related
13-133
-------
increase in methemoglobin and mtrosylhemoglobm and decreased feme catalase and iron
transfernn activities
Effects on Red Blood Cell Membranes
Several studies have examined changes in RBC membranes of rats after NO2 exposure
(see Table 13-16) In a preliminary report, Mersch et al (1973) showed that RBC
D-2,3-diphosphoglycerate was increased in all four guinea pigs continuously exposed to
<3
677 j«g/m (0 36 ppm) NO2 for 1 week However, as previously mentioned, Azoulay et al
*>
(1978) reported no changes in 2,3-diphosphoglycerate levels after a 3,760-jttg/m (2 0-ppm)
continuous NO2 exposure lasting between 1 day and 6 weeks The authors attribute the
difference between their results and the results of Mersch et al (1973) to their use of a more
precise enzymatic assay and to a larger study population Additionally, there may be
species-related differences because Azoulay et al (1978) examined rats and Mersch et al
(1973) examined guinea pigs
Changes in the contents of RBC membranes were detected after exposure to
3
7,520 /tg/m (4 0 ppm) NO2 (Kaya et al, 1980) Increased amounts of sialic acid were
noted in rats exposed between 1 and 10 days to 7,520 /-ig/m3 NO2 Increased sialic acid, a
glycosidic residue distributed on the outer surface of the RBC, is found in younger RBCs
(Durocher et al., 1975), suggesting that NO2 inhalation may have stimulated renewal of the
RBC population (increased population of immature cells) An increased amount of
lyso-phosphatidylethanolamine, known to increase cell membrane fragility, was found on
-3
Days 5, 7, and 10 after exposure to 7,520 /*g/m and after 1,5, and 7 days of exposure to
18,800 /tg/m3 (10 ppm) NO2 The protein content of RBCs was slightly decreased at
o
18,800 /tg/m after 1, 5, and 7 days of exposure, but not after 3 days
The possibility of a younger circulating RBC population was investigated by Kummoto
et al (1984), who showed that after 1 and 4 days of exposure to 7,520 ^g/m3 (4 0 ppm)
NO2, the activity and content of Na+, K+-ATPase, and the amount of siakc acid were
increased in RBC membranes These changes have also been associated with a younger
population of RBCs (Cohen et al, 1976)
In contrast, Mochitate and Miura (1984) found that after 7 days of continuous exposure
o
to 7,520 /ig/m (4 0 ppm) NO2, there was a decreased population of younger RBCs
13-134
-------
TABLE 13-16. EFFECTS OF NITROGEN DIOXIDE ON RED BLOOD CELL MEMBRANES3
NC>2 Concentration.
jttg/m ppm
677 0 36
940 05
3,760 2 0
7,520 4 0
V1 7,520 4 0
H- '
OJ
Ul
7,520 4 0
18,800 10
7,520-37,780 4-20
"NS = Not stated
RBCs = Red blood cells
Exposure Gender
Continuous, NS
7 days
8 h/day, NS
7 days
8 h/day,
4 mo
Continuous, M
up to 6 weeks
Continuous, M
1-10 days
Continuous, M
1-10 days
Continuous, M
10 days
Continuous,
7 days
Continuous, M
1-10 days
M = Male
GSH = Glutathione
Species
Age (Strain)
NS Guinea pig
(NS)
NS Guinea pig
(Hartley)
8 weeks Rat
(Wistar)
16-21 Rat
weeks (JCL Wistar)
16-20 Rat
weeks (JCL Wistar)
8-18 weeks Rat
(JCL Wistar)
8-14 weeks Rat
(JCL Wistar)
Effects
Increased RBC
D-2,3-diphosphoglycerate
Decrease in RBC GSH
peroxidase
No change in RBC GSH
peroxidase
No effect on RBC
2,3-diphosphoglycerate
Increased sialic acid, Na , and
K+-ATPase in RBC membranes
associated with an increased
proportion of younger RBCs
At Day 7, the fraction of young
RBCs was reduced and the
fraction of older RBCs increased
Activities of pyruvate kinase and
phosphofructokinase were
increased in young RBCs
Increased arachidomc acid in
membranes and serum Stearic
palmatic acid decreased at 10 and
4 ppm in RBC membranes
Decreased RBC membrane
protein at Day 1, 5, and 7, but
not 3 Lyso-phosphatidyl-
ethanolamine, sialic acid, and
hexose increased at 4 and
10 ppm
Reference
Merschetal (1973)
Menzeletal (1976)
Azoulay et al (1978)
Kummotoetal (1984a)
Mochitate and Miura (1984)
Kaya and Miura (1982)
Kayaetal (1980)
-------
However, the activity of two glycolytic enzymes (pyruvate kinase and phosphofructokinase)
was elevated in NO2-exposed animals on Days 5 and 7, but returned to control levels on
Day 10 The authors concluded that there was not a corresponding reduction in the activity
of the glycolytic pathway with the NO2-induced increase in the apparent aging of the RBCs
Kaya and Miura (1982) investigated the effects of NO2 on fatty acids in RBCs, sera,
and liver They found a net increase in unsaturated fatty acids (predominately arachidomc
o
acid) occurred in RBC membranes from rats after exposure to 7,520 /*g/m (4 0 ppm) NO2
for 10 days.
Effects on Serum and Plasma
As mentioned in the previous section, Kaya and Miura (1982) showed that arachidomc
acid was increased in the RBC membrane after exposure to NO2 Because de novo synthesis
of fatty acids is not possible in the mature RBC, composition changes in the membrane
usually reflect exchange with serum fatty acids, probably originating in the liver In fact,
Kaya and Miura (1982) found that after exposing rats to 7,520 /*g/m3 (4 0 ppm) NO2 for
10 days, arachidomc acid was elevated in serum and in liver homogenates However,
*}
exposure to 18,800 j^g/m (10 ppm) NO2 for 7 days increased RBC and serum arachidomc
acid, but liver concentrations were decreased The authors suggested that the rat cannot
>a
completely overcome the consequences of a 18,800-/*g/m NO2 exposure, but could
•^
metabohcally compensate for the effects of exposure to 7,520 jwg/m NO2
Menzel et al (1977) demonstrated that acute effects do not necessarily predict chronic
injury by contrasting the serum changes of guinea pigs after short-term (7-day) and long-term
•o
(4 mo) continuous exposure to 940 jttg/m (0 5 ppm) NO2 Plasma ChE was elevated after a
7-day exposure, but was decreased compared to control values with a long-term exposure
(4 mo) This depression in ChE is suggestive of a hepatic lesion (Moore et al, 1957)
A depression in RBC GSH peroxidase activity was also initially observed, but the effect did
not persist after 4 mo of exposure Similarly, several indices of nonspecific tissue damage
(serum creatuie phosphokinase, LDH, serum glutamic oxaloacetic transammase, and serum
glutamic pyruvic transammase) were also increased after 7 days, but were not altered by
long-term exposure
13-136
-------
The following studies all indicate a general decrease in serum proteins and kpoproteins
and an increase in serum globulins, thus suggesting hepatic damage Drozdz et al (1976)
reported decreased serum total protein, albumin, and seromucoid concentrations in guinea
-3
pigs exposed to 2,000 jug/m (1 05 ppm) NO2, 8 h/day for 180 days However, serum
levels of a}- and /3-globulins were increased These authors also found that serum alanine
and aspartate aminotransferase activity was increased in the mitochondnal fraction, but was
decreased in the cytoplasmic fraction In agreement with the Menzel et al (1977) subchromc
data, Drozdz et al (1976) also observed decreased plasma ChE levels However, the
meaning of the cytoplasmic and mitochondnal fraction of serum is not clear from the
translation of the article
Kosmider et al (1973a) reported a general decrease in protein synthesis evidenced by
2
decreased serum proteins in guinea pigs after continuous exposure to 1,880 jug/m (1 0 ppm)
3
NO2 for 6 mo Following exposure to 1,000 /xg/m NOX (mainly NO2, »0 5 ppm), 8 h/day
for 120 days, Kosmider (1975) reported decreased serum cholesterol, total lipids, /3 (low
density kpids) and gamma lipoproteins, and sodium, and increased serum a. (high density
lipids)-kpoproteins in guinea pigs Similarly, Mitina (1962), after exposing rabbits to
2,400 to 5,680 /ig/m (1 3 to 3 0 ppm) NO2 for 15 and 17 weeks, found reduced amounts of
albumin, but increased serum globulins Table 13-17 summarizes the data on NO2-induced
changes in serum proteins and clinical chemistries
13.2.3.3 Hepatic Function
As described in the above section, changes in serum chemistries suggest that NO2
exposure may affect the liver Several studies have examined hepatic function either directly
or by indirect means These studies are cataloged in Table 13-18
One important function of the liver is detoxification of xenobiotic compounds
Measurement of the duration of barbiturate-induced sleeping tune has been used as an
indirect measurement of hepatic mixed-function oxidase activity, the enzymes responsible for
xenobiotic metabolism Nitrogen dioxide has been shown to mcrease pentobarbital-induced
sleeping times in mice (Miller et al , 1980, Graham et a] , 1982) The effect was observed
m female mice, but not in males, occurred only at specified time intervals after exposure,
and usually did not persist beyond 1 day postexposure Bowever, the effects reliably
13-137
-------
TABLE 13-17. EFFECTS OF NITROGEN DIOXIDE ON SERUM PROTEINS AND CLINICAL
STRIES3
oo
N(>2 Concentration
3
/tg/m ppm
600 032
940 05
1,000 NOX 0 5
(mainly
NQj)
1,880 1 0
1,880 1 0
2,000 1 05
1,880- 1-30
56,400
11,700 6 2
Exposure
Continuous,
3 mo
8 h/day,
7 days,
8 h/day,
4 mo
8 h/day,
120 days
Continuous,
6 mo
Continuous,
16 mo
8 h/day,
180 days
18 h
Continuous,
8 days
Gender Age
M NS
NS NS
NS NS
NS NS
M NS
M NS
NS NS
M 8 weeks
Species
(Strain)
Rat
(NS)
Guinea pig
(Hartley)
Guinea pig
(NS)
Guinea pig
(NS)
Monkey
(Squirrel)
Guinea pig
(NS)
Mouse
(NS)
Rat
(Sprague-
Dawley)
Effects
Cholinesterase was not affected
At 4 days serum LDH, total creatimne
phosphokinase, SGOT and SGPT, plasma
cholinesterase, and lysozyme elevated At
4 mo lysozyme and plasma cholinesterase
depressed
Serum cholesterol and total hpids
depressed
Protein synthesis inhibited, total serum
proteins and immunoglobulins decreased
Animals challenged with virus No effect
on clinical biochemical parameters
Plasma changes decreased albumin,
seromucoid, choline-sterase, alanine, and
aspartate transminases Increased al and
^2 globulins
Decreased catalase and iron transfernn
activity
No effect on serum lysozyme
Reference
Yalomchuk and Chelikanov
(1972)
Menzel et
Kosmider
Kosmider
Fenters et
Drozdz et
Case et al
al (1977)
(1975)
etal (1973a)
al (1973)
al (1976)
(1979)
Chowetal (1974)
*M = Male
NS = Not stated
LDH = Lactate dehydrogenase
SGOT = Serum glutamic oxaloacetic transammase
SGPT = Serum glutamic pyruvic transammase
-------
TABLE 13-18. EFFECTS OF NITROGEN DIOXIDE ON THE LIVER3
NC>2 Concentration
/ig/m
50
100
1,000
10,000
470-9,400
235
752
2,256
7,520
752
3,260
7,520
7,520
18,800
1,000 NOX
(mainly NO^
940
1,880
ppm
003
005
05
53
0 25-5 0
0 125
04
12
40
04
1 2
40
40
10
05
)
05
10
Exposure Gender
6 h/day, M/F
7 days/week,
21 days
3 h/day, 1, 2, or M/F
3 days
3 h/day,
1-2 days
Continuous, M
14 weeks
Continuous, M
7 days
10 days
7 days
8 h/day, NS
120 days
Continuous F
17 mo
Age
in utero
5-7 weeks
22-24
weeks
23-26
weeks
21-24
weeks
NS
weanling
Species
(Strain)
Rat
(Wistar)
Mouse
(CD-I)
Rat
(JCL Wistar)
Rat
(Wistar)
Guinea pig
(NS)
Mouse
(NS)
Effects Reference
Increased hexobarbital-rnduced sleeping Tabacova et al (1985)
time at 0 5 and 5 0 ppm Cytochrome
P-450 level and aminopynne
AT-demethylase activity were decreased,
whereas hpid peroxides and 0%
consumption were increased at 5 0 ppm
Increase in pentobarbital-induced Miller et al (1980)
sleeping time in female mice only,
repeated daily exposures caused no
effect
No effect on pentobarbital-induced
sleeping time
Cytochrome P-450 level decreased Takahashi et al (1986)
during first 8 weeks of exposure, but
returned to control levels with continued
exposure (4 0 ppm)
Succinate-cytochrome c reductase Mochitate et al (1984)
activity was reduced at 10 ppm only, on
Days 3 and 5 Cytochrome P-450 level
was reduced at 10, 4 0, and 0 4 ppm but
not at 1 2 ppm NADPH-cytochrome c
reductase activity was reduced at
4 0 ppm only
Depleted liver magnesium and zinc, Kosinuder (1975)
swollen liver mitochondria
No effect on lipofusin pigment in liver Azaz and Csallany (1977, 1978)
or other organs Csallany and Ayaz (1978)
-------
TABLE 13-18 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON THE LIVER*
NC-2 Concentration
2
leg/in ppft^
2,000 1 05
7,520 4 0
9,400 50
Exposure
8h/day,
180 days
Continuous,
1, 14, or
30 days
3h
Species
Gender Age (Strain)
M NS Guinea pig
(NS)
M 4 weeks Rat
(Wistar)
F 6-7 weeks Mouse
(CD-I)
Effects Reference
Decreased liver protein and Drodz et al (1976)
cytoplasmic aspartate
transaminase activity, increased
mitochondnal alamne and
aspartate transaminase activities,
intracellular edema, and
inflammatory and parenchymal
changes observed
Cytochrome P-450 level and Takano and Miyazaki (1984)
armnopynne JV-demethylase
activity increased and aniline
hydroxylase activity was
decreased No effect at Day 1
No decrease in cytochrome Graham et al (1982)
P-450 levels or mixed function
oxidase activities
"M = Male
F = Female
C>2 = Oxygen
NADPH = Reduced mcotmamide aderune dmucleotide phosphate
NS = Not Stated
-------
3
occurred after a 3-h exposure to concentrations as low as 470 /xg/m (0 25 ppm) NO2
3
No significant effects were detected after 1- or 2-day exposure to 235 /ttg/m (0 125 ppm)
In an attempt to examine the mechanism of this response, the level of hepatic cytochrome
P-450 and the activities of aminopyrine Af-demethylase, p-nitroamsole 0-demethylase, and
3
anihne hydroxylase were measured in the livers of mice exposed for 3 h to 9,400 /*g/m
(5 0 ppm) NO2, however, no NO2-related effects were found (Graham et al , 1982)
Increased hexobarbital-induced sleeping tunes have also been reported in the progeny of
maternally exposed rats (Tabacova et al, 1985) This effect was measured in the offspring
exposed to 10,000 jwg/m3 (5 3 ppm) NO2 at 7, 14, and 21 days postexposure Additionally,
lipid peroxides increased and O2 consumption decreased in liver homogenates Cytochrome
P-450 content and aminopynne-A^-demethylase activity were decreased on Postnatal Day 30
'J
In the animals exposed to 1,000 jwg/m (0 5 ppm) NO2, increased hexobarbital-induced
sleeping tunes occurred on Days 7 and 21, but not on Day 14 Liver lipid peroxides were
also increased on Postnatal Day 30 in this exposure group No exposure monitoring method
was cited and the details of the biological methods used were not available
Components of the rat microsomal electron-transport system, especially cytochrome
-------
Drozdz et al (1976) found decreased total liver protein and siahc acid, but increased
protein-bound hexoses in guinea pigs exposed to 2,000 jug/m3 (1 05 ppm) NO2, 8 h/day for
180 days Liver alanine and aspartate aminotransferase activity was increased in the
mitochondria! fraction In contrast to the effect seen in the cytoplasmic fraction of the
serum, aspartate aminotransferase activity was decreased in the cytoplasmic fraction of the
liver Electron micrographs of the liver showed intracellular edema and inflammatory and
parenchyma! degenerative changes
Kosmider (1975) reported liver magnesium and zinc stores were depleted in guinea pigs
following exposure to 1,000 j^g/m3 NOX (mainly NO2, »0 5 ppm), 8 h/day for 120 days
Swollen liver mitochondria were also observed
Ayaz and Csallany (1978) and Csallany and Ayaz (1978) exposed weanling mice to
3
940 or 1,880 /*g/m (0 5 or 1 0 ppm) NO2 continuously for 17 mo Animals were divided
into three groups receiving the basal diet with either a normal supplement of vitamin E
(30 mg/kg), 300 mg/kg vitamin E, or 30 mg/kg of the synthetic antioxidant A^A^-diphenyl-
phenylenediamine After 17 mo of exposure, the presence of kpofuscin pigment in the liver,
lungs, spleen, heart, brain, kidney, and uterus was determined No effect could be ascribed
to NC>2 exposure
13.2.3.4 Effects on the Kidney and on Urine Content
The direct effects of NO2 exposure on the kidney and spleen have been described, and
several studies have explored the composition of urine during and after exposure These
studies are summarized in Table 13-19 and are discussed below
Takahashi et al (1986) found that continuous exposure to 2,256 and 7,520 )tig/m3
(1 2 and 4 0 ppm) NO2 increased the amount of cytochrome P-450 and cytochrome b5 in the
kidney after 8 weeks of exposure Continued exposure for 12 weeks resulted in less
substantial increases in the amount and activity of the microsomal electron-transport
enzymes This is in contrast to the decreased activity these authors reported for the liver, as
discussed in Section 13 2 3 3
Yakimchuk and Chelikanov (1972) reported that during a 3-mo continuous exposure to
•a
600 /jg/m (0 32 ppm) NO2, rats showed a significant increase in the urinary excretion of
13-142
-------
TABLE 13-19. EFFECTS OF NITROGEN DIOXIDE ON THE KIDNEY AND ON URINE CONTENTS2
NO2 Concentration
i— '
i
i— '
•£»
jUg/m
600
940
752
1,000
2,260
7,520
2,260-
16,500
aM = Male
NS = Not stated
ppm
032
05
04
«05
12
40
12-
88
Exposure
Contmuous,
3 mo
Continuous,
7 or 14 days
8 h/day,
120 days
Continuous,
3 mo
24 h
Gender Age
M NS
NS NS
NS NS
M 22-24
weeks
M 6 weeks
Species
(Strain)
Rat
(NS)
Guinea pig
(NS)
Guinea pig
(NS)
Rat
(JCL Wistar)
Rat
(Sprague-
Dawley)
Effects
Increased urinary
coproporphyrins
Increased urinary protein and
specific gravity Proteins
characteristic of the nephrotic
syndrome
Increased urinary nitrite, nitrate,
and coproporphynn
Increased cytochrome P-450 and
b5 after 8 weeks of exposure,
less of an effect after 12 weeks
Increased urinary nitrate,
urinary nitrite increase appeared
to be artifactual
Reference
Yakimchuk and Chelikanov
(1972)
Sherwin and Layfield (1974)
Kosmider (1975)
Takahashi et al (1986)
Saul and Archer (1983)
-------
coproporphynns Kosmider (1975) also reported increased levels of urinary coproporphynn
in guinea pigs exposed to 1,000 /*g/m3 NOX (mainly NO2, «0 5 ppm), 8 h/day for
120 days Increased coproporphynns can indicate increased heme synthesis, which might
occur if an increased number of RBCs were synthesized As discussed in Section 13 2 3 2,
NO2 exposure has been reported to cause polycythemia and an increase in the number of
RBCs m the red pulp of the spleen
Increases in urinary protein and specific gravity of the unne were reported by Sherwin
<>
and Layfield (1974) in guinea pigs exposed continuously to 940 jwg/m (0 5 ppm) NO2 for
14 days Proteinuna was detected in another group of animals when the exposure was
reduced to 752 jtg/m3 (0 4 ppm) NO2 for 4 h/day Disc electrophoresis of the urinary
proteins demonstrated the presence of albumin and alpha, beta, and gamma globulins The
presence of high molecular weight proteins in unne is characteristic of the nephrotic
syndrome. Differences in water consumption or in the histology of the kidney were not
found
In a more comprehensive study of the relationship between inhaled NO2 and urinary
nitrite and nitrate, Saul and Archer (1983) exposed rats for 24 h to 2,256 to 16,544 /*g/m3
(1 2 to 8 8 ppm) NO2 They demonstrated that mostly nitrate, with very little nitrite, was
excreted in the unne The small amount of urinary nitrite appeared to be an artifact that
onguiated from an in vitro reaction with unne The rate and linearity of the conversion of
NO2 to urinary nitrate suggested that NO2 does not form nitrate by reacting with respiratory
water, but reacts with oxidrzable tissue to form nitrite Nitnte is then further oxidized in the
blood by oxyhemoglobin (Kosaka et al, 1979) to form nitrate, which is excreted in the
•3
urine. Nitrite and nitrate were also found in the unne of guinea pigs exposed to 1,000 /xg/m
NOX (mainly NO2, «0 5 ppm), 8 h/day for 120 days (Kosmider, 1975)
13.2.3.5 Cardiovascular Effects
Few papers have reported the effects of NO2 exposure on the heart Potential changes
in hemoglobin and RBCs as well as lung edema could reduce oxygen uptake and affect
cardiovascular performance Because many of the NO2-induced cardiovascular effects are
secondary to pulmonary edema or stimulation of sensory receptors in the respiratory tract,
13-144
-------
some of the studies addressing effects on the cardiovascular system are addressed in the
discussion on pulmonary function (see Section 13 2 2 3)
Suzuki et al (1981) exposed rats for up to 3 mo to between 752 and 7,520
(0 4 and 4 0 ppm) NO2 After 3 mo of exposure to 7,520 /itg/m NO2, anesthetized rats,
artificially ventilated at high frequencies, had a significant reduction in PaO2 A reduction in
heart rate was reported in unanestheteed mice exposed to 2,250 or 7,520 jwg/m3 (1 2 or
4 0 ppm) NO2 for 1 mo (Suzuki et al , 1984)
Messiha et al (1983) examined rats, which previously had been maintained on 10%
ethanol or drinking water as the sole drinking fluid, after 3 days of exposure to 9,400 /-cg/m3
(5 0 ppm) NO2 Heart LDH was significantly elevated in NO2-exposed rats maintained on
water, but not in NO2-exposed rats maintained on etharol Because no changes in LDH
were found in liver or serum, the authors suggested that NO2 may be responsible for the
induction of LDH, however, induction by lactate could not be excluded
Tsubone and Suzuki (1984) examined the effects of NO2 exposure on phenyl diguamde-
-3
induced cardiopulmonary changes Rats were preexposed to 18,800 jttg/m (10 ppm) NO2
for 24 h, 7,520 /ig/m (4 0 ppm) for 1 week, or 752 pg/m (0 4 ppm) for 4 weeks pnor to
phenyl diguamde injection The cardiopulmonary effects of phenyl diguamde (decreased
o
heart rate and respiratory rate) were enhanced by exposure to 18,800 |ug/m NO2, but not by
lower NO2 exposures
Dowell et al (1971) showed decreased cardiac output, blood pressure, PaO2, and pH in
dogs after a 1-h exposure to between 13,160 and 30,080 /*g/m3 (7 and 16 ppm) NO2 This
is in contrast to the findings in an other experimental animal species exposed to higher
concentrations of NO2 (Abraham et al , 1980) However, exposures in the Dowell et al
(1971) study were delivered via an endotracheal tube in anesthetized dogs, thereby bypassing
any scrubbing effects of the upper airways
13.2.3.6 Effects on the Central Nervous System and Behavioral Effects
Information regarding the effects of NO2 on development and animal behavior is
limited to a few studies (see Table 13-20), most of which have uncertain relationships to
humans Shalambendze (1969) exposed rats to 100 /tg/m3 (0 05 ppm) NO2 for 3 mo with no
demonstrated effects on the central nervous system Yakimchuk and Chelikanov (1972)
13-145
-------
TABLE 13-20. EFFECTS OF NITROGEN DIOXIDE ON THE CENTRAL NERVOUS SYSTEM AND BEHAVIOR3
NO2 Concentration
o
jig/m
50-10,000
100
600
845
1,000
1,880
6,580
37,600
6,580
9,400-75,200
ppm
0 03-5 3
005
032
045
053
10
50
20
35
5-40
Exposure
6 h/day,
7 days/week
Continuous,
3 mo
Continuous,
3 mo
7 h/day,
4 weeks
8h/day,
180 days
20 nun/day,
up to 6 mo
6 h/day,
8 weeks
24 h
Species
Gender Age (Strain)
M/F in utero Rat
(Wistar)
NS NS Rat
M NS Rat
(NS)
M NS Mouse
(Swiss Webster)
M NS Guinea pig
(NS)
F 12 weeks Rat
(Wistar)
M 15-16 Mouse
weeks (JCL ICR)
Effects
Significant defects in posture and gait were
detected at 9 and 14 days at 0 05 ppm,
additional effects at higher levels, no
monitoring method was described
No effect on CNS
Decreased conditioned reflexes to sound and
light
Increased 5-HT, 5-HIAA, and turnover
Decreased malate, sorbitol, LDH, alamne
aminotransferase, ATPase, and
5'-nucleotidase homogenate, increased
1,6-diphosphofmctose aldolase, isocitrate,
a-hydroxybutyrate dehydrogenase,
phosphocreatine kinase, and chohnesterase
More or less constant swimming
performance in only 1 0-ppm group, with a
slight tendency to detenoration, decrease of
25% by fifth and sixth week of exposure to
5 0 ppm, declined from first month at
20 ppm
Decreased swimming performance at
3 5 ppm
Decreased swimming performance at
10 ppm, increased blood lactate compared to
similarly exercised controls at 5 0 ppm
Reference
Tabacovaetal (1985)
Shalambendze (1969)
Yakimchuk and
Chehkanov (1972)
Sherwinetal (1986)
Drozdzetal (1975)
Tusletal (1973)
Suzuki etal (1982a)
-------
TABLE 13-20 (cont'd). EFFECTS OF NITROGEN DIOXIDE ON THE CENTRAL NERVOUS SYSTEM
AND BEHAVIOR3
NO2 Concentration
jug/m
14,000
9,400
18,800
9,400
18,800
ppm
77
50
10
50
10
Exposure Gender Age
6 h NS NS
2 h/day, M NS
5 weeks
2 h/day, M NS
5 weeks
Species
(Strain)
Mouse
Guinea pig
(NS)
Guinea pig
(NS)
Effects
Decreased voluntary running
activity, return to normal within
24 h postexposure
Depleted total hpids,
phosphohpids, and cholesterol in
all brain regions, except
increased cholesterol in spinal
cord, increased lipid
peroxidation in all brain regions
Decreased total and protein
bound sulfhydryls, increased
nonprotein bound sulfhydryls
Reference
Murphy et al (1964)
Farahani and Hasan (1990)
Faraham and Hasan (1991)
M = Male
F = Female
NS = Not stated
CNS = Central nervous system
5-HT = 5-hydroxyfryptamiiie
5-HIAA = 5-hydroxymdole acetic acid
LDH = Lactate dehydrogenase
-------
'a
reported that during a 3-mo continuous exposure to 600 jug/m (0 32 ppm) NO2, rats
developed an increased latency of response to conditioned sound and light stimuli
Exposure of guinea pigs to 1,000 jwg/m3 (0 53 ppm) NO2, 8 h/day for 180 days
affected brain enzyme activity levels (Drozdz et al, 1975) Decreased activities in brain
protein metabolism enzymes were seen in brain malate dehydrogenase, alanine
aminotransferase, sorbitol dehydrogenase, LDH, ATPase, 5'-nucleotidase, and asparagine
aminotransferase Increases ui braui glycolytic enzyme activities were seen in
1,6-diphosphofructose aldolase, isocitrate dehydrogenase, alpha-hydroxybutyrate
dehydrogenase, phosphocreatuie kmase, and ChE
A study by Sherwin et al (1986) indicated that the brain content of serotonin (5-HT)
and 5-hydroxyindoleacetic acid (5-HIAA, the primary metabolite of 5-HT) increased in mice
exposed to 846 /*g/m3 (0 45 ppm) NO2, 7 h/day for 4 weeks The ratio of 5-fflAA 5-HT
was also increased The authors did not speculate as to what these observations mean,
however, they noted that increased turnover, as reflected in the increased 5-HIAA 5-HT
ratio, have also been observed in trtmethyltin and chlordecone exposure
Vyskocil et al (1985) measured a variety of hormone levels and organ weights after
-a
continuous exposure to 6,580 /ig/m (3 5 ppm) NO2 for 1 or 2 mo The only significant
effect reported was a decrease in the hypothalamic concentration of noradrenaline at both
exposure durations
Two recent papers by the same group of authors (Farahani and Hasan, 1990, 1991)
reported neurotoxic changes in guinea pigs exposed 2 h/day for 35 days to 9,400 or
o
18,800 ^g/m (5 or 10 ppm) NO2 Although the report contains insufficient information to
adequately evaluate the exposure and t-tests were used for all comparisons, the effects were
substantial, as well as brain region- and concentration-dependent In the first study
(Farahani and Hasan, 1990), total lipids, cholesterol, and phospholipids were found to be
\
decreased in a concentration-dependent manner by 49 to 41 1 % in three brain regions
cerebral hemisphere, cerebellum, and midbram Similar decrements in lipids were observed
in the spinal cord, except that cholesterol was significantly increased Lipid peroxidation, as
measured by malonaldehyde formation, was increased in all four brain regions from 7 5 to
46 5 %, again in a region-dependent and concentration-dependent manner In the second
report (Farahani and Hasan, 1991), using the same exposure regimen, total nonprotein bound
13-148
-------
(mostly GSH) and protein bound sulfhydryl groups in the same four brain regions were
affected by NO2 exposure in a concentration-dependent manner Nonprotein bound
-3
sulfhydryls significantly increased after 9,440 /tg/m (5 0 ppm) exposure, whereas protein
bound sulfhydryls decreased in the cerebellum and especially in the midbrain The increased
nonprotein sulfhydryls may be a protective, compensatory response to the kpid peroxidation
described above, further corroborating that finding, however, the degree of protection from
neurotoxic injury was not evaluated
As discussed in the section on reproductive, developmental, and heritable mutagemc
effects (Section 13 2 3 7), Tabacova et al (1985) reported significant postnatal deficits in the
onset of normal neuromotor development and reduced open field activity in the progeny of
o
maternally exposed rats 2 mo after the dams were exposed to 1,000 or 10,000 jitg/m (0 5 or
5 3 ppm) NO2, 6 h/day for 7 days/week Postural and gait defects were also reported at
100 j«g/m3 (0 05 ppm) No NO2 monitoring method was specified
Tusl et al (1973) exposed rats to 9,400 jttg/m3 (5 0 ppm) NO2 for 8 weeks The
influence of NO2 on forced swimming endurance time was measured By the fifth and sixth
weeks of exposure, swimming performance had decreased 25% In rats exposed to
a
1,880 jttg/m (1 0 ppm) NO2, performance was maintained with a slight tendency toward
deterioration
A concentration-dependent decrease in forced swimming endurance time after a single
24-h exposure to between 9,400 and 75,200 /*g/m3 (5 and 40 ppm) NO2 was reported by
Suzuki et al (1982a) Significant decrements in performance were reported at exposure
2
concentrations as low as 18,000 j^g/m (10 ppm) Recovery from exposure required 5 to
6 days, 7 to 8 days, and over 9 days for the 9,400, 18,800, and 37,600 /*g/m3 (5, 10, and
20 ppm) groups, respectively In an attempt to examine the mechanism that produced the
decrement in performance, it was observed that as forced swimming endurance time
decreased, lung edema increased Furthermore, compared to similarly exercised control rats,
-q
blood lactate concentration was increased in rats exposed to 9,400 jttg/m both immediately
and 24 h after exposure These two findings suggest that lung edema prevented sufficient
O2 from entering the blood during exercise to meet aerobic demands See Section 13 2 2 3
on pulmonary function
13-149
-------
13.2.3.7 Reproductive, Developmental, and Heritable Mutagenic Effects
As summarized in Table 13-21, few studies have examined the effects of NO2 on
reproduction and development or the heritable mutagemc potential of NO2 in vivo Exposure
to 1,880 jig/m3 (1 0 ppm) NO2, 7 h/day, 5 days/week for 21 days resulted in no alterations
in spermatogenesis, germinal cells, or interstitial cells of the testes in six rats (Kripke and
Sherwin, 1984) Additionally, the level of vitamin B12, a coenzyme in Mate metabolism
that is used for DNA synthesis, was not affected by NO2 exposure Similarly, breeding
studies by Shalambendze and Tsereteh (1971) found that long-term NO2 exposure had no
effect on fertility However, there was a decrease in litter size and neonatal weight when
-3
male and female rats exposed to 2,360 jttg/m (1 3 ppm) NO2, 12 h/day for 3 mo were bred
In utero death due to NO2 exposure resulted in smaller litter sizes, but no direct teratogemc
effects were observed in the offspring In fact, after several weeks, NO2-exposed litters
approached weights similar to controls
In the only study that has examined postnatal development, a significant delay in eye
opening and incisor eruption was observed in the progeny of maternally exposed Wistar rats
(Tabacova et al , 1985) The dams were exposed to 50, 100, 1,000, or 10,000 /-tg/m3 (0 03,
0.05, 0 5, or 5 3 ppm) NO2 for 6 h/day, 7 days/week throughout gestation and the offspring
were studied for 2 mo postexposure Significant effects were detected in the offspring of
dams exposed to 1,000 and 10,000 jttg/m3 NO2 There were also concentration-related
increases in neurobehavioral development reported in the offspring of the maternally exposed
animals These findings are discussed in the section on NO2 effects on the central nervous
system and behavior (Section 13236) The method of monitoring NO2 was not reported
Balabaeva and Tabakova (1985) exposed pregnant and nonpregnant albino rats to
1,000 or 10,000 /*g/m3 (0 5 or 5 3 ppm) NO2, 5 h/day for 21 days and examined lipid
peroxidation in lung, liver, and placenta Nonpregnant rats had greater lipid peroxidation in
the liver than in the lung, whereas the opposite was true in pregnant rats Even more
surprising was a fourfold increase in lipid peroxidation in the placenta of 10,000-)wg/m
NO2-exposed rats compared to unexposed pregnant controls The authors then examined the
offspring of the pregnant rats The 1-mo-old Fj nonpregnant rats, exposed to air or the same
concentrations of NO2, showed similar changes as were observed in their mothers
13-150
-------
TABLE 13-21. EFFECTS OF NITROGEN DIOXIDE ON REPRODUCTION, DEVELOPMENT,
AND HERITABLE MUTAGENESIS3
NC<2 Concentration
j«g/m
50
100
1,000
10,000
188
1,880
9,400 '
18,800
1,880
2360
18,800
ppm
003
005
05
53
0 1
10
50
10
10
13
10
Exposure Gender Age
6 h/day, M/F in utero
7 days/week,
21 days
6h M NS
7 h/day, M NS
5 days/week,
21 days
12 h/day, F NS
3 mo
Continuous, M/F in utero
from pregnancy
to 3 mo after
delivery
Species
(Strain)
Rat
(Wistar)
Mouse
(C3H)
Rat
(LEW/fmai)
Rat
(NS)
Rat
(NS)
Effects
Concentration-dependent delay
in eye opening and incisor
eruption in progeny of dams
exposed to 0 5 or 5 3 ppm
during gestation Monitoring
method not described
No increase in chromatid- or
chromosome-type alterations in
leukocytes or primary
spermatocytes immediately and
1 to 2 weeks postexposure No
mutagemc effects
No alterations in
spermatogenesis, germina, or
interstitial testicular cells, no
effect on vitamin B12
No effect on fertility, but litter
size and weight were decreased
No teratogemc effects
Decreased litter size and
mortality of neonates up to
15 days postdehvery No
teratogemc effects noted
Reference
Tabacovaetal (1985)
Goochetal (1977)
Knpke and Sherwin (1984)
Shalambendze and Tsereteh
(1971)
Freeman et al (1974b)
"M = Male
F = Female
NS = Not stated
-------
However, pregnant F^ rats, exposed to 1,000 or 10,000 /*g/m3, had a 9- or 17-fold increase
in placental lipid peroxides, respectively The authors report that increased placental
formation of toxic lipid peroxides in the Fx rats could be due to decreased blood GSH
(no measurements presented) and that such levels of lipid peroxides could be fetotoxic
However, the methods of monitoring NO2 and lipid peroxides were not reported, nor were
the statistical methods
Potential mutagenic effects were investigated by Gooch et al (1977), who reported that
exposure to 188, 1,880, 9,400, and 18,800 /*g/m3 (0 1, 1 0, 5 0, and 10 ppm) NO2 for
6 h did not increase either chromatid or chromosome aberrations in the leukocytes of mice
Blood samples were obtained immediately after exposure and 1 and 2 weeks postexposure
Similarly, no increase in the number of translocations in primary spermatocytes was detected
Therefore, the authors concluded that NO2 exposure did not induce mutagenesis in these
experiments
13.2.3.8 Potential Carcinogenic or Cocarcinogenic Effects
No direct evidence indicates that tumors may be produced by NO2 exposure alone
Several studies have evaluated the issue of carcinogenesis and cocarcinogenesis, but results
are often unclear or conflicting Insofar as we are aware, there are no published reports on
studies using classical carcinogenesis whole-animal bioassays An excellent critical review
and discussion of some of the important theoretical issues in interpreting these types of
studies was written by Witschi (1988) Table 13-22 summarizes information on the
carcinogenic or cocarcinogenic potential of NO2
Studies of Hyperplasia and Enhanced Retrovirus Expression
Hyperplasia of the lung epithelium, although a common response to lung injury, could
•s
be construed as suggesting a potential carcinogenic or cocarcinogenic effect of NO2
However, the relatively frequent reports of hyperplasia, as discussed in Section 13 2 2 4 on
morphological effects, did not include the observation of any tumors It should be noted that
these studies were not designed to detect tumors, so it is not surprising that none were found
Nakajuna et al (1972) found hyperplastic foci due to proliferation of epithelial cells of the
terminal bronchioles and alveoli in mice exposed to 940 to 1,504 jug/m3 (0 5 to 0 8 ppm)
13-152
-------
TABLE 13-22. EFFECTS OF NITROGEN DIOXIDE ON CARCINOGENESIS OR COCARCINOGENESIS2
NO2 Concentration
/ig/m
75
752
7,520
188
1,880
18,800
470
658
752
1,504
940-1,504
1,504
1,800
9,400
18,800
2,000
3,000
ppm
004
04
40
0 1
10
10
025
035
04
08
05-08
08
10
50
10
1 1
16
Exposure Gender Age
Continuous,
17 mo
0 5-4 h M 7 weeks
7 h/day, F 5 weeks
5 days/week, up
to 181 days
7 h/day, M 5 weeks
5 days/week,
6 or 12 weeks
8 h/day, M NS
5 days/week,
10-12 weeks
Continuous, F 4 weeks
30 days
8 h/day, M/F 6 and 10
5 days/week, weeks
18 weeks
6 h/day,
5 days/week,
6 weeks
Continuous, M/F NS
lifetime
Species
(Strain)
Rat
Mouse
(ICR)
mice
(AKR/cum)
Mouse
(C57BL/6J)
Mouse
(Swiss Webster,
C57BL/6J)
Mouse
(ICR JCL)
Mouse
(Swiss Webster,
AKR)
Mouse
(AJ)
Rat
(NS)
Effects
Nonsignificant increase in
BHPN-induced tumors with
exposure to 4 0 ppm
Mice exposed to DMA had
whole-body concentration-related
increase in DMN
Fewer spontaneous lymphomas
and increased survival time
Significant increase in lung
tumors in mice injected with
melanoma cells at 6 weeks
Increased lung tumors in mice
injected with melanoma cells
after NO2 exposure
Hyperplastic foci identical to
those observed after exposure to
known carcinogens
Enhanced retroviras expression
in two strains of mice
No effect at 1 0 or 5 0 ppm At
10 ppm, spontaneous adenomas
in strain A/J mice increased only
when compared to pooled
control group
DMA plus NO2 did not produce
tumors Nitroso-DMA, DMA,
and NO2 produced excess
tumors
Reference
Ichinoseetal (1991)
Iqbaletal (1981)
Richters and Damji (1990)
Richters and Richters (1989)
Richters and Kuraitis (1981)
Nakajimaetal (1972)
Roy-Burman et al (1982)
Adkinsetal (1986)
Benemanskii et al (1981)
-------
TABLE 13-22. EFFECTS OF NITROGEN DIOXIDE ON CARCINOGENESIS OR COCARCINOGENESIS8
NC>2 Concentration
Mg/m
9,400
9,400-
18,800
18,800
28,200-
94,000
31,000-
38,500
ppm Exposure Gender Age
5 0 Continuous, NS 5 weeks
up to 11 weeks
5-10 2h/day, NS NS
5 days/week,
50 weeks
10 2h/day, NS 4 weeks
5 days/week,
50 weeks
15- 1-4 h M NS
50
165-205 5-6h/day, M NS
4 days, plus 3 h
on fifth day
Species
(Strain)
Rat
(NS)
Mouse
(NS)
Mouse
(NS)
Mouse
(ICR)
Mouse
(CD-I)
Effects
Hyperplastic foci at 3 weeks
Decreased ciliated cells
Extensive hyperplasia, cuboidal
metaplasia by 5 weeks
Decreased bronchiolar lumen
and polymorphous epithelium by
7 weeks Increased ciliated cells
and decreased epithelial layers at
9 weeks By 11 weeks, return
to one-layer epithelium
4-Nitroquinoline-l -oxide during
NO2 exposure had no effect on
tumor production
Mice given 4-nitroquinoline-l-
oxide and NO2 NO2 decreased
incidence of lung tumors
Mice gavaged with morpholrne
had concentration-dependent
increase in whole-body content
ofNMOR
In vivo production of NMOR
when 1 g/kg of morpholrne was
administered each day prior to
exposure
Reference
Rejthar and Rejthar (1975)
Ide and Otsu (1973)
Otsu and Ide (1975)
Iqbaletal (1980)
VanSteeetal (1983)
aBHPN = JV-bis(2-hydroxypropyl) mtrosamine
M = Male
DMA = Dimethylamine
DMN = Dimethylmtrosamme
F = Female
NS = Not stated
NMOR = Af-nitrosomorphohne
-------
NO2 for 30 days The authors reported that these lesions were completely identical to early
changes that appeared in the development of pulmonary adenomas after administration of
known carcinogenic chemicals such as isomazid, urethane, and 4-nitroquinoline-l-oxide
(4NQO) However, no adenomas were detected
Rejthar and Rejthar (1975) exposed rats to 9,400 jwg/m3 (5 0 ppm) NO2 continuously
for periods of 3, 5, 7, 9, or 11 weeks After 3 weeks of exposure, the bronchioles had
a uniform cuboidal one-layer epithelium composed of nonciliated cells The cells showed
vacuolization, and hyperplastic foci appeared in the bronchiolar epithelium The foci were
two- to four-layer pyramidal formations By 5 weeks, extensive hyperplasia composed of
three to four layers of epithelial cells was apparent Centers of cuboidal metaplasia were
found in adjacent alveoli By 7 weeks, hyperplasia was apparent in all bronchioles, thereby
narrowing the bronchiolar lumen The polymorphous epithelium was extensive with a few
ciliated cells in hyperplastic areas After 9 weeks, the terminal bronchiolar epithelium
generally showed two or three irregular layers The number of ciliated cells increased, but
cilia were often located atypically in intercellular spaces A return to a single layer of
epithelium without cilia was observed after 11 weeks Seven weeks after exposure to NO2,
the lungs appeared to be in a slate of repair, moving towards reversal of the lesions
The possibility that NO2 may facilitate the production of tumors has been suggested and
examined by several authors Endogenous retrovirus expression was enhanced in the spleen
of low-expressor Swiss Webster mice after exposure to 1,500 jttg/m3 (0 8 ppm) NO2,
8 h/day, 5 days/week for 1 or 18 weeks (Roy-Burman et al , 1982) However,
measurements taken at intermediate time points were not different from controls High-
expressor AKR mice also showed an increase in the concentration of virus-specific RNA in
the spleen after 8, 12, or 15 weeks of exposure to 564 /,ig/m (0 3 ppm) NO2 The authors
suggest that such data may indicate inappropriate or inoidinate expression of genes that could
potentially influence genetically controlled diseases, such as cancer
Studies with Nitrogen Dioxide Plus Known Carcinogens
Ide and Otsu (1973) studied tumor production in conventional mice receiving five
weekly injections of 0 25 mg 4NQO (a lung-mmor-specific carcinogen) when the animals
2
were exposed from birth to a NO2 concentration between 9,400 and 18,800 jwg/m (5 and
13-155
-------
10 ppm), 2 h/day, 5 days/week for 50 weeks There was no difference in the number of
tumors produced by 4NQO alone (6 of 10) and the number produced in combination with
NO2 (6 of 13) Mice exposed to NO2 alone had a similar number of tumors as the air
controls Thus, NO2 did not facilitate the production of tumors
One of the goals of a study by Benemansky et al (1981) was to evaluate the potential
of NO2 to influence the production of tumors during coexposure to a known carcinogen,
nitrosodimethylamine (NDMA) or its precursor, dimethylamine (DMA) No excess tumors
3
were observed in rats during a continuous lifetime exposure to the combination 0 07 mg/m
DMA + 2,000 jig/m3 (1 1 ppm) NO2 This suggested that NO2 did not convert DMA to
NDMA, which alone was shown to produce tumors However, when the rats were exposed
3
to NDMA at a concentration that alone did not produce tumors (0 06 mg/m ), an excess of
tumors, especially in males, was observed when DMA (0 05 mg/m ) plus NO2
(3,000 jttg/m3, 1 6 ppm) was added to the exposure Appropriate statistical techniques and
control groups were not incorporated into the design, and the methods of exposure and
monitoring of NO2 were not reported, making the study difficult to evaluate
In a similarly designed study, Ichinose et al (1991) evaluated rats injected with
W-bis(2-hydroxypropyi) nitrosamine (BHPN) and continuously exposed to 75, 752, or
7,520 /*g/m3 (0 04, 0 4, or 4 0 ppm) NO2 for 17 mo Although their data indicated five
times as many lung adenomas or adenocarcinomas in the rats injected with BHPN and
*2
exposed to 7,520 ng/m (4 0 ppm) NO2, the results failed to achieve statistical significance
using a Chi-square test Nitrogen dioxide exposure alone caused no significant increase in
tumors
Facilitation ofMetastases
Richters and Kuraitis (1981) performed two experiments in which mice were exposed to
either 752 or 1,500 /*g/m3 (0 4 or 0 8 ppm) NO2, 8 h/day, 5 days/week for 10 or 12 weeks,
respectively After exposures were terminated, the mice were injected mtravenously with a
cultured-derived melanoma cell line (B16) The first experiment suggested that there was an
increased tumor yield if tumors were counted at 21 days postinjection, however, they did not
observe a significant interaction or main tune effect in the analysis of variance For the
second experiment, they chose a 3-week tune penod to count tumors The results indicated
13-156
-------
an increased number of tumors in the NO2 group compared to filtered chamber and room air
control groups The authors concluded that NO2 might facilitate the metastases of tumors,
again, these conclusions were based on inappropriate statistics In more recent experiments,
consistent effects have not been observed For example, tumor facilitation was observed
when mice were exposed to 564 or 752 /*g/m (0 3 or 0 4 ppm) NO2 for 12 weeks (Richters
<3
and Kuraitis, 1983) However, when mice were exposed to 940 /tg/m (0 5 ppm) for
8 weeks (Richters and Kuraitis, 1983) or 752 jug/m3 (0 4 ppm) for 12 weeks with
intermittent air exposures (Richters and Richters, 1983), facilitation was not observed
Richters et al (1985) attempted to extend their findings by showing that, if allowed, the
increased metastases from exposure to 752 jug/m3 (0 4 ppm) NO2 for 12 weeks led to
increased mortality in the mice However, their post hoc analysis of the data precludes this
q
conclusion More recently, Richters and Richters (1989) exposed mice to 658 /*g/m
(0 35 ppm) NO2 for 6 or 12 weeks, and examined tumor facilitation or lung injury after B16
melanoma injection The authors reported increased facilitation at 6 weeks (p = 0 04,
t-test), however, no statistical evaluation of the 12-week results were reported The authors
further claim that NO2-induced injury to pulmonary endothelium may facilitate the retention
of the injected melanoma Again, this result (number of microthrombi in lung, p = 0 10,
t-test) was found only after 6 weeks of exposure (not at 12 weeks) and only when examined
24 h after melanoma injection (not at 4 h) Pulmonary endothelial injury from NO2 alone
was not examined Furthermore, the actual experimental design used in these studies
probably did not evaluate metastases formation, as the term is generally understood, but
more correctly, evaluated colonization of the lung by tumor cells Studies in a true tumor
metastases model, such as the Lobin Wistar rat, should be performed
An abstract by Weinbaum et al (1987) indicated tliat NO2 could inhibit metastases
formation if exposure occurred after injection of the B16F10 tumor cell suspension Thus,
studies showing facilitation of tumor colonization in the lung after NO2 exposure should be
viewed with caution because NO2 may inhibit metastases as well as facilitate their
colonization
13-157
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Studies in Animals with Spontaneously High Tumor Rates
A study by Wagner et al (1965) suggested that NO2 may accelerate the production of
tumors in CAF^/JAX mice (a strain that is genetically susceptible to pulmonary tumors) after
<3
continuous exposure to 9,400 jwg/m (5 0 ppm) NO2 At the 12-mo evaluation, 7 of 10 mice
had tumors in the exposed group, compared to 4 out of 10 in the controls The number of
tumors per animal was not reported At the 14- and 16-mo evaluation, no differences in
tumor production were observed A statistical evaluation of the data was not presented
The frequency and incidence of spontaneously occurring pulmonary adenomas was
found to increase in strain A/J mice after exposure to 18,800 jug/m3 (10 ppm) NO2 for
6 h/day, 5 days/week for 6 mo (Adkins et al , 1986) These small, but statistically
significant, increases were only detectable when the control response from nine groups
(N=400) were pooled Exposure to 1,880 and 9,400 jug/m3 (1 0 and 5 0 ppm) NO2 did not
increase the number of spontaneous adenomas in this in vivo short-term model for predicting
carcuiogenicity
A study by Richters and Damji (1990) evaluated the effect of exposure to 470 /xg/m
(0.25 ppm) NO2, 7 h/day, 5 days/week for up to 181 days on the development and
progression of spontaneous T-cell lymphomas in AKR/cum mice Their results indicated that
control animals developed lymphomas earlier and their survival time was less than NO2-
exposed mice The reason for the increased incidence and progression of the lymphoma in
control animals over that seen in NO2-exposed animals was attributed to the decrease in
T-helper/inducer (CD4+) lymphocytes, which produce growth factors for lymphomas, in the
spleen of NO2-exposed mice A discussion of NO2-induced effects on host lymphocyte
populations appears in Section 13 2 2 1 on host defense mechanisms
Production ofN-Nitroso Compounds
Because of evidence that NO2 could produce nitrite and nitrate in the blood, and nitrite
is known to react with amines to produce animal carcinogens (mtrosamines), the possibility
that NO2 could produce cancer via nitrosoamine formation has been investigated
Iqbal et al (1980) was the first to demonstrate a linear tune-dependent and
concentration-dependent relationship in the amount of Af-nitrosomorpholine (NMOR), an
animal carcinogen, found in whole-mouse homogenates after the mice were gavaged with
13-158
-------
2 mg of morpholine (an exogenous amine that is rapidly mtrosated) and exposed for between
1 and 4 h to 28,200 to 94,000 /*g/m3 (15 to 50 ppm) NO2 Thus, because NMOR
(a mtrosamine) is an animal carcinogen, these studies aie sometimes used to suggest that
NO2 exposure could theoretically react with amines in the body to produce tumors
Iqbal et al (1981), using DMA, an amine that is slowly mtrosated to
dimethylnitrosamine (DMN), found a concentration-related increase in biosynthesis of DMN
**
at NO2 concentrations as low as 188 fj-g/m. (0 1 ppm), however, the rate was significantly
greater at concentrations above 18,800 /ig/m (10 ppm) NO2 Increased length of exposure
also increased DMN formation between 0 5 and 2 h, but synthesis of DMN was less after
3 and 4 h of exposure than after 0 5 h
Mirvish et al (1981) concluded that the results of Iqbal et al (1980) were technically
flawed According to these researchers, the Iqbal et al (1980, 1981) method, which
involved homogemzation of the whole frozen mouse, did not use an adequate stopping
solution to prevent in vitro production of nitrosamines According to Mirvish et al (1981),
they could verify the results of Iqbal et al (1981) by eliminating the use of the stopping
solution, but found no in vivo production of NMOR when in vitro production was
eliminated However, they did find that in vivo exposure to NO2 could produce a mtrosating
agent (NSA) that would nitrosate morpholine when morpholine was added in vitro
Additional experiments showed that NSA was localized in the skin (Mirvish et al, 1983) and
that mouse skin cholesterol was a likely NSA (Mirvish et al, 1986) It has also been
reported that only very kpid soluble amines, which can penetrate the skin, would be available
to the NSA Compounds such as morpholine, which is not lipid-soluble, could only react
with NO2 when it was painted directly on the skin (Mirvish et al , 1988)
Iqbal (1984), responding to the criticisms of Mirvish et al (1981), concluded after the
completion of several control experiments that in vitro nitrosation could only account for
between 1 to 2 % of the total amount of NMOR collected using his previous technique (Iqbal
et al , 1980). Several control experiments further suggested that the effects in the original
experiments were due to in vivo nitrosation One experiment showed that nitrosamine
biosynthesis could be inhibited in vivo with the addition of sulfamate, ascorbate, or
a-tocopherol prior to NO2 exposure Another experiment indicated that the rapid half-life of
morpholine (48 to 54 min) might explain why significant levels were not found by Mirvish
13-159
-------
et al (1981) because they transferred the NO2-exposed rats to room air for 30 min pnor to
sacrifice In vivo mtrosation was also demonstrated by Norkus et al (1984) after morpholine
administration and a 2-h exposure to 84,600 /tg/m3 (45 ppm) NO2
Postlethwait and Mustafa (1983) examined this problem of in vivo production of
mtrosamines using an isolated perfused rat lung Rat lungs were ventilated with
37,400 /ig/m3 (19.9 ppm) NO2 and the perfusion media was supplemented with 10 mM of
morpholine An excess of NMOR was found in the NO2-exposed group when lung tissue
and perfusate were combined Control experiments could not exclude the possibility that
NMOR was produced in the perfusate
Another study (Van Stee et al , 1983) reported that NMOR was produced in mice
gavaged with 1 g of morpholine/kg of body weight/day and then exposed to 31,020 to
38,540 jiig/m3 (16 5 to 20 5 ppm) NO2, 5 to 6 h/day for 5 days The single site containing
the greatest amount of NMOR was the gastrointestinal tract Regardless of whether in vivo
mtrosation can occur, the relative significance of nitrite from NO2 compared to nitrite
resulting from food, tobacco, and nitrate-reducing oral bacteria is questionable (Murdia
et al , 1982)
Aside from mtrosamines, other evidence suggests the possibility that inhaled NO2 may
be involved in the production of other potentially hazardous W-mtroso compounds Protein
and peptides may undergo mtrosation to produce diazo derivatives, most of which are
mutagemc and/or carcinogenic Challis et al (1987) suggested, based on in vivo studies,
that diazopeptides could be produced from inhaled NO2 that is absorbed into the blood
These diazopeptides would be relatively stable at blood pH so as to allow them to act as
circulating carcinogens
Summary
Exposure to NO2 produce a wide array of health effects beyond the confines of the
lung. Evidence suggests that NO2 and/or some of its reactive products penetrate the lung
and enter the blood, producing alterations in the blood and other organs
Conflicting results have been reported on whether NO2 affects body weight gam in
experimental animals One study reported that NO2 did not affect body weight gam in
rabbits, guinea pigs, rats, hamsters, and mice at exposure concentrations of up to
13-160
-------
47,000 jwg/m3 (25 ppm) and dogs at 9,400 /*g/m3 (5 0 ppm) for up to 18 mo (Wagner et al,
1965) However, a decline in body weight in guinea pigs exposed continuously to
1,880 jttg/m3 (1 0 ppm) for 6 mo (Kosmider et al , 1973b) and in rabbits exposed to
2,400 jwg/m3 (1 3 ppm) for 15 to 17 weeks (Mitina, 1962) has been reported Newborn mice
appear to be more sensitive to NO2 exposure than adult mice (Kuraitis et al, 1981, Richters
et al , 1987), but based on limited data, juvenile rats appear to be less sensitive to the effects
of NO2 exposure than young adult rats (Stevens et al , 1988)
Nitrogen dioxide-induced changes in blood constituents may result from the direct effect
of NO2, formation of nitrate and nitrite, or secondary effects emanating from other organs
such as the lung, liver, heart, kidneys, and spleen No effect on hematocnt and hemoglobin
have been reported in squirrel monkeys exposed to 1,880 /ig/m3 (1 0 ppm) NO2 for 16 mo
(Fenters et al, 1973) and in dogs exposed to up to 9,400 jug/m3 (5 0 ppm) for 18 mo
(Wagner et al , 1965) There was, however, polycythemia and an increased ratio of PMNs
to lymphocytes found in rats exposed to 3,760+1,880 /tig/m3 (2 0±1 0 ppm) NO2 for 14 mo
(Furiosi et al, 1973) There have also been reported changes in the RBC membranes of
experimental animals following NO2 exposure Red blood cell D-2,3-diphosphoglycerate was
a
reportedly increased in guinea pigs exposed to 677 /*g/m (0 36 ppm) NO2 for 1 week
(Mersch et al, 1973) An increase in RBC sialic acid, indicative of a younger population of
RBCs, was reported in rats exposed to 7,520 /*g/m3 (4 0 ppm) continuously for 1 to 10 days
(Kunimoto et al, 1984), but in another study, exposure to the same concentration of NO2
produced a decrease in RBCs (Mochitate and Miura, 1984)
Decreases in serum proteins and lipoproteins and increases m serum globulins,
indicating NO2-induced hepatic damage, have also been reported (Drozdz et al, 1976,
Menzel et al , 1977, Kosmider et al, 1973a, Kosmidei, 1975) Nitrogen dioxide increased
pentobarbital-mduced sleepmg tunes in female mice after a 3-h exposure to 470 jwg/m
(0 25 ppm) (Miller et al , 1980), suggesting effects on hepatic xenobiotic metabolism The
effects only occurred at specified tune penods after exposure ended and did not persist
beyond 1 day. Similar effects (increased hexobarbital-mduced sleeping tune) were reported
in the progeny of maternally exposed rats on Postexposure Days 7 and 21, but not on
Day 14, after being exposed to 1,000 jwg/m3 (0 5 ppm) NO2 (Tabacova et al , 1985)
Decreases in cytochrome P-450 levels in rat liver microsomes have been found after 7 days
13-161
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*5 <5
of exposure to 752 or 7,520 /ig/m (0 4 or 4 0 ppm), but not after exposure to 2,260 /tg/m
(1.2 ppm) NO2 (Mochitate et al , 1984)
Contrary to the finding of decreased amounts of cytochrome P-450 in liver homogenate
following NO2 exposure, cytochrome P-450 and cytochrome b5 levels were increased in the
<»
kidney of rats after 8 weeks of exposure to both 2,260 and 7,520 jug/m (1 2 and 4 0 ppm)
NO2 (Takahashi et al , 1986) Nitrogen dioxide has also been reported to increase urinary
concentrations of coproporphyrrns, indicating a possible increase in heme synthesis, at NO2
exposure concentrations of 600 /xg/m3 (0 32 ppm) over a 3-mo penod (Yakimchuk and
Chehkanov, 1972) and has increased urinary alpha, beta, and gamma globulins in guinea pigs
o
exposed to 752 /Ag/m (0 4 ppm) NO2, 4 h/day for 14 days (Sherwin and Layfield, 1974)
Only limited information is available on the effect of NO2 on the heart Nitrogen
dioxide-induced effects on cardiac performance are suggested by a significant reduction in
PaO2 in rats exposed to 7,520 jug/m (4 0 ppm) NO2 for 3 mo When exposure was
o
decreased to 752 /tg/m (0 4 ppm) over the same exposure penod, PaO2 was not affected
(Suzuki et al , 1981) Also, a reduction in heart rate has been shown in mice exposed to
both 2,250 and 7,520 j^g/m3 (1 2 and 4 0 ppm) NO2 for 1 mo (Suzuki et al , 1984)
However, whether these effects are the direct result of NO2 exposure or secondary to lung
edema and changes in blood hemoglobin content, is not known
From the limited information available, it would appear that NO2 affects the central
nervous system Decreased activity of protein metabolizing enzymes, increased glycolytic
enzymes; changes in neurotransmitter levels (5-HT and noradrenaline), and increased lipid
peroxidation, accompanied by lipid profile and antioxidant changes, have been reported
(Faraham and Hasan, 1990, 1991, Sherwin et al , 1986, Drozdz et al , 1975)
Unfortunately, none of these effects have been replicated and all reports lack sufficient
methodological rigor, thus, the implications of these findings, albeit important, are not clear
and require further investigation
The available data do not support the possibility that NO2 is a direct acting carcinogen
The data that suggest that NO2 may act as a promoter or facilitator of neoplastic disease are
fraught with methodological and interpretive problems The evidence suggests that further
study may be warranted
13-162
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13.3 EFFECTS OF MIXTURES CONTAINING NITROGEN DIOXIDE
Exposure to pollutant mixtures in ambient air provides a basis for possible toxicologic
interactions, whereby combinations of pollutants may behave differently than would be
expected from consideration of the action of each constituent separately In many cases, the
study of mixtures containing NO2 involved exposures to only two pollutants, and the role
played by each can be elucidated with the appropriate experimental design However, there
is a fairly large data base that involves mixtures of more than two components, often with no
single pollutant control, so the contribution of each individual agent to overall response is
often obscure In some cases, the NO2 (or NOX) may have varied between exposure groups,
or NO2 was present in one group and not in another, so its relative influence could be
assessed This section focuses on those studies where the role of NO2 (or NOX) can be
elucidated
Simple Mixtures Containing Nitrogen Dioxide
Table 13-23 outlines those studies in which experimental animals were exposed to
constant levels of an atmosphere containing NO2 with only one other pollutant (binary
mixtures) The table is categorized by pollutants in the mixture and further subdivided by
class of effect By far, the largest data base is for NO2 plus O3 Examination of these
studies mdicates that various degrees and types of interaction may occur The morphologic
response to an NO2/O3 mixture, as reported by Freeman et al (1974a) and Yokoyama et al
(1980), was primarily that due to O3 alone, although in these studies, the levels of NO2 used
would have produced only small changes (by light microscopy) that would easily be obscured
by the more potent O3 Acute lethality and some biochemical responses to NO2/O3
mixtures involve synergism (e g , Diggle and Gage, 1955, Mustafa et al , 1984, Sagai and
Ichinose, 1991, Lee et al, 1989), some have ascribed this interaction to the production of
new reaction products in the exposure atmosphere On the other hand, antagonism has also
been reported for effects of NO2 and O3 in some enzyme systems (Takahashi and Miura,
1989)
In terms of host antimicrobial defense, toxicologic interactions involving NO2 and
O3 are generally additive after acute exposures It seems that each pollutant contributes to
13-163
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TABLE 13-23. TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES CONTAINING NITROGEN DIOXIDE3
o\
Pollutant
Concentration
N02 (3,390-
30,200 /ig/m ,
1 8-4 75 ppm)
+ 03 (11,000-
23,300 jMg/m3,
5 0-10 7 ppm)
N02 (4,700 /ig/m3,
2 5 ppm) + O3
(490 jug/m3,
0 25 ppm)
NO2 (1,690 jug/m3,
0 9 ppm) + O3
(1,760 /ig/m3,
0 9 ppm)
NO2 (10,300 ng/m,
5 5 ppm) + O3
(2,160/Bg/nf,
1 1 ppm)
NO2 (10,200 jig/m3
5 4 ppm) + O3
(1,960 /tg/m ,
1 0 ppm)
Exposure Gender
4h M
6 mo M
60 days
3 h/day, M
14 days
3 h/day,
14 or
30 days
Species
Age (Strain)
NS Rat
(NS)
4 weeks Rat
(Sprague-
Dawley)
8 weeks Rat
(Wistar)
7 weeks
End Points
NO2
Mortality,
dyspnea
Morphology
Enzyme activity
Morphology
pulmonary
mechanics
Response
to Mixture
+ 03
Increase
Hypertrophy of
alveolar duct
epithelium
Emphysema
Increase
Increase
no change
Interaction Remarks
Synergistic Interaction due to
production of
nitrogen
pentoxide
None Lesion due
primarily to O3
None Lesion due
primarily to O3
Synergistic
None or additive
Reference
Diggle and
Gage (1955)
Freeman et al
(1974)
Yokoyama
etal (1980)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
Pollutant
Concentration
NO2 (75 pg/m3,
0 04 ppm) + O3
(98 /*g/m , 0 05 ppm)
N02 (752 /ig/m3,
0 4 ppm) + O3
(98 fig/m3, 0 05 ppm)
NO2 (752 jug/m3,
0 4 ppm) + O3
(784 /tg/m3, 0 4 ppm)
NO2 (2,260 ng/m,
I 2 ppm) + O3
(588 /ttg/m3, 0 3 ppm)
NO2 (3,380 liglm,
1 8 ppm) + 03
(882 jttg/m ,
0 45 ppm)
Exposure Gender
N02 M
continuous,
5-22 mo
(O3 was
intermittent)
N02
continuous,
5-22 mo
(O3 was
mtermittent)
24h/dayfor M
2 weeks
M
Continuous, M
3 days
Continuous, M
3 days
Species
Age (Strain)
7 Rat
weeks (Wistar)
10 Rat
weeks (Wistar)
10 Guinea pig
weeks (Hartley)
3 mo Rat
(Sprague-
Dawley)
3 mo Rat
(Sprague-
Dawley)
End Points
N02
Lipid
peroxidation,
antioxidants,
antioxidant
enzyme activity
Lipid peroxide
production and
activity of
antioxidant
enzymes
Lung weight,
activity of
various enzymes
Lung enzymes,
lung weight
Response
to Mixture
+ 03
Increase in lipid
peroxides, no
change in
enzyme of
activity
Increased lipid
peroxides only in
guinea pigs
Increased levels
of antioxidants
only in rats
Increases in
enzyme activity
and lung weight
Increases in
enzyme activity
Interaction
Synergistic (for
peroxides up
to 9 mo exposure
only)
Synergistic
Synergistic
None or synergistic
(end point dependent)
Synergistic (for some
enzymes, additive for
others, and same as
O3 for lung weight
and G-6-P
dehydrogenase
activity)
Remarks
NO2 or O3 alone
showed no change
in peroxides
Relation between
antioxidant
production and
peroxide
formation
Synergistic for
some end points,
additive for
others, same as
O3 for others
Reference
Sagai and
Ichinose
(1991)
Ichinose
and Sagai
(1989)
Lee et al
(1990)
Lee et al
(1989)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
Ui
ON
Pollutant
Concentration Exposure
NO2 (7,520 jig/m , Continuous,
4 0 ppm) + O3 1-2 mo
(392/rig/m3, 02ppm)
N02 (13,200 and 3 h/day,
28,200 uglm, 7 and 7 days
15 ppm) + 03 (980
and 1,960 jtig/m ,
0 5 and 1 0 ppm)
NO2 (9,020 /tg/m3, 8 h/day,
4 8 ppm) + O3 7 days
(880 /ig/m3,
0 45 ppm)
Species Response
Gender Age (Strain) End Points to Mixture Interaction
NO2 + O3
M 22 weeks Rat Xenobiotic (see Remarks) Antagonistic
(Wistar) metabolizing
system in lungs
M 4 weeks Mouse Level of Increase Additive
(ICRJCL) reduced
glutattuone in
lung
M 8 weeks Mouse Lung weight, Increase Synergistic
(Swiss rate of O2
Webster) consumption
(in lung
homogenate),
sulfhydryl
metabolism in
lung, activity of
NADP reducing
enzymes
Lung DNA No change None
content,
lung protein
content
Remarks
Mixture with
NO2 reduces
any increased
metabolism
produced by 63
alone for
various enzyme
activities
No significant
difference seen
between effects
of NO2 and O3,
each alone
produced no
change
No effect with
mixture or
either alone
Reference
Takahashi and
Miura (1989)
Watanabe et al
(1980)
Mustafa et al
(1984)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
Pollutant
Concentration
N02 (5,640 jag/m3,
30ppm) + 03
(588 jtig/m , 0 3 ppm)
NO2 (5,640 pg/m3,
3 0 ppm) + O3
(588 /tg/m3, 0 3 ppm)
N02 (3,760-
18,800 jug/rn3,
2-10 ppm) + O3
(980 and
1,960 jtig/m3, 0 5 and
1 0 ppm)
NO2 (6,770-
27,100/ig/m3, 36-
14 4 ppm) + O3 (392-
1,570 /tg/m3, 0 2-
0 8 ppm)
Exposure Gender Age
2 h M 5 mo
2 h/day, M 5 mo
up to
14 days
1-2 h NS NS
6-24 h/day, M 10-12
3 days weeks
(depending
on concen-
tration)
Species
(Strain)
Rabbit
(New
Zealand)
Rabbit
(New
Zealand)
Mouse
(NS)
Rat
(Sprague-
Dawley)
End Points
NO2
Effects on
arachidomc acid
metabolites
Prostanoids in
lavage
Creatimne
phosphokmase in
plasma
BAL protein,
cell types in
BAL
Response
to Mixture Interaction
+ 03
Increase Synergistic
Decrease in None
selected
prostanoids
Increase or None
decrease,
depending on
concentration
Increased protein Additive or
at > 10 8 ppm synergistic
NO2 + >0 6 ppm
O3, increased
epithelial cells at
all concentrations,
increase
neutrophils at
> 10 8 ppm NO2
+ >0 6 ppm O3
Remarks
Increases in
PGE2and
PGE^
(Response
driven by O3 )
Depending on
prostanoid, and
number of days
of exposure
mixture was
additive or
similar to O3 or
NO2 when
given alone
Effect due to
°3
Additive at low
dose-rate
(3 6 ppm NO2
+ 02 ppm O3)
and synergistic
at higher dose-
rate
Reference
Schlesinger
etal (1990)
Schlesinger
etal (1991)
Veninga et al
(1982)
Gelzleichter
etal (1992a)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
ON
00
Pollutant
Concentration Exposure Gender Age
NO2 (6,770- 6h/day, M 10-12
27,100 pig/m3, 36- 3 days weeks
14 4 ppm) + 03 (392-
1,570 Mg/m3, 0 2-
0 8 ppm) (concurrent
and sequential)
NO2 (7,520 jtig/m3, Contrnuous, M 8-10
4 0 ppm) + O3 3, 7, 14, or weeks
(1,568 /tg/m3, 56 days
0 8 ppm)
Species Response
(Strain) End Points to Mixture Interaction
N02 + 03
Rat BAL protein, Increased in all Additive,
(Sprague- cell types endpoints synergisbc, or
Dawley) antagonistic
Mouse Organ weights, Decrease in spleen None
BALB/c antibody response weight and
to SRBCs and to increase in lung
DNP-Ficoll weight, no effect
on response to
DNP, but response
to SRBCs was
depressed with
3-14 day exposure
only
Remarks
For BAL proteui
and PMNs,
additivity with
sequential
exposure,
synergism for
concurrent
mixture For
epithelial cells,
O3 and then NO2
caused additivity,
N02 then 03
caused
antagonism,
concurrent
mixture caused
synergism
Additive for
spleen weight,
similar to O3 for
lung weight,
antibody response
similar to O3
Reference
Gelzleichter
etal (1992b)
Fujimaki
(1989)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
UJ
I
Pollutant
Concentration Exposure Gender Age
NO2 (2,820 /ig/m3, 4 h M NS
1 5 ppm) + O3
(200 /tg/m , 0 1 ppm)
NO2 (2,800- 17 h
7,860 /ig/m3, 1 49-
4 18 ppm) + O3
(220-530 /tg/m3,
0 11-0 27 ppm)
NO2 (2,820- 3h F 6-10
9,400 /ig/m3, 1 5- weeks
5 0 ppm) + O3 (100-
980 /tg/m3, 0 05-
0 5 ppm)
NO2 (3,760 /tg/m3, 3 h/day,
2 0 ppm) + O3 5 days/week,
(100 /tg/m3, 1-4 weeks
0 05 ppm)
Species
(Strain)
Mouse
(Swiss
albino)
Mouse
(CF-1,
CD2Fj)
Response
End Points to Mixture Interaction
N02 + 03
Bactericidal Decrease in
activity bactericidal
activity when
NO2 >40ppm
+ O3
>0 36 ppm
Decrease in Additive
bactericidal
activity when
NO2 > 1 8 ppm
+ O3
>0 2 ppm
Bacterial Decreased Additive
infectraty survival time
with 0 5 ppm
O3 and any
NO2, and with
0 1 ppm O3 and
3 5 ppm NO2
Excess Synergistic
mortality
at all times
Remarks Reference
Bacterial Goldstein et al
challenge after (1974)
exposure
Bacterial
challenge
before
exposure
Bacterial Ehrhch et al
challenge after (1977)
exposure
No effect with
either alone,
except for NO2
at 2 weeks
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
Pollutant Species Response
Concentration Exposure Gender Age (Strain) End Points to Mixture Interaction
NO2 + O3
3
NO2 (2,260-/Kg/m , 15 days F 4-6 Mouse Bacterial Increased Synergistic
1 2-ppm base with baseline, weeks (CD-I) infectivity infectivity
4,700-fig/m , twice daily
2 5 ppm peak) + 63 1-h peak
(196-/ig/m3, 0 1-ppm
base with 588-/ig/m ,
0 3-ppm peak)
3
NO2 (940-/ig/m , Synergistic
0 5-ppm base with
l,880-/ig/m3,
1 0-ppmpeak) + 63
(98-pg/m , 0 05-ppm
base with 196-jttg/m ,
0 1-ppm peak)
NO2 (94-jng/m3, None
0 05-ppm base with
188-/tg/m3, 0 1-ppm
peak) + 03
(98-/tg/m , 0 05-ppm
base with 196-jttg/m ,
0 1-ppm peak)
Remarks
NO2and
03 alone
increased
infectivity
NO2 alone
increased
infectivity,
03 alone did
not
NC^or
O3 alone had
no effect
Reference
Graham et al
(1987)
Gardner et al
(1982)
Gardner (1980)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
Pollutant
Concentration
NO2 (3,760 /tg/m3,
2 0 ppm) + SO2
(5,240 /tg/m3,
2 0 ppm)
NO2 (8,000-
11, 000 /tg/m3, 42-
5 8 ppm) + SO2
(9,000-1 1,000 /tg/m3,
3 4-4 2 ppm)
NO2 (3,760 /tg/m3,
2 0 0pm) + NaCl
(33d"/ig/m3)
N02 (3,760 /tg/m3,
2 0 ppm) + NaCl
(330 /tg/m3)
Exposure Gender Age
Continuous, NS 8 weeks
up to
13 weeks
24 h/day, F NS
6 days/week,
6 mo
Continuous, M/F NS
14 mo
Continuous,
18 mo
Continuous, M 4 weeks
6 mo
Species
(Strain)
Rat
(Wistar)
Guinea
pig
(BFA-
ZH-
Kisslegg)
Monkey
(Macaca
speciosa)
Rat
(Sprague-
Dawley)
End Points
N02
Morphology
Blood variables
(oxyHb
dissociation
curve, RBC
count, metHb,
enzymes)
Respiratory
mechanics
(frequency, flow
rate, minute
volume)
N02 +
Morphology
Hematology
Hematology
Response
to Mixture Interaction
+ SO2
No change None
No change None
,
No change None
Particles
Respiratory None
bronehiokr
epithelial
hypertrophy
Polycythemia None
Polycythemia None
Remarks
No effect of
either SO2 or
N02
No effect of
either SO2 or
N02
Effect due to
N02
Effect due to
N02
Effect due to
N02
Reference
Azoulay et al
(1980)
Antweiler and
Brockhaus (1976)
Funosi et al
(1973)
Funosi et al
(1973)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE
Pollutant
Concentration
Oig/m3, ppm)
N02 (9,400 jug/m3,
5 0 ppm) + NaCl
(1,000 /tg/m3, 0 4 jKin)
N02 (9,400 jig/m3,
5 0 ppm) + NaCl
(1,000 pg/m3, 0 4 fim)
NO2 {47,000-56,400
/tg/m , 25-30 ppm) +
carbon
NO2 {9,400-47,000
/tg/m , 5-25 ppm) +
(NH4)2S04
(5,000 jug/m , 0 8-1 jinn
MMAD)
NO2 (3,760 and
9,400 /tg/rn3, 2 0 and
5 0 ppm) + H2SO4
(890 fig/m, 0 4 /tm
MMAD)
Exposure Gender Age
7 days M NS
1, 3 days
6 h/day, NS NS
5 days/week,
3 mo
23 5 h/day, M 10-11
7 days weeks
23 5 h/day, M NS
7 days
Species Response
(Strain) End Points to Mixture
NO2 + Particles
Rat Rate of collagen Increase
(Sprague- synthesis by lung
Dawley) minces
Protein content Increase at
of lung lavage 1 and 3 days
fluid
Mouse Morphology Focal
(Swiss parenchymal
albino) lesions
Rat Rate of collagen Increase
(Sprague- synthesis by lung
Dawley) minces
Rat Rate of collagen Increase
(Sprague- synthesis by lung
Dawley) minces
Interaction Remarks
Synergistic
Synergistic at
3 days
Carbon acted as
earner for localized
NO2 deposition
Synergistic
Synergistic
Reference
Last and
Warren
(1987)
Boren (1964)
Last et al
(1983)
Last and
Warren
(1987)
Last (1989)
-------
TABLE 13-23 (cont'd). TOXICOLOGIC INTERACTIONS TO SIMPLE MIXTURES
CONTAINING NITROGEN DIOXIDE3
Pollutant
Concentration
NO2 (560 and
1,880 jtig/m3, 0 3 and
1 0 ppm) + H2SO4
(500 ftg/m3, 0 4 jam
MMAD)
NO2 (560 and
1,880 /tg/m3, 0 3 and
1 0 ppm) + H2SO4
(500 jttg/m3, 0 3 ^m
MMAD)
Exposure Gender
2 h/day, M
5 days/week,
14 days
2 h/day, M
5 days/week,
up to 14 days
Species
Age (Strain)
5 mo Rabbit
(New
Zealand)
5 mo Rabbit
(New
Zealand)
End Points
N02 +
Response
to Mixture
Particles
Particle clearance Decrease
from respiratory
region
Mucociliary
clearance
Alveolar
macrophage
function and
numbers
Variable,
depending on
N02
concentration
and end point
Interaction Remarks
None at 0 3 ppm,
response due to
H2SO4, at
1 0 ppm,
response different
from both NO2
and H2SO4
With 0 3 ppm
NO2 in mixture,
clearance faster,
no change with
1 ppm NO2
Additive or
synergistic
depending on
N02
concentration and
end pomt
Reference
Schlesinger
and Gearhart
(1987)
Schlesinger
etal (1987a)
Schlesinger
(1987a)
M = Male
NS = Not stated
NADP = Nicotmamide ademne
PGE2 = Prostaglandm %
PGEja = Prostaglandm E2el,
F = Female
SOj = Sulfur dioxide
oxyHb = Oxyhemoglobm
RBC = Red blood cell
MetHb = Methemoglobm
NaCl = Sodium chlonde
(NH4)2SO4 = Ammonium sulfate
MMAD = Mass median aerodynamic diameter
H2SO4 = Sulfunc acid
G-6-P dehydrogenase = Glucose-6-phosphatedehydrogenase
SRBCs = Sheep red blood cells
BAL = Bronchoalveolar lavage
-------
the observed response when its concentration reaches a level at which it would have affected
bacterial resistance when administered alone (Goldstein et al, 1974) The mouse subchronic
infectivity study conducted by Ehrlich et al (1977) provided a suggestion of synergism with
exposure to 3,760 pig/m3 (2 0 ppm) NO2 and 97 5 /*g/m3 (0 05 ppm) O3
Simulation of urban patterns involving NO2 and O3 have also been performed by
examining the effects, on bacterial resistance, of a continuous baseline exposure, with
superimposed short-term peaks to a higher level Ehrlich et al (1979) exposed mice for 1 to
*a
6 mo (24 h/day, 7 days/week) to a baseline concentration of 0 (air) or 188 /wg/m (0 1 ppm)
NC>2, upon which was superimposed 3-h/day, 5-day/week peak exposures of 940 /*g/m3
(0.5 ppm) NO2, or a combination of 940 jwg/m NO2 and 200 /xg/m O3, bactenal exposure
followed pollutant exposure, and animals were then observed for 14 days A significant and
similar increase in percentage mortality was found by 6 mo in all groups, with no evidence
that exposure to the NO2/O3 peaks altered the response, there was also no change in survival
time. In another experiment, mice were reexposed for 14 days (after 1- to 3-mo pollutant
exposure and bactenal challenge) to the same pollutant concentrations as above, and
mortality was examined during this tune Animals preexposed for a least 2 mo either to
NO2/O3 peaks over the air baseline or to NO2/O3 peaks over the 188 /jg/m3 NO2 baseline
showed significant reductions in survival time Although no conclusions were drawn as to
the efficacy of the mixture, the investigators concluded that the sequence of peak exposure
was important in altering resistance to infection
Ehrkch et al (1979) also examined the effect of the same baseline and peak exposures
(for 1 to 3 mo) on AMs Cell viability was decreased after 3 mo of exposure only when the
NO2/O3 peaks were superimposed on continuous exposure to clean air There also was a
sy
general increase in blood enzyme activity, but continuous exposure to 188 jug/m (0 1 ppm)
NO2 with superimposed peaks of NO2 and O3 was the most effective in this regard
In another study, Ehrkch (1983) examined the effects of continuous exposure (24 h/day,
O
5 days/week) to a baseline level of 376 jwg/m (0 2 ppm) NO2, with two daily peaks given
5 days/week as follows 1,880 /*g/m3 (1 0 ppm) NO2 for 1 h in the morning and a mixture
3 3
of 200 /ng/m (0 1 ppm) O3 plus 1,880 jug/m NO2 given for 1 h in the afternoon
Exposures lasted for 9 mo, followed by bactenal challenge Other groups were exposed
3 3
continuously to 376 /tg/m NO2 either with no peak or with a 1,880-jKg/m peak given for
13-174
-------
1 h in both the morning and the afternoon The only group that showed a significant
o
increase in mortality was that exposed for 9 mo to 376 /^g/m NO2 with daily peaks of NO2
in the morning and NO2 and O3 in the afternoon In addition, only this group showed a
change (increase) in cellular ATP levels in AMs By 8 mo of exposure, this group also
showed an increase in counts of RBCs, leukocytes, and lymphocytes, and a decrease in mean
hemoglobin concentration The other pollutant exposure groups showed increases only in
leukocyte count
Gardner et al (1982), Gardner (1980), and Graham et al (1987) examined bacterial
resistance in mice continuously exposed (15 days, 24 h/day) to a baseline level of an NO2/O3
mixture with two daily 1-h peaks of the mixture, as follows (1) high exposure level
2,260 jtig/m3 (1 2 ppm) NO2 plus 196 /*g/m3 (0 1 ppm) O3 baseline with 4,700 /^g/m3
•3
(2 5 ppm) NO2 plus 588 /xg/m (0 3 ppm) O3 peak, (2) intermediate exposure level
940 jwg/m3 (0 5 ppm) NO2 plus 98 /*g/m3 (0 05 ppm) O3 baseline with 1,880 /wg/m3
3 3
(1 0 ppm) NO2 plus 196 /xg/m (0 1 ppm) O3 peak, or (3) low exposure level 94 /-cg/m
(0 05 ppm) NO2 plus 100 ^g/m3 (0 05 ppm) O3 baseline with 188 ^g/m3 (0 1 ppm) NO2
a
plus 196 jwg/m (0 1 ppm) O3 peak Animals were also exposed to the same baseline levels
of either NO2 or O3 onto which were superimposed twice daily, 1-h peaks of either NO2 or
O3 at the concentrations described above The low concentrations of either given alone, or
in combination, did not significantly increase mortality At the intermediate exposure levels,
the mixture was synergistic, whereas NO2 alone increased mortality and O3 had no effect
At the high exposure level, the combined exposure was again synergistic, exposure to each
pollutant given separately also increased mortality
Sagai et al (1987) exposed mice, hamsters, guinea pigs, and rats to a mixture of
750 Acg/m3 (0 4 ppm) NO2 and 780 jwg/m3 (0 4 ppm) O3, 24 h/day for 2 weeks, to assess
effects on kpid peroxidation in the lungs Although the two gases were not also administered
singly to allow assessment of effects due to each alone, the study showed species differences
in lipid peroxide formation following exposure that were related to the relative content of
antioxidants and the specific composition of phospholiplds and their fatty acids The guinea
pig was the most sensitive animal and the hamster was the most resistant In follow-up
studies, Sagai and Ichinose (1991) exposed rats for 22 mo to mixtures of NO2 and O3 The
13-175
-------
increase in hpid peroxidation was synergistic and maximal at 9 mo, later examinations
revealed no effects
Ichinose and Sagai (1989) also observed a species dependence in the interaction of NO2
(752 Aig/m3, 0 4 ppm) and O3 (784 jug/m3, 0 4 ppm) after 2 weeks of continuous exposure
Guinea pigs, but not rats, had a synergistic increase in lung hpid peroxides, expressed as
TEA reactants Rats, but not guinea pigs, had synergistic increases in nonprotein
sulfhydryls, vitamin C, G-6-P dehydrogenase, and GSH peroxidase
Duration of exposure can also have an impact Schlesinger et al (1990) observed a
synergistic increase in prostaglandins E^ and F2a in the lung lavage of rabbits exposed for
o --3
2 h to 5,640 jitg/m (3 0 ppm) NO2 plus 588 pg/m (0 3 ppm) O3, the response appeared to
have been driven by O3 However, with 7 of 14 days of repeated 2-h exposures, only
prostaglandin E^ was decreased, apparently due to NO2, there was no synergism (Schlesinger
et al., 1991).
The studies described above involved simultaneous exposure to both NO2 and another
gas However, "real world" exposures to these pollutants typically have temporal patterns,
and exposure to one agent may then alter the response to another subsequently inhaled
Thus, order of exposure to inhaled NO2 may be important in toxic interactions
Yokoyama et al (1980) exposed rats to either NO2 or O3 for 3 h or to NO2 for 3 h followed
by O3 for 3 h, and assessed lung mechanics in postmortem lungs, lung histology, and
enzyme activity in subcellular fractions of lung tissue In one series of exposures, rats were
exposed for 7 or 14 days to NO2 and O3 at concentrations of 10,300 jwg/m3 (5 5 ppm) and
o
2,160 jtig/m (1 1 ppm), respectively The activity of phospholipase A2 in the mitochondnal
fraction of lung homogenate was only increased in those animals exposed to O3 after NO2,
for 14 days A decrease in activity of lysolecithin acyltransferase in the supernatant fraction
was found after 7 and 14 days in all groups of animals In a second study, rats were
% 3
exposed for 14 or 30 consecutive days to 10,200 /*g/m (5 4 ppm) NO2 followed by
o
1,960 /itg/m (1.0 ppm) O3 Pulmonary mechanics tests performed on the postmortem lung
indicated an increase in pulmonary flow resistance in the O3- and sequential NO2/O3-exposed
animals There were no changes in volume-pressure curves in any of the groups
Histologically, the lungs of the animals exposed to both NO2 and O3 appeared similar to
those exposed to O3 alone However, a slight degree of epithelial necrosis in the medium
13-176
-------
bronchi, not found with either NO2 or O3 alone, was seen in the animals exposed to both
pollutants In addition, damage at the bronchioloalveokr junction appeared to be somewhat
more marked in animals exposed to both gases than in those exposed to O3 alone These
studies suggest that sequential exposures produced responses that were, in most cases, not
greatly different from those due to O3 alone
Gelzleichter et al (1992b) also evaluated sequential exposure Rats were exposed for
3 days for 6 h/day to O3 (392 to 1,570 /tg/m3, 0 2 to 0 8 ppm) or NO2 (6,770 to
27,100 /tg/m , 3 6 to 14 4 ppm) or their combinations Combinations were either concurrent
or sequential (O3 first and then NO2 or vice versa) For either of the sequential exposures,
the increase in BAL protein and PMNs was additive, it was synergistic for the concurrent
mixture For lavageable epithelial cells, the O3 then NO2 group showed additivity, whereas
the NO2 then O3 group displayed antagonism, this end point exhibited synergism when the
O3 and NO2 were concurrent The synergisms observed were concentration-dependent
Effects on epithelial cell numbers were most sensitive, showing synergism at 392 jwg/m3
^
(0 2 ppm) O3 with 27,100 jttg/m (14 4 ppm) NO2 The authors postulate that the synergism
may be due to chemical reactivity of O3 and NO2 within the exposure chamber and the
subsequent formation of nitrogen pentoxide
An important consideration in examining responses to air pollutants is the relative roles
of exposure C and T on response The roles of C and T in responses to mixtures of NO2
and O3 were examined by Gelzleichter et al (1992a) Rats were exposed to various
concentrations of each gas (6,770 to 27,100 j^g/m3 [3 6 to 14 4 ppm] NO2 and 392 to
1,570 /tg/m3 [0 2 to 0 8 ppm] O3) for various durations, such that the product of C x T was
constant in all cases They found that the response to these mixtures could not be related to
the product of C x T but, rather, seemed to be more dependent upon actual concentration
than exposure duration The responses were disproportionately greater at the higher
concentrations of these gases
Some limited data exist for combinations of NO2 with gases other than O3 In the two
reported studies with sulfur dioxide (SO^, neither SO2 nor NO2 given alone, or together,
produced any response The concentrations used by Azoulay et al (1980) were quite low,
and the respiratory mechanical end points assessed by Antweiler and Brockhaus (1976) were
likely not very sensitive to pollutant-induced changes
13-177
-------
Trzeciak et al (1977) exposed guinea pigs to 940 /*g/m3 (0 5 ppm) NO2 plus 61
(0.05 ppm) NO, or this NOX mixture plus an equal amount of ammonia, for 8 h/day for a
total of 122 days and analyzed lung phospholipids There was no difference in the
phospholipid content, expressed as milligrams per gram of wet tissue, of exposed versus
control lungs Significant alterations were found in the individual phospholipid classes
Decreases were noted in phosphatidylethanolamine, sphingomyelin, phosphatidylserine,
phosphatidylglycerol- 3-phosphate, and phosphatidic acid Increases were noted in the lyso-
phosphatidyl-ethanolamine content, whereas the lecithin content remained constant or was
slightly depressed Such changes could be indicative of changes in the permeability of the
cell wall and subsequently changes in the cell content The presence of ammonia did not
significantly influence the results
One major interaction that may occur in ambient air is that between NO2 and particles
Particle contact may result in gas adsorption and subsequent transport to target sites where
the gas normally would not deposit in concentrated amounts Boren (1964) adsorbed NO2
onto carbon to determine whether this earner changed the toxicity of the NQ2 Mice were
exposed 6 h/day, 5 days/week for 3 mo to carbon (38% of particles were <2
16,000 particles/cm ) onto which 553 mg NO2 was adsorbed per gram, the exposure air also
contained 47,000 to 56,400 /*g/m3 (25 to 30 ppm) free NO2 as well The exposed animals
showed focal changes in the lung parenchyma These lesions contained carbon particles, and
were characterized by enlarged airspaces and loss of alveolar walls Exposure solely to NO2
resulted in edema and inflammation, but no parenchymal lesions, and no lesions were found
due to carbon-only exposure Thus, Boren (1964) concluded that the carbon particles served
as a carrier for NO2, delivering high concentrations of NO2 to localized areas in the lungs
where the carbon deposited
The role of adsorbed NO2 in the toxicity of mineral dusts was addressed by Robertson
et al. (1982). They examined the effects of NO2 adsorption on the cytotoxicity of coal,
quartz, or kaolirnte on P388D! cells exposed in vitro to mineral dusts for 48 h, viability and
enzyme release (e g , LDH) were used as end points The amount of NO2 absorbed was 5 to
10 jwg/mg dust. Although a small decrease in cytotoxicity was found after adsorption of
NO2, the investigators concluded that there was no systematic or significant difference in
biochemical measures of toxicity from cells exposed to dust with or without NO2 On the
13-178
-------
other hand, Shevchenko (1971) noted an increase in the fibrogemcity of quartz dust in albino
rats following adsorption of 0 36 pg NO2/mg dust, a lower level than that used by Robertson
et al (1982) These different results may be due to differences in particle residence time
Robertson et al (1982) exposed the cells for only 48 h, whereas dust was present in the
lungs in the Shevchenko (1971) study for months, allowing a greater time for gas desorption
Other aerosols, although not necessarily acting as earners, may potentiate response to
NO2 by producing local changes in the lungs that enhance the toxic action of co-inhaled
NO2 Last et al (1983) and Last and Warren (1987) have examined the effects of inhalation
of acidic sulfate aerosols plus NO2 on biochemical end points, using minces prepared from
the lungs of rats after various exposure regimes Last et al (1983) exposed rats to 9,400 to
3 3
47,000 jiig/m (5 to 25 ppm) NO2 alone, or in combination with 5,000 /tg/m (ammonium
sulfate (NH4)2SO4) (1 jum mass median aerodynamic diameter [MMAD]), for up to 7 days,
and examined the rate of collagen synthesis by lung minces Ammonium sulfate alone
caused no effects Analysis of the slope of the exposure concentration-response curve for
NO2 indicated an approximate doubling of the synthesis rate when the mixture was employed
compared to NO2 alone, examination of responses at individual NO2 concentrations showed
that the mixture clearly began to increase the synthesis rate (above NO2 alone) when NO2
v
concentrations exceeded 18,800 /tg/m (10 ppm) The investigators also noted that there was
a tendency towards a reduction in lethal concentration for 75 % of the animals when
exposures were to (NH4)2SO4 plus NO2, compared to that for NO2 alone On the other
hand, there was no difference in the level of pulmonary edema between animals exposed to
NO2 alone or to NO2 in combination with (NH4)2SO4
>3
In a later study, Last and Warren (1987) exposed rats to 9,400 jitg/m (5 0 ppm) NO2
o
alone or in combination with either 1,000 /*g/m sulfunc acid (H2SO4) (0 4 j»m MMAD) or
sodium chloride (NaCl) (0 4 ju,m MMAD) for up to 7 days A synergistic interaction for
collagen synthesis rate was found when either aerosol was used with NO2 Reduction of the
NO2 level to 3,760 /ig/m3 (2 0 ppm) also resulted in a synergistic increase in the collagen
O
synthesis rate when combined with 1,000 /*g/m H2SO4 (Last, 1989) Changes in protein
content of the lavage fluid (an index of lung edema) showed evidence of synergism at 1 day
with H2SO4 or 3 days with NaCl The investigators suggested that the interaction with NaCl
was due to the formation of acids (e g , hydrogen chloride, HNO3, HONO) from mtrosyl
13-179
-------
chloride following its hydrolysis after deposition in the deep lung, the latter may be formed
from a chemical reaction between NO2 and NaCl Similarly, potentiation with the acid
sulfate aerosols was likely due to localized effects following their deposition It has been
proposed that the acid aerosols would produce a shift in local pH within the alveolar milieu
This shift would result in a change in the reactivity or residence tune of reactants involved in
oxidant-induced pulmonary effects (Last et al , 1984)
The effects of exposure to mixed atmospheres of NO2 and H2SO4 on lung host defenses
have been examined by Schlesinger and Gearhart (1987) and Schlesinger (1987a) In the
former study, rabbits were exposed for 2 h/day, 5 days/week for 14 days to either 564 or
1,880 jwg/m3 (0.3 or 1 0 ppm) NO2 or 500 jwg/m3 H2SO4 (0 3 ^m) alone, or to mixtures of
the low and high NO2 concentrations with the acid After the first exposure, an inert tracer
aerosol was administered to assess clearance from the respiratory region of the lungs In the
single-pollutant groups, both concentrations of NO2 accelerated clearance, whereas H2SO4
retarded clearance, compared to air-exposed controls Exposure to the combination of
o
564 jtg/m NO2 plus H2SO4 resulted in a response that was not different from that due to the
acid alone However, exposure to 1,880 ^g/m NO2 plus H2SO4 resulted in a clearance
pattern that differed from that of both NO2 and H2SO4, but was more similar to that of the
H2S04
Schlesinger (1987a) exposed rabbits to the same NO2/H2SO4 atmospheres as above, but
then examined the animals 24 h after 2, 6, or 13 exposures and recovered cells from the
lungs by bronchopuhnonary lavage Exposure to 1,880 jwg/m3 (1 0 ppm) NO2 with acid
resulted in an increase in PMNs at all tune points (not seen with either pollutant alone), and
an increase in the phagocytic capacity of AMs after two or six exposures In contrast,
o
exposure to 564 /*g/m (0 3 ppm) NO2 with acid resulted in depressed phagocytic capacity
and mobility A comparison of responses due to exposure to the NO2/H2SO4 mixture with
those due to either pollutant alone showed that the effects of the combined atmospheres were
generally either additive or synergistic, depending on the specific cellular end point being
examined
Funosi et al (1973) exposed rats and monkeys continuously to a combination of
3,760 (Ag/m3 (2.0 ppm) NO2 and 330 /*g/m3 NaCl Histological response after 14 mo of
exposure in monkeys (respiratory bronchiolar epithelial hypertrophy) was similar in groups
13-180
-------
exposed to NO2 alone or to NO2 with NaCl Hematologic changes (polycythemia) ui both
monkeys, after 18 mo, and rats, after 6 mo, were similar for groups exposed to NO2 with or
without NaCl Thus, in this study, the NaCl did not potentiate response to NO2 Perhaps
the end points were not sensitive to the effects of any reaction products of NO2 and NaCl, or
the concentration of NaCl was too low to allow production of significant amounts of such
products
Complex Mixtures Containing Nitrogen Dioxide
Although many studies have examined the response to NO2 with only one additional
pollutant, the atmosphere in most environments is a complex mixture of more than two
materials. A number of studies have attempted to examine the effects of multicomponent
atmospheres containing NO2 But, as mentioned, in many cases, the exact role played by
NO2 in the observed responses is not always clear
Klemman et al (1985a,b) exposed rats for 4 h to atmospheres consisting of various
combinations of NO2 (4,700 /xg/m3, 2 5 ppm), O3 (1,180 jwg/m3, 0 6 ppm), SO2
3 3
(13,100 jwg/m ; 5 0 ppm), and particles The particles consisted of 1 mg/m (0 2 fim
MMAD) of either H2SO4 or (NH4)2SO4, laced with iron and manganese sulfates The
metallic salts act as catalysts for the conversion of sulfur IV into sulfur VI and the
incorporation of gases into the aerosol droplets The respiratory region was examined for
morphological effects A confounding factor in these studies was the production of HNO3 in
atmospheres that contained NO2 and O3, and nitrate in atmospheres that contained O3 and
(NH4)2SO4, but not NO2 Nevertheless, a significant enhancement of tissue damage was
produced by exposure to atmospheres containing H2SO4 or HNO3, compared to those
containing (NH4)2SO4 In addition, it was suggested that the former atmospheres resulted in
a greater area of the lung becoming involved in lesions, which were characterized by a
thickening of alveolar walls, cellular infiltration in the mterstitium, and an increase in free
cells within alveolar spaces Exercise seemed to potentiate the histological response to the
complex mixtures containing acids (Klemman et al, 1980)
One of the more common complex mixtures studied is that of combustion exhaust
emissions from automobiles In some cases (see below), the exhaust was irradiated to
produce a reactive mixture that is a model for photochemical smog Coffin and Blommer
13-181
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(1967) exposed mice for 4 h to irradiated gasoline-engine exhaust to assess the effects on
bacterial resistance Levels of NOX in the atmosphere were as follows NO2, 200 to
1,600 /tg/m3 (0 11 to 0 85 ppm), and NO, 20 to 180 ^g/m3 (0 02 to 0 15 ppm) Exposure
was found to result in an increase in bacterial-induced mortality, but the investigators were
not able to clearly ascribe the results to any one pollutant However, they noted that the
exposure levels of NO2 were less than those that were known to alter resistance when NO2
was given alone and, thus, they suggested that the effect of the exhaust mixture was due to
other oxidants, such as O3
Stupfel et al (1973) exposed rats for 6 h/day, 5 days/week for 2 5 mo to 2 years to
gasoline-engine exhaust mixtures for morphologic analysis The atmosphere contained CO2,
aldehydes, carbon monoxide (CO), and either 0 2 or 23 ppm NOX Only the mixture with
the higher NOX concentration produced any significant toxic response, namely a decrease in
body weight and increase in spontaneous tumors However, the latter was ascnbed to the
hydrocarbon component of the exhaust mixture
Cooper et al (1977) exposed rats continuously for 38 or 88 days to three gasoline-
engine exhaust atmospheres that differed in their component concentrations, all contained
H2SO4, SO2, and CO, as well as NO (8,700 to 13,300 /xg/m3, 7 1 to 10 8 ppm) and NO2
3
(564 to 9,590 jwg/m , 0 3 to 5 1 ppm) All exposures resulted in a significant depression of
spontaneous locomotor activity not seen with exposure to either H2SO4 or CO alone, the
investigators concluded that this response was due to either the hydrocarbon or the NOX
components of the mixture
The results of a long-term exposure of dogs to gasoline-engine exhaust emissions have
been described by several investigators (Stara et al , 1980) Animals were exposed for
68 mo (16 h/day) to various atmospheres, which included raw exhaust, irradiated exhaust, or
-------
dynamic compliance, or total expiratory resistance to flow after 18 mo of exposure
However, by 36 mo, a significant number of animals exposed to high NO2/low NO had an
abnormally low CO diffusing capacity (as a ratio of total lung capacity) (Lewis et al, 1974)
Additional changes were observed after 61 mo of exposure, in the dogs breathing low
NO2/high NO or raw auto exhaust, residual volume was increased compared to animals
exposed to control or high NO2/low NO The common treatment factor causing this effect
appeared to be the higher concentration of NO A significant number of dogs exposed to
high NO2/low NO had a lower mean CO diffusing capacity/total lung capacity ratio, and a
lower peak flow rate, compared to controls The investigators attributed the change in
diffusing capacity to an alteration in the alveolocapillary membrane Bloch et al (1973)
reported no significant change in hematocnt, blood viscosity, or level of methemoglobin due
to any of the exposure atmospheres after 48 mo of exposure
After all exposures were terminated, the animals were allowed to recover for 2 years
before pulmonary function measurements were made again (Stara et al, 1980) In all
pollutant-exposed dogs, total lung capacity was increased relative to the control group of
animals Those animals that received the NO2/NO mixtures experienced modest increases in
inspiratory volume, vital capacity, and total lung capacity
Orthoefer et al (1976) evaluated biochemical alterations 2 5 to 3 years after the end of
all exposures In groups exposed to irradiated exhaust or high NO2/low NO, there was a
rise in lung propyl hydroxylase, an enzyme involved in collagen synthesis In addition, a
correlation was found between lung weight and hydroxyproline content in animals exposed to
the NOX atmospheres
Lung morphology of the dogs was evaluated by Hyde et al (1978) 32 to 36 mo after
68 mo of exposure In the high NO2/low NO group, there were increases in total lung
capacity and lung volume, and decreases in the surface density of the alveoli and the
volumetric density of parenchymal tissue Alveoli were enlarged in both the high NO2 and
high NO groups In the high NO2, but not the high NO group, there was cilia loss and
hyperplasia of nonciliated bronchiolar cells In the high NO group, there were lesions in the
interalveolar pores In the most severely affected dogs in the high NO2 group,
morphological changes considered to be analogous to centnlobular emphysema were present
(see Section 13 2 2 4 discussing NO2-induced emphysema in experimental animals) Because
13-183
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these morphologic measurements were made after a 2 5- to 3-year holding penod in clean
air, it cannot be determined with certainty whether these disease processes abated or
progressed during this tune However, indications were that the long-term exposures
produced persistent damage that was indeed progressive even after exposures ended
Another complex mixture involving NO2 is diesel-engine exhaust Like gasoline-engine
exhaust, this contains a number of gases and particles Numerous toxicologic studies have
been performed with acute, subchromc, and chronic exposure protocols (U S Environmental
Protection Agency, 1991) In acute exposures, toxic effects appear to be associated with
high concentrations of CO, NO2, and various other gases On the other hand, comparison of
responses in laboratory animals repeatedly exposed to whole diesel exhaust or filtered
exhaust containing no particles appears to demonstrate that the particles are the principal
etiologic agent of noncancerous health effects resulting from exposure (U S Environmental
Protection Agency, 1991) Whether these particles act additively or synergistically with the
gases in the exhaust mixture cannot, however, be determined from the designs of the
available studies Thus, the diesel studies do not provide additional information concerning
the toxicity of NO2 over and above that which is already available in the data base
Summary
Exposures to mixtures containing NO2 are quite common and provide a basis for
toxicological interactions whereby combinations of pollutants may behave differently than
would be expected from consideration of the action of each constituent separately The
largest data base exists for the combination of NO2 and O3 Morphologic response to
exposure to this mixture is generally due to O3 (Freeman et al, 1974a, Yokoyama et al,
1980), but biochemical effects may involve synergism (Yokoyama et al, 1980, Ichinose and
Sagai, 1989, Sagai and Ichinose, 1991, Mustafa et al , 1984, Schlesinger et al , 1990)
Reactions of host defenses, specifically antibacterial activity, may be additive or synergistic
(Goldstein et al, 1974, Graham et al, 1987, Ehrkch et al, 1977) Mixtures of NO2 and
acid sulfates result in additive to synergistic effects (Last, 1989, Last et al, 1983, Last and
Warren, 1987, Schlesinger and Gearhart, 1987, Schlesinger, 1987a, Schlesinger et al ,
1987a). Although many studies examined responses to simple mixtures of NO2 with one
other material, the atmosphere in most environments is a complex mix of more than two
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pollutants The effects of complex mixtures have been examined to some extent, however,
the exact role played by NO2 in the observed responses is not always clear
13.4 NITRIC OXIDE
The toxicologic data base for NO is not extensive, except for those studies examining
its interaction with blood One problem is that it is often difficult to obtain pure NO in air
without some contamination with NO2 In recent years, much research has increased the
understanding of the role of endogenous NO as a mediator of vascular tone, macrophage
cytotoxicity of microorganisms and tumors, and platelet disaggregation (Moncada et al ,
1991) However, this research and findings are not directly related to NO as an air
pollutant
Little is actually known about NO absorption in the respiratory tract, and nothing is
known on its subsequent intrapuhnonary distribution Because NO is less water soluble and
less reactive than NO2, it follows that its absorption from inhaled air should be less
Yoshida et al (1981) found that < 10% of the NO "inhaled" by isolated, perfused lungs of
rabbits was absorbed On the other hand, absorption in normal breathing humans in vivo
was 85 to 92% for NO concentrations ranging from 400 to 6,100 /tg/m3 (0 33 to 5 0 ppm)
(Wagner, 1970, Yoshida and Kasama, 1987), values foi NO2 were 81 to 90% (Wagner,
1970) Absorption of NO with exercise was 91 to 93% in humans (Wagner, 1970) Yoshida
et al (1980a) found the percentage of NO absorbed in rats acutely exposed to 169,300 /ig/m3
(138 ppm), 331,300 ^g/m3 (270 ppm), and 1,079,800 jug/m3 (880 ppm) to be 90%, 60%,
and 20 %, respectively The lower absorption at the two highest concentrations was ascribed
to an exposure-induced decrease in ventilation Vaughan et al (1969) exposed dogs to auto
exhaust mixtures and found that 73 % of the constituent NO was removed when the mixture
was passed in through the nose and out through a tracheostomy tube, this compared to 90%
removal for NO2 Thus, respiratory tract absorption of NO has some similarities to that of
NO2 in spite of solubility differences The lower solubility of NO may, however, result in
greater amounts reaching the pulmonary region, where it then diffuses into blood and reacts
with hemoglobin (Yoshida and Kasama, 1987) In fact, exposures in vivo do seem to
13-185
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indicate that NO has a faster rate of diffusion through tissue than does NO2 (Chiodi and
Mohler, 1985)
High exposure levels of NO are apparently needed to be lethal Pflesser (1935)
reported that mice exposed to 380,400 jug/m3 (310 ppm) NO for 8 h showed no mortality,
whereas 50% mortality was seen during similar exposures to 392,600 jwg/m (320 ppm),
however, possible NO2 contamination was not accounted for Greenbaum et al (1967)
reported that dogs exposed to 2% NO (24,540 mg/m3, 20,000 ppm) for 7 to 50 mm all died
within 15 mm after exposures ended, a single dog exposed to 0 5% (5,000 ppm) NO also
died Death was due to pulmonary edema No increase in death rate over control was found
in mice exposed to 12,270 /*g/m3 (10 ppm) NO for 6 5 mo (Oda et al , 1976), or to
2,940 jttg/m3 (2 4 ppm) NO for their lifetime (23 to 29 mo) (Oda et al , 1980b)
The few studies that have examined histologic response to nonlethal levels of NO are
outlined in Table 13-24 With chronic exposure, the morphologic changes seen are similar
to those discussed in the section on the morphological effects of NO2, except that the NO
levels needed to produce them are higher In terms of pulmonary effects with high level
acute exposure, NO is estimated to be approximately 30 tunes less toxic than NO2 (Stavert
and Lehnert, 1990) Additionally, Hugod (1979) noted that the absence of NO-mduced
alterations in the alveolar epithelium suggested that the observed responses occurred after
absorption of NO, that is, they were not due to direct action of deposited NO Perhaps
higher exposure concentrations of NO are needed for direct toxic action (e g , results of Holt
et al., 1979) Some of the effects seen by Oda et al (1976) with 12,270 /*g/m3 (10 ppm)
NO may be due to the presence of 1,880 to 2,820 /*g/m3 (1 0 to 1 5 ppm) NO2 in the
exposure atmosphere
Data concerning the physiological effects of inhaled NO are sparse Murphy (1964)
found no changes in respiratory function of guinea pigs exposed for 4 h to NO at 19,600 or
3
61,300 jtg/m (16 or 50 ppm) Yoshida et al (1980b) reported that guinea pigs exposed
twice per week (30 mm each) for 7 weeks to 5,900 /*g/m (5 02 ppm) NO and challenged
during exposure to aerosolized albumin exhibited dyspneic breathing patterns and an
increased responsiveness to acetylcholine These results were not substantially different from
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TABLE 13-24. EFFECT OF NITRIC OXIDE ON RESPIRATORY TRACT MORPHOLOGY3
oo
NO Concentration
jug/m ppm
2,460 2 0
(NO2 = 0 08 ppm)b
2,950 2 4
(NO2 = 0 01-0 04 ppm)
6,150 50
(NO2 = <0 1 ppm)
12,300 10
12,300 10
(NO2 = 1-1 5 ppm)
Exposure Gender
Continuous, NS
6 weeks
Continuous, F
23-29 mo
Continuous, M
14 days
2 h/day, F
5 days/week, up
to 30 weeks
Continuous, F
6 5 mo
Species
Age (Strain)
8 weeks Rat
(Wistar)
12 weeks Mouse
(JCL-ICR)
NS Rabbit
(Danish)
6-8 Mouse
weeks (BALB/c)
12 weeks Mouse
(JCL-ICR)
Effects
Slight emphysema-like alterations of
alveoli
No difference from control
Edema, thickening of alveolo-
capillary membrane due to fluid in
interstitial space, fluid-filled
vacuoles seen in artenolar
endothehal cells and at junctions of
endothelial cells, no changes in
alveolar epithelium, no
inflammation
Enlarged airspaces in lung
periphery, paraseptal emphysema,
some hemorrhage, some congestion
in alveolar septa, increased
concentration of goblet cells in
bronchi
Bronchiolar epithelial hyperplasia,
hypererma, congestion, enlargement
of alveolar septum, increase in ratio
of lung to body weight
References
Azoulayetal (1977)
Odaetal (1980a)
Hugod (1979)
Holtetal (1979)
Odaetal (1976)
aNS = Not stated
F = Female
M = Male
Represents reported NC>2 levels measured during exposure
-------
those in guinea pigs exposed to 9,400 jug/m3 (5 0 ppm) NO2 (details of the study are
reported in Section 13 2 2 3 addressing NO2 exposure-related effects on pulmonary
function)
The effects of NO on defense function of the lungs has been examined in two studies
o
Holt et al. (1979) examined immunological end points in mice exposed to 12,270 jwg/m
(10 ppm) NO, 2 h/day, 5 days/week for up to 30 weeks Leukocytosis was evident by
5 weeks of exposure, and a decrease in mean hemoglobin content of RBCs was found by
30 weeks A decrease in RBC count at Week 15 was not found at 30 weeks An
enhancement of the humoral immune response to SRBCs was seen at 10 weeks, but this was
not evident at the end of the exposure series Spleen cell response to phytohemagglutimn
was decreased after 15 weeks of exposure, but mitogenesis then recovered and became
greater than control The ability of spleen cells to mount a graft versus host reaction was
stimulated by 20 weeks of exposure, but was suppressed by 26 weeks Finally, the ability of
mice to reject virus-induced tumors was assessed, only 40% of the NO-exposed animals
survived tumor challenge, compared with 66% for control animals This study suggests that
NO exposure may have affected the immunologic competence of exposed animals
Effects of NO on bacterial defenses were examined by Azoulay et al (1981) Male and
female mice were exposed continuously to 2,450 /*g/m3 (2 0 ppm) NO for 6 h to 4 weeks, to
assess the effect on resistance to infection induced by a bacterial aerosol (Pasteurella
multoada) administered after each NO exposure Although there appeared to be somewhat
of an increase in bacterial-induced mortality in each group of females exposed to NO for at
least 1 week, there was no statistically significant difference for either sex Likewise, each
group of females exposed to NO for at least 1 week showed a slight decrease in mean
survival tune, but this change was not statistically significant, nor was there any observable
difference in males exposed to NO When the data for those groups exposed for 1 to
4 weeks were combined, NO-exposed females showed a significant increase in percentage
mortality and a significant decrease in survival tune, this was not seen for males Thus, this
study suggests some gender-related difference in response, at least to the one level of NO
examined
One possible mechanism of toxic action of NO is lipid peroxidation The GSH
transferase system serves to protect vital molecules from peroxidative damage Thus,
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changes in constituents of this system may serve as a marker of effects from inhaled NO
However, mice exposed to 12,300 to 25,800 /xg/m3 (10 to 21 ppm) NO, 3 h/day for 7 days
showed no change in lung levels of reduced GSH, a cofactor for GSH peroxidase (Watanabe
et al, 1980)
There is some evidence that NO may alter the activity of other enzymes A number of
in vitro studies (Arnold et al, 1977, Braughler, 1982, Katsuki et al , 1977) have indicated
that NO may affect guanylate cyclase, the enzyme that catalyzes the formation of cyclic-
guanasine monophosphate and guanosine tnphosphate They have shown, based upon
exposure of purified enzymes or tissue minces from various organs, that NO increases
enzyme activity in a concentration-dependent fashion, and that the activation is reversible
when NO is removed from the preparation Although variable degrees of activation were
seen in different tissues, lung tissue showed one of the highest degrees of activation It is,
however, not known whether NO would alter guanylate cyclase activity with in vivo
exposure
The bulk of the toxicologic data base for NO biochemistry concerns its reaction with
hemoglobin Inhaled NO that enters the bloodstream through the lungs binds to hemoglobin,
producing nitrosylhemoglobin (Oda et al , 1975, 1980a, 1980b, Case et al, 1979, Nakajima
et al, 1980) This may, in fact, be a major mechanism of action, and in vitro studies have
suggested that NO may severely reduce the ability of KBCs to carry O2 These studies have
shown that the affinity of hemoglobin for NO is very high, much higher even than that for
O2 (Gibson and Roughton, 1957; Moore and Gidson, 1976) In addition, in vitro
measurements of O2-dissociation curves for partially NO-liganded human hemoglobin have
shown that NO binding tends to reduce dissociation of bound O2 on the molecule (Kon et al ,
1977) Finally, nitrosylhemoglobin is easily and rapidly oxidized to methemoglobin in the
presence of O2 (Chiodi and Mohler, 1985, Kon et al., 1977), further reducing the ability of
KBCs to transport O2
Following in vivo exposures, a linear relationship was found between the exposure
concentration of NO (24,500 to 98,200 jwg/m3, 20 to 80 ppm) for 1 h in mice and blood
content of nitrosylhemoglobin, however, levels of methemoglobin were found to increase
exponentially with NO concentration, resulting in greater blood levels of methemoglobin than
^
nitrosylhemoglobin (Oda et al, 1980b) After exposure of mice to 49,100 jtg/m (40 ppm)
13-189
-------
for 1 h, concentrations of both methemoglobin and mtrosylhemoglobin decreased rapidly,
with half-times of only a few minutes (Oda et al, 1980b) Thus, the steady-state
concentration of mtrosylhemoglobin during NO exposure would be fairly low, whereas that
for methemoglobin would be somewhat higher (Maeda et al, 1987)
Studies of animals exposed to NO in vivo have shown that the amount of
mtrosylhemoglobin in blood was much less than would be expected from in vitro exposure
data (Oda et al, 1980b, 1975) Lifetime (23 to 29 mo) exposures of mice to 2,940 jug/m3
(2.4 ppm) NO resulted in the blood content of nitrosylhemoglobin remaining relatively steady
at 0.01%, whereas the maximum amount of methemoglobin was 03% (Oda et al , 1980a)
Mice exposed to 12,300 jttg/m3 (10 ppm) NO for 6 5 mo showed mtrosylhemoglobin at
0.13% and methemoglobin at 0 2% (Oda et al, 1976) These results suggest that a steady-
state concentration of mememoglobin may be reached with exposures to different
concentrations Furthermore, although the results of various studies have shown that the
final product of NO reaction with hemoglobin is methemoglobin, with some persistent
nitrosylhemoglobin, this effect of NO is not generally lethal because of a number of factors,
these include the conversion of inhaled NO to NO2 in the airways, the rapid oxidation of
nitrosylhemoglobin into methemoglobin, and the subsequent reduction of methemoglobin into
ferrous hemoglobin by methemoglobin reductase, an enzyme present in RBCs (Kon et al ,
1980, Maeda et al, 1984b, 1987) As long as the activity of methemoglobin reductase is
maintained, the conversion of nitrosylhemoglobin to methemoglobin should mitigate any
potentially toxic effect on hemoglobin due to NO inhalation (Kon et al, 1980) In long-term
exposure studies, Oda et al (1976, 1980a) exposed mice to 4,512 or 18,800 /xg/m3 (2 4 or
10 ppm) NO and after examination of organs sensitive to O2 depletion (e g , brain and
heart), found no evidence of hypoxic damage, which would have been expected if
methemoglobin levels were substantially increased
Azoulay et al (1977) exposed rats to 2,450 jug/m (2 0 ppm) NO continuously for
6 weeks to examine various hematologic parameters, including blood-O2 affinity
No exposure-related changes were found in hemoglobin content, hematocnt, RBC count, red
cell glucose metabolism, or in the oxyhemoglobin dissociation curve In addition, no
methemoglobin was detected in either exposed or control animals This showed that low-
level NO exposure did not alter the blood-O2 affinity On the other hand, the same
13-190
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investigation reported that in vitro studies had shown that blood-O2 transport was altered by
3
high levels of NO (> 12,300 jWg/m , 10 ppm) in both human and rat blood
In addition to interaction with hemoglobin, exposure to NO may alter other aspects of
blood Case et al (1979) exposed mice to 11,070 ^g/m3 (9 0 ppm) NO for 16 h and found a
decrease in the level of iron transfemn Mice exposed to 12,300 jwg/m (10 ppm) NO for
6 5 mo showed increased WBC counts and an increase in the ratio of PMNs to lymphocytes
(Oda et al , 1976) These investigators noted that 11 % of the RBCs obtained from NO-
exposed mice contained Heinz bodies, whereas the control group showed none Coupled
with an increase in spleen weight and bilirubin, the investigators suggested that this indicated
that NO facilitated the destruction of RBCs
3
A slight increase in RBC hemolysis was seen in mice exposed to 2,940 /ig/m
(2 4 ppm) NO for their lifetime (Oda et al , 1980a) Rat RBCs exposed to NO in vitro,
showed oxidative cross-linking between cell membrane proteins and hemoglobin (Maeda
et al , 1984a), an alteration that could change the cells' Theological properties However, in
an in vivo exposure study, no cross-linking of membrane proteins was detected in rats
exposed to 30 7 to 254 4 mg/m3 (25 to 200 ppm) NO for 1 h (Maeda et al , 1987), the
investigators suggested that this may have been due to rapid repair mechanisms operating
in vivo
The pH of blood has been shown to be reduced by NO, but only with very high
exposure levels (e g , 0 5 to 2 0%, 5,000 to 20,000 ppm) (ToothiU, 1967, Greenbaum et al ,
•7
1967) Rats exposed to 2,450 /ug/m (2 0 ppm) NO continuously for 6 weeks showed no
change in blood pH (Azoulay et al , 1977)
An examination of the mutagemcity of NO was performed by Arroyo et al (1992)
*}
Salmonella typhimunum TA1535 was exposed for 30 mm to 6,150 to 110,700 jug/m (5 to
90 ppm) NO, and mutagemc potential was assessed using a modified Ames reversion assay
The number of revertant colonies increased roughly in proportion to the square of the NO
concentration up to 24,600 pg/m (20 ppm), and then remained relatively constant or slightly
decreased at > 24, 600 /*g/m It was also noted that the observed mutagemcity required that
the bacteria were actively dividing at the tune of exposure to NO These results suggested
that NO can act as a direct-acting mutagen
13-191
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A few studies have examined the response to inhalation of mixtures of NO plus one
other component Watanabe et al (1980) exposed mice to NO (12,300 Mg/m3, 10 ppm) plus
-------
have various biological functions as an inter- and intracellular messenger (Curran et al ,
1991)
13.5 NITRIC ACID AND NITRATES
13.5.1 Nitric Acid
There are only a few toxicologic studies of HNO3, which exists in ambient air
generally as a highly water soluble vapor In an early study, Diggle and Gage (1954) noted
-3
that a single exposure to HNO3 vapor at 63,000 jwg/m (25 ppm) had no "obvious effect on
rats", exposure duration and end points examined were unspecified
More recent studies have examined the histological response to instilled HNO3 (usually
1 %), a procedure used in developing models of bronchiolitis obliterans in various animals,
namely the dog, rabbit, and rat (Totten and Moran, 1961, Greenberg et al, 1971, Mink
et al , 1984) The major changes noted were degeneration of alveolar Type 2 cells and
alveolar cell hyperplasia In a somewhat similar study, Peters and Hyatt (1986) delivered
1 % HNO3 into a catheter positioned in the main bronchi of the dog, however, in this case,
the acid was delivered via nebubzation, alternately (every other day) as either a coarse spray
or as a fine mist, for 2 h/day for 4 weeks Pulmonary function testing after 4 weeks of
exposure indicated decreases in expiratory flow rate, dynamic compliance, total lung
capacity, and vital capacity, and increases in pulmonary resistance, closing capacity, the ratio
of functional residual capacity to total lung capacity, and phase IH of the single breath
nitrogen washout curve Histologically, there was widespread chronic inflammation of
conducting airways, especially medium and small ones, penbronchiolar fibrosis, focal
hemorrhage, edema, and hyperplasia of goblet cells in the trachea and bronchi
Gardiner and Schanker (1976) examined the effect of HNO3-induced damage on drug
absorption from the lungs of rats Instillation of 1 % HNO3 produced bronchioktis and
alveolitis and increased the rate of pulmonary absorption of various drugs up to 1 6 tunes
control values This change was ascribed to an increase in the permeability of the
alveolocapillary barrier
Only two studies were designed specifically to examine the pulmonary response to pure
HNO3 vapor Abraham et al (1982) exposed both normal sheep and allergic sheep (i e ,
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those having airway responses similar to that occurring in humans with allergic airway
disease) for 4 h to 4, 120 ^g/m (1 6 ppm) HNO3 vapor The exposure, which was
performed using a "head-only" chamber, resulted in a decrease in specific pulmonary flow
resistance, compared to preexposure control values, in both groups of sheep, this indicated
the absence of any bronchoconstnction To assess airway reactivity, pulmonary resistance
was also measured after challenge with a bronchoconstnctor aerosol (carbachol) Allergic
sheep showed increased reactivity, both immediately and 24 h after HNO3 exposure
Although there was no significant change in reactivity in the normal groups as a whole, two
of the animals showed an increase in reactivity to carbachol after HNO3 exposure, according
to the investigators, this suggested that some individuals in the normal population may be
more sensitive than others
Nadziejko et al (1992) exposed rats for 4 h to HNO3 at either 644 /*g/m3 (0 25 ppm)
3 3
or 2,575 jwg/m (1 mg/m ) Exposures were nose-only, and bronchopulmonary lavage was
performed 18 h after the exposure ended Exposure to HNO3 had no effect on total number
of cells recovered, numbers of macrophages recovered, or protein content in lavage fluid
Exposure to the lower acid level did result in a reduction in respiratory burst activity of
macrophages (which was not similarly measured at the higher concentration), and exposure
to the higher concentration resulted in an increase in the lavage fluid elastase inhibitory
capacity
13.5.2 Nitrates
The toxicologic data base for inhaled nitrates is quite sparse Ehrlich (1979) examined
the effect of nitrates on resistance to respiratory infection Mice were exposed for 3 h to
various nitrate salts at maximal concentrations as follows lead nitrate, 2,000
<> -2 -a
calcium nitrate, 2,800 /*g/m , NaNO3, 3,100 jug/m , potassium nitrate, 4,300 jiig/m ,
3 3
ammonium nitrate (NH4NO3), 4,500 jwg/m , and zinc nitrate (Zn[NO3]2), 1,250 ^g/m
Following exposure, the animals were challenged with a bacterial aerosol, and mortality
determined after 14 days Only the Zn(NO3)2 exposure resulted in any significant mortality
increase, the extent of which seemed to be concentration related, the highest concentration
increased mortality by =20% However, since the response was similar to that seen with
zinc sulfate, the investigator ascribed the effect to the zinc ion, rather than to the nitrate
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Busch et al (1986) exposed rats and guinea pigs with either normal lungs or lungs with
-5
elastase-induced emphysema to 1 mg/m NH4NO3, 6 h/day, 5 days/week for 4 weeks
Using both LM and electron microscopy, the investigators concluded that there were no
significant effects of exposure on lung structure due to the nitrate exposure
Charles and Menzel (1975) examined the effects oi nitrate on the release of histamine
by guinea pig lung fragments, response to some pollutants may be a function of their ability
to elicit histamine release Lung fragments were incubated for 30 mm with 20 to 200 mM
NH4NO3 Histamine was released in proportion to the concentration of salt present
However, the response was not totally due to nitrate, ammonium ion was also a possible
contributor
Summary
Inhalation studies with HNO3 are limited and no conclusions can be reached Likewise,
the toxicologic data base for inhaled nitrates is sparse, with no conclusions possible
13.6 SUMMARY
The vast majonty of animal toxicology studies of NOX are on NO2, which apparently is
more toxic than other nitrogen species (NO, HNO3, and particulate nitrates) that commonly
occur in the ambient air However, direct comparative studies of NOX species are rare,
more new information could challenge assumptions of relative potency Given the current
weight of evidence, this summary will only address NO?, alone and in mixtures Ambient
and indoor levels of NO2 are ordinarily below 1,880 /4g/m (1 0 ppm) acutely and 94 jwg/m3
(0 05 ppm) chronically, making high-concentration animal studies difficult to interpret for
assessment of ambient air Thus, with rare exception, only studies below 9,400 /*g/m3
(5 0 ppm) are summarized here Although a wide array of systemic effects have been
observed after NO2 exposure, their interpretation for risk assessment is quite difficult and
unclear (with the^possible exception of immune system effects) compared to respiratory tract
effects Thus, for more discussion of systemic effects, j>ee the summaries within the chapter
This summary is organized to focus attention on key issues pertaining to respiratory tract
effects They include animal-to-human extrapolation, mechanisms of effects, effects on
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host defenses, relative influences of concentration and time (duration) of exposure and
exposure patterns, impact of exposure duration on effects, and effects of pollutant mixtures
For summaries of each end point, see the summaries within the main text
13.6.1 Animal-to-Human Dosimetric Extrapolation Estimates
Qualitatively, most experts would agree that a class of effects of NO2 observed m
several animal species could also occur in humans, if exposures were adequate to induce the
effect Such a qualitative extrapolation is founded on the interspecies commonality in
molecular mechanisms of toxicity and in targets of toxicity For example, small laboratory
animals, nonhuman primates, and humans all have AMs with susceptible membrane
components However, quantitative extrapolation requires quantitative knowledge of
interspecies commonalities and differences in dosimetry and species sensitivity Although
some information is available on these two elements, it is not yet sufficient for quantitative
extrapolation Nevertheless, the state of knowledge facilitates the interpretation of animal
studies in terms of potential human risks
Total NO2 respiratory tract uptake in humans depends on the experimental methods
used, the health status of the subjects, and breathing state (Wagner, 1970, Bauer et al,
1986). Total respiratory tract uptake ranged from 81 to 90% m normally breathing healthy
subjects and increased to 91 to 92% during maximum respiration (Wagner, 1970) The
average total uptake in resting asthmatics was 72 %, and as respiration increased, the percent
total uptake of NO2 increased to 87% (Bauer et al , 1986) Roughly similar findings were
made in dogs (Kleinman and Mautz, 1991) At rest, total respiratory tract uptake was 78%,
during exercise, it was 94% Exercise obviously increases the total uptake of NO2, but it
also alters the regional distribution of dose Generally, increased ventilation decreases the
percent uptake in the upper respiratory tract and increases the percent uptake m the total and
lower respiratory tract Theoretical models based on O3 predict that the increased dose to
the lower respiratory tract is predominantly to the pulmonary region (Miller et al , 1985)
A wider range of lower respiratory tract uptake values has been observed m animals as
a result of differences in species and methods (Postlethwait and Mustafa, 1981, 1989,
Kleinman and Mautz, 1991) However, mathematical modeling of lower respiratory tract
uptakes in humans and animals (rats, guinea pigs, and rabbits) revealed that the greatest dose
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is delivered to the centnacinar region (i e , junction between the conducting airways and the
gas-exchange region) in all these species (Miller et al , 1982, Overton, 1984) This is the
site where NO2-induced lesions are observed morphologically in animal species, lending
credence to the mathematical model Once deposited and bound, NO2 reacts with fluids and
tissues, forming other products that can be transported systemically Various theories and
experimental findings are available suggesting that HONO and HNO3 can be produced in the
lung from NO2 exposure (Goldstein et al , 1977b) or that nitrite is produced in the lungs,
enters the bloodstreams, and reacts with hemoglobin to form nitrate and methemoglobin
(Postlethwait and Mustafa, 1981, 1989, Saul and Archer, 1983)
13.6.2 Biochemical and Cellular Mechanisms
Acute exposure to NO2 at or below 9,400 jwg/m (f» ppm) can oxidize unsaturated fatty
acids in cell membranes as well as functional groups of proteins (either soluble proteins in
the cell, such as enzymes, or structural proteins, such as components of cell membranes),
producing cell injury or death and the toxic symptoms associated with NO2 inhalation
(Menzel, 1976 Freeman and Mudd, 1981) Such a proposed mechanism of action is
supported by data showing an initial increase in lipid peroxidation products and some
protective lung antioxidant enzymes after NO2 exposure (Sagai et al, 1984), as well as an
increased susceptibility to NO2 in animals deficient in vitamins C and E (Selgrade et al ,
1981, Sevaman et al , 1982) The direct cytotoxic effect of NO2 on epithelial cell
membranes may be the fundamental mechanism of edemagenesis in response to NO2
exposure, whereas the direct cytotoxicity of NO2 to membranes of AMs could well be the
mechanism underlying increased infectivity of bacteria and viruses in the lungs of exposed
animals A large number of studies have suggested that various enzymes in the lung,
including GSH peroxidase, SOD, and catalase, may also serve to defend the lung against
oxidant attack One may speculate that were there to be a threshold level for NO2 toxicity to
the lung, it would be that concentration of NO2 that was able to overwhelm these endogenous
defense systems of the lung
The biochemical study using the lowest concentration of NO2 was conducted by
Sagai et al (1984), who reported that 9 and 18 mo of exposure to >75 /ig/m (0 04 ppm)
NO2 increased ethane exhalation (exhaled ethane is an in vivo indicator of lipid peroxidation)
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*3
in rats; at 752 /ng/m (0 4 ppm), they observed this response at 6 mo Although this chronic
study showed increases in lipid peroxidation with increasing concentration and duration of
exposure, a shorter term (4-mo) exposure revealed that ethane exhalation increased after
1 week of exposure, decreased to control levels by 4 weeks, and then rose again (Ichinose
and Sagai, 1982) Sagai et al (1984) also observed that lipid peroxidation had an inverse
relationship with changes in lung antioxidant metabolism
13.6.3 Effects on Host Defenses
Host defenses are a broad category of functions, encompassing defenses against
infectious (bacterial and viral) disease, neoplastic disease, and inhaled paniculate matter
The immune system, a major component of host defense, is compartmentalized
physiologically (e g , pulmonary and systemic immune system) Effects of NO2 on the
respiratory tract defense mechanisms will be presented first, followed by a discussion of
systemic effects
Studies of respiratory tract host defenses have shown that NO2 enhances susceptibility
to bacterial and viral disease, probably through effects on AMs and possible through changes
in the immune system and other defense mechanisms not yet adequately investigated The
mucociliary escalator, an important component of defenses, is not functionally affected in
rabbits exposed for 14 days (2 h/day) to 1,880 jug/m3 (1 ppm) or for 2 h to 18,800 /*g/m3
(10 ppm) NO2 (Schlesinger et al, 1987a,b), although there are numerous reports of
structural changes in the ciliated epithelium at levels below 9,400 jwg/m (5 ppm)
(Rombout et al, 1986, Stephens et al, 1972, Yamamoto and Takahashi, 1984)
3
Various acute and subchromc exposure regimens, generally > 1,889 /xg/m (1 0 ppm)
NO2, increase the number of AMs in the lung (Mochitate et al, 1992, Gregory et al, 1982,
Rombout et al, 1986) Structure, function, and metabolic activity of AMs are also affected
by NO2 exposure Pulmonary bactericidal activity, often interpreted as representative of AM
activity, is decreased in mice by a 17-h exposure to > 4,320 /jg/m3 (2 3 ppm) NO2
(Goldstein et al , 1973), however, effects on AM phagocytosis are complex For example,
3
exposure to 560 /-tg/m (0 3 ppm) NO2, 2 h/day for 13 days, initially decreased AM
phagocytosis in rabbits, whereas AMs exposed to 1,880 /ig/m (1 0 ppm) showed an initial
increase in phagocytosis (Schlesinger, 1987b) However, exposure of rabbits to these NO2
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concentrations for 2 h/day for 14 days increased alveolar clearance, which also represents
AM function (Schlesinger and Gearhart, 1987, Vollmuth et al , 1986) Acute NO2 exposure
decreases superoxide amon radical production by AMs, and longer exposures cause
morphological changes in AM membranes, metabolic changes, an increase in AM numbers, a
decreased responsiveness to migration inhibitory factor, and a decrease in AM random
mobility (Mochitate et al, 1986, Aranyi et al, 1976, Greene and Schneider, 1978, Amoruso
et al, 1981, Schlesinger, 1987b)
One of the most widely applied methods to investigate effects on defenses in
experimental animals is the infectivity model Using this model, experimental animals are
exposed to NO2 and then are challenged with viable bacteria or viruses, microbial-induced
mortality is measured The mortality reflects the net impairment of host defense
mechanisms The sensitivity of this model to detect NO2-induced changes in host
susceptibility to infectious disease is influenced significantly by the microbial species, the
animal species, and exposure regimen After acute exposure, the sensitivity ranking was
mice > hamsters > monkeys (Ehrlich, 1975) In mice exposed for 2 to 3 h, the lowest
3
concentration that enhanced mortality was 6,580 /*g/m (3 5 ppm) using Klebsiella
pneumonia and 3,760 pg/m (2 0 ppm) using Streptococcus sp (Ehrlich, 1975, Purvis and
•3
Ehrlich, 1963, Ehrlich et al , 1977) Long-term, intermittent exposure of mice to 940 /jg/m
(0 5 ppm) has been reported to decrease resistance to bacterial infections within 6 mo,
however, continuous exposure decreases resistance to bacterial infections within 3 mo
(Ehrlich and Henry, 1968) Extensive studies of C x T relationships observed in the
infectivity model are summarized in Section 13 6 4
Few studies with other microbes have been conducted, but they show that repeated
exposures can increase susceptibility to influenza virus or cytomegalovirus infection in mice
and monkeys (Ito, 1971, Henry et al, 1970, Rose et al 1988, 1989) Acute, high-
concentration exposure (9,400 /xg/m , 5 0 ppm) of mice increases the incidence and seventy
of Mycoplasma pulmoms lesions (Parker et al, 1989)
The pulmonary immune system is rarely investigated, and NO2 reports with modern
methods and appropriate experimental designs and analyses have not appeared Systemic
immune responses to antigens delivered via the respiratory tract are altered by NO2
For example, monkeys exposed to 1,880 j^g/m3 (1 0 ppm) NO2 for 16 mo or 9,400
13-199
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(5.0 ppm) for 6 mo and immunized with influenza experienced alterations in circulating
antibody titers (Fenters et al, 1973, Ehrlich and Fenters, 1973) Several other investigations
show that NO2 can alter systemic humoral and cell-mediated immunity Using examples
from studies at lower concentrations, a 7-week intermittent exposure to 470 /xg/m
(0 25 ppm) altered percentages of splenic T-cell subpopulations in mice (Richter and Damji,
o
1988, 1990), a 4-week exposure to 752 /ig/m (0 4 ppm) decreased splenic primary PFC
responses in mice (Fujunaki et al, 1982), and a 12-mo exposure to 940 /xg/m3 (0 5 ppm)
caused a linear decrease in PHA-induced mitogenesis of mouse spleen cells with NO2
duration (Maigetter et al, 1978) Selgrade et al (1991) found no effects on splenic or
circulating lymphocytic responses to B- or T-cell mitogens after up to 78-weeks of exposure
3 3
of mice to an urban exposure pattern of NO2 (940-/xg/m baseline with 2,820-jwg/m peaks,
0.5 ppm and 1 5 ppm)
13.6.4 Influence of Concentration, Duration, and Exposure Regimen
An extensive body of research on the exposure-response of NO2 indicates the
importance of understanding the complexity of the exposure used in the study Two classes
of studies have contributed to this topic C x T examinations and investigation of other
exposure patterns, both of which are discussed here
Studies directly comparing C x T responses focus on host defenses and, to a limited
degree, lung morphology and are discussed here Other work that allows interpretation about
the progression of effects with exposure duration is summarized Section 13 6 5 Most of the
C X T findings described here are a result of using the mouse streptococcal infectivity
model. In one series of studies, Gardner et al (1977a,b, 1979), Gardner et al (1982), and
Coffin et al (1977) varied the concentration of NO2 from 1,880 to 26,320 jwg/m3 (1 to
14 ppm) and the exposure duration from 0 5 to 7 h so that the C x T product was 7 ppm-h
The bacterial-induced mortality was enhanced more by concentration than by time
-------
constant C X T of 14 ppm-h, a 9 3-h exposure to 2,820 /*g/m3 (1 5 ppm) increased mortality
O
by 10 2%, whereas a 1 0-h exposure to 27,300 /tg/m (14 0 ppm) enhanced mortality by
44 9% Intermittent (7-h/day) and continuous (22- to 24-h/day) exposures were also
compared in the streptococcal mfectivity model (Gardner et al, 1979) Mice were exposed
to 2,820 or 6,580 jwg/m3 (1 5 or 3 5 ppm) NO2 for up to 15 days All exposures increased
mortality At the higher concentration, there were no significant differences between the two
exposure groups However, when the concentration was reduced, a longer duration of
exposure (14 days) was required for intermittent exposure to produce a level of effect
equivalent to continuous exposure
In mice exposed to 940 /ttg/m3 (0 5 ppm) for 6 mo, intermittent exposure (6 or
18 h/day) was equivalent to continuous exposure (24 h/day) in increasing mortality due to
Klebsiella pneumomae However, after 12 mo of exposure, effects were only observed in
the continuous exposure As duration increased to 12 mo, the decreased bacterial clearance
was equivalent in the two intermittent and continuous groups (Ehrkch and Henry, 1968)
Rombout et al (1986) evaluated C x T impacts on lung morphology Rats were
exposed from 1,000 to 20,000 /*g/m3 (0 53 to 10 6 ppm) for up to 28 days Epithelial
changes were more related to exposure concentration than to duration
In ambient air, there is a low baseline concentration of NO2 on which are superimposed
one or two peaks of higher concentrations (primarily Monday through Friday) resulting from
the influence of vehicular traffic The impacts of such patterns have been investigated using
the mfectivity model, pulmonary function, and lung morphology/morphometry Using the
mouse streptococcal mfectivity model, mice were exposed to a series of regimens, with and
without a continuous baseline of 2,820 Atg/m3 (1 5 ppm) and with and without peaks (1, 3 5,
O
or 7 h) of 8,460 jug/m (4 5 ppm), mice were challenged with bacteria immediately or
18 h after the peak exposures, total exposure durations varied between 1 day and 2 weeks
(Graham et al, 1987, Gardner, 1980, Gardner et al , 1982) The baseline exposure alone
caused no effects, whereas peaks alone enhanced mortality when the bacterial challenge was
immediately after the peak exposure With both the baseline and peak exposures, the effect
persisted 18 h after the peak exposure When these data were compared to a 2-week
o
continuous exposure to 2,800 jwg/m (1 5 ppm), there was no apparent trend towards a
•^
C x T relationship In a 1-year exposure study, continuous exposure to 376 /ig/m
13-201
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(0.2 ppm) did not affect streptococcal-induced mortality (Miller et al , 1987) However,
-------
exposures are of more interest Longer term exposures result in lesions in some species with
NO2 concentrations as low as 560 to 940 jwg/m3 (0 3 to 0 5 ppm) (Sherwin and Richters,
1982, Kubota et al , 1987, Yamamoto and Takahashi, 1984; Hayashi et al , 1987) Lesions
are characterized by epithelial damage similar to that described following acute exposure to
<9,400 jwg/m (5 0 ppm), but with the involvement of more proximal airways, the thickness
of the basal lamina and interstitmm can increase Many of these changes, however, will
resolve even with continued exposure, and long-term exposures to levels above about
•^t
3,760 jtg/m (2 0 ppm) are required for more extensive and permanent changes in the lungs
Some effects are relatively persistent, for example, bronchiolitis and collagen deposition,
whereas others, such as epithelial cell hyperplasia, tend to be reversible and limited even
with continued exposure (Kubota et al , 1987, Yamamoto and Takahasi, 1984) In any case,
it seems that for both acute or longer term exposure regimes, the response is more dependent
on concentration than on exposure duration (Rombout et al , 1986)
Results from rats exposed to 940, 1,880, and 3,760 jwg/m3 (0 5, 1 0, and 20 ppm)
NO2 with two daily 1-h peaks at three tunes the baseline concentration provide an example of
subchronic effects of NO2 on pulmonary function and lung morphometry (at the electron
microscopic level) (Stevens et al , 1988, Chang et al , 1986, 1988) Pulmonary function
(decreased respiratory system compliance) was only affected by 6 weeks (and not 1 or
3 weeks) or exposure to the highest concentration, recovery occurred by 3 weeks after
exposure ceased Morphometnc measurements were only made at 6 weeks of exposure
Animals exposed to the 940-jwg/m3, but not the 3,760-/*g/m3, base (plus peaks) had a
thickening of the alveolar interstitium in the proximal alveolar region due to the increase in
total volume of fibroblasts At the lowest concentration of NO2, Type 2 cells were spread
over more surface area and exhibited hypertrophy, the number of AMs increased There
were no effects in the terminal bronchiolar region At the highest NO2 concentration, the
proximal alveolar region had similar changes in Type 2 cells as well as an increase in the
number of Type 1 cells, which were smaller in size, the terminal bronchiolar region had
fewer ciliated cells and alterations in nonciliated (Clara) cells A study by Hayashi et al
(1987) provides an example of chronic effects of rats exposed to 940 jttg/m3 (0 5 ppm)
continuously for up to 19 mo At 4 mo of exposure, Type 2 cell hypertrophy was observed,
13-203
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by 6 mo, the thickness of alveolar septa had increased, and at the end of exposure, there was
fibrous pleural thickening
One of the major factors determining responsiveness within a particular species is age
at time of exposure Compared to adults, neonatal animals seem to be more resistant to
pulmonary function or structural changes caused by NO2, however, interpretation is
confounded by difficulty in exposing animals prior to weaning (Stevens et al , 1988, Chang
et al , 1986, 1988, Mauderly et al , 1987, Azoulay-Dupuis et al , 1983, Kyono and Kawai,
1982) Kyono and Kawai (1982) observed a complex interrelationship between NO2
concentration and age that cannot be interpreted clearly However, for some end points
(e g., air-blood barrier thickness), there was a decrease from 1 to 12 mo of age and an
increase in 21-mo-old rats exposed for 1 mo to >207 jwg/m (Oil ppm)
There is very substantial evidence that long-term exposure of several species of
laboratory animals to high concentrations of NO2 (>9,400 /ttg/m , 5 0 ppm) results in
morphologic lung lesions, which meet the current NHLBI criteria for an animal model of
emphysema (National Institutes of Health, 1985) Those criteria are "An animal model of
emphysema is defined as an abnormal state of the lungs in which there is enlargement of the
airspaces distal to the terminal bronchiole Airspace enlargement should be determined
qualitatively in appropriate specimens and quantitatively by stereologic methods "
Destruction of alveolar walls, an essential additional criterion for human emphysema, has
been reliably reported in lungs from animals in a limited number of studies (Haydon et al ,
1967, Freeman et al , 1972, Hyde et al , 1978) The only one of these studies conducted at
3
NO2 exposure levels of less than 9,400 /tg/m (5 0 ppm) involved coexposure of Beagle dogs
to 1,210 jttg/m3 (0 6 ppm) NO2 and 310 /xg/m3 (0 16 ppm) NO or to 270 |iig/m3 (0 14 ppm)
NO2 with 2,050 jttg/m3 (1 1 ppm) NO (Hyde et al , 1978) Animals were exposed 16 h/day
for 68 mo and then breathed clean air during a 32- to 36-mo postexposure penod The dogs
exposed to the higher level of NO2 had emphysema of the type seen in human lungs
Although the lowest NO2 concentration and the shortest exposure duration that will result in
emphysematous lung lesions cannot be reliably determined from these published studies, the
NO2 concentrations and exposure durations used are far greater than those currently reported
in ambient air
13-204
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The susceptibility to NO2 of animals with experimentally induced emphysema has also
'-}
been examined Nitrogen dioxide (3,760 /xg/m , 2 0 ppm, intermittent, 8 weeks) appeared to
exacerbate emphysema in hamsters using morphological methods, pulmonary function was
not affected (Lafuma et al , 1987) However, elastase-mduced emphysema in rats was not
affected, even though the exposure was high (17,900 /xg/m3, 9 5 ppm, 7 h/day, 5 days/week,
24 mo, Mauderly et al [1990])
The literature provides no evidence that NO2 is a direct-acting carcinogen, but no
classical chronic inhalation bioassays have been reported Other reports that NO2 may act as
a promoter or facilitator of neoplastic disease are fraught with methodological and
interpretative problems
13.6.6 Effects of Pollutant Mixtures
Nitrogen dioxide exists in the ambient air with other pollutants, especially NO and 0$,
which are part of the photochemistry of NOX Animal toxicology research has addressed
complex mixtures (e g , exposure to ambient air containing NO2, automobile exhaust), but
the contnbution of NO2 to the mixtures effect(s) cannot be determined due to the study
designs used Binary mixture studies primarily include O3 and, to a lesser extent, H2SO4
Even with the numerous studies available (descnbed in Section 13 3), interpretation of
interactions is unclear In binary mixtures, NO2 either makes no contnbution, is additive, or
is synergistic, depending on exposure regimen and end point For example, in a 6-mo
•3
exposure study of rats, 4,700 /ttg/m (2 5 ppm) NO2 did not affect the lung lesion induced by
490 jwg/m3 (0 25 ppm) O3 (Freeman et al , 1974a) Mustafa et al (1984) found no effect of
an O3-NO2 mixture on lung DNA or protein content of mice exposed for 1 week, however,
the mixture caused a synergistic increase of oxygen consumption, sulfhydryl metabolism, and
activities of NADP-reducmg enzymes Ehrlich et al (1977) reported that a 3-h exposure to
O3 plus NO2 caused an additive response in the mouse mfectivity model, whereas longer
exposure (4 weeks) appeared to result in synergism When mixtures of NO2 and H2SO4
were examined, Schlesinger and Gearhart (1987) and Schlesinger (1987a) found additive or
synergistic effects on host defense mechanisms, depending on the NO2 concentration and end
point
13-205
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The findings of either additivity or synergism are of concern because of the ubiquitous,
cooccumng nature of O3 and NO2 and the type of effects observed For example, if one of
these pollutants is causing a decrease in host defenses, even an additive response to the other
pollutant would likely increase the incidence or seventy of the effect Precise interpretation
of these findings to ambient scenarios is confounded, however In the ambient air, the
common diurnal pattern is a series of peaks of the photochemical oxidants and their
precursors (e g , NO, NO2, O3), there is some mixing between the peaks Such a
"real-world" pattern has been approximated by Gelzleichter et al (1992b), who examined the
effects of O3 and NO2 in mixture and in sequence Acute exposure of rats to the mixture
caused a synergistic increase in lavage fluid protein, PMNs, and epithehal cells Sequential
exposures generally caused an additive response, with one exception When the sequence
was NO2 first and O3 second, there was an antagonistic response for the number of lavagable
epithelial cells. The body of work with NO2 and NO2-O3 mixtures illustrates the importance
of exposure patterns, so extrapolating laboratory binary mixture study results to ambient
patterns raises concern, but does not allow precise conclusions
13-206
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14. EPIDEMIOLOGY STUDIES
OF OXIDES OF NITROGEN
14.1 INTRODUCTION
This chapter discusses the epidemiological evidence for the effects of nitrogen oxides
(NOX) on human health Major emphasis is placed on discussion of the effects of nitrogen
dioxide (NO2) because it is the NOX compound measured in most epidemiological studies and
because it is the NOX compound currently of greatest concern from a public health
perspective Human health effects associated with exposure to NO2 have been the subject of
several literature reviews since 1970 National Research Council (1971, 1977), World
Health Organization (1977), Samet et al (1987, 1988), and Graham et al (1990) Oxides of
nitrogen have also been reviewed previously by the U S Environmental Protection Agency
(1982a), which presented a comprehensive review of studies conducted up to 1980 This
chapter focuses mainly on studies conducted since 1980, while also utilizing some key
information from earlier literature
Studies discussed in the chapter text are those that provide useful quantitative
information on exposure-effect relationships for health effects associated with ambient air
levels of NO2 likely to be encountered in the United States In addition, some studies that do
not provide quantitative information are briefly discussed in the main text as appropriate to
help elucidate particular points concerning NO2 health effects Both the quantitative studies
discussed in the main text and the additional qualitative studies evaluated here but found to
be of limited usefulness for present criteria development purposes are concisely summarized
in Appendix 14A.
The present chapter is organized as follows First, studies of respiratory symptoms and
illnesses that meet criteria (see Section 14 6 4) for use in a quantitative analysis are
discussed, followed by studies that provide qualitative information The respiratory illness
section is divided into indoor and outdoor subsections Next, studies are described that
examine effects of NO2 exposure on pulmonary function Then, a short discussion of
occupational studies is provided Finally, a quantitative analysis is presented that synthesizes
the available evidence on respiratory illness
14-1
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In U S. Environmental Protection Agency (1982a), a group of studies examining the
relationship between respiratory illness and exposure in the home to gas (cooking fuel)
combustion products, notably including NO2, were evaluated At that time, those studies
inferred the presence of NO2 by the presence of gas combustion emission sources Since
then, new studies and updates of earlier ones have been conducted Many of these studies
provide data on NO2 concentrations and estimates of human exposure
14.2 METHODOLOGICAL CONSIDERATIONS
Studies assessed here were evaluated for several factors noted earlier by Hill (1965) and
U.S Environmental Protection Agency (1982b) to be of importance for interpreting
epidemiological studies Factors considered here in evaluating epidemiological studies of the
health effects of NO2 include (1) exposure measurement errors, (2) misclassification of health
outcomes, (3) adjustments for covanates, (4) selection bias, (5) internal consistency, and
(6) plausibility of observed effects, based on other evidence Because these factors are
common to the evaluation of all epidemiology studies, a brief discussion follows
14.2.1 Measurement Error
Measurement error in exposure is potentially one of the most important methodological
problems in epidemiological studies of NO2 Ideally, personal monitors would be placed on
all subjects for the entire period of a study Even then, some error associated with the
monitoring device itself would remain Such intensive personal monitoring is not feasible
Even personal monitoring, because of the integrated multiday sampling, does not adequately
measure short-term peaks nor long-term chronic exposures Instead, NO2 exposure may be
estimated by source description, personal monitors, in-home monitors, and fixed-site outdoor
monitors. In most of the early studies, gas stove presence was related to health outcomes
without any direct exposure estimates Additionally, a by-product of nitrogen dioxide,
nitrous acid (HONO), may be a factor contributing to observed effects, however, very
limited aerometnc or health effects data are available that examine this possibility
The effect of exposure measurement error on estimation has been studied by several
authors, including Shy et al (1978), Gladen and Rogan (1979), Clark (1982), Stefanski and
14-2
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Carroll (1985), Walker and Blettner (1985), Fuller (1987), Lebret (1987), Schafer (1987),
Whittemore and KeUer (1988), Samet and Utell (1990), and Yoshimura (1990) In general,
exposure measurement error that is independent of the health outcome results in estimated
effects being biased towards the null For example, Whittemore and Keller (1988)
specifically consider the data of Melia et al (1980) as described by Florey et al (1979) and
show that a 20% misclassification rate of the exposure category would result in an
underestimate of the logistic regression coefficient by as much as 50% Also, Stefanski and
Carroll (1985) have shown that even without the independence of error related to outcome,
the bias is towards the null in situations where the probabilities of response are not extremely
close to 0 or 1 The use of the presence of a gas stove as a surrogate for an actual NO2
exposure can introduce measurement misclassification error Clark (1982) studied the effect
of measurement error, which was towards the null in logistic regression when that error was
sampled from a normal or logistic regression, and also found bias towards the null for certain
multiple logistic models
If the observed health effects (see Chapter 13 and Section 14 3) result from peaks
(1-h or less) generated during source use rather than longer term averages, then the use of
estimated averages creates another source of exposure measurement error However,
inadequate data are available to adequately evaluate the relative contnbutions of personal
exposures to peak versus average NO2 values to health effects studied in epidemiology
studies Peak levels in bedrooms and other locations are not as high as in kitchens (see
Chapter 7), and most indoor activity occurs in locations other than the kitchens (see
Chapter 8) Harlos et al (1987) state that NO2 concentrations in the kitchen are different for
each cooking event in a 12- or 24-h penod To improve exposure measurement estimates,
NO2 concentrations during room occupancy are needed The average bedroom NO2
concentration already contains most of the tune-location information by virtue of being a
primary daily location, especially for infants In most homes, peak values may be related to
average values such that reducing peaks reduces the average concentration Average values
may serve as surrogates for the peaks, however, if effects are associated with the peaks, then
the use of averages will increase measurement error
14-3
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14.2.2 Misclassification of Health Outcomes
Misclassification of the health outcome can occur whether the outcome is continuous,
such as a measure of pulmonary function, or dichotomous, such as the presence or absence
of respiratory symptoms Lung function is typically measured with spirometry, a well
standardized (Ferns, 1978) technique The measurement errors of the instruments collecting
the data have also been carefully estimated, and random errors will simply add to the error
variance On the other hand, respiratory symptoms and disease are usually measured by a
questionnaire Responses to symptom questions are typically positively correlated and
depend on the interpretation of the respondent As noted later in the chapter, a specific
respiratory disease is likely to be reflected by reporting of a constellation of symptoms, it is
therefore appropriate to consider aggregate rather than, or as well as, single specific
symptom reports Obviously, questionnaire measurements that depend on recent recall are
better than those based on recall of events that occurred several years in the past
Questionnaires for cough and phlegm production have been standardized, such as the Bntish
Medical Research Council (BMRC) questionnaire (American Thoracic Society, 1969) and
revisions of the BMRC questionnaire (Ferns, 1978, Samet, 1978) These questionnaires and
modifications of them have been used extensively
14.2.3 Adjustments for Covariates
It is common when analyzing a data set to discover that one or more key covanates for
the analysis were not measured Schenker et al (1983) discuss socioeconomic status (SES),
passive smoking, and gender as important covanates in childhood respiratory disease studies
Other covanates include age, humidity, and pollutants, such as particulate matter The
concern is that, had an omitted covanate been measured, then the estimate of the regression
coefficient for a dependent vanable of interest would have been significantly different
Although the problem is faced by many investigators, the literature on the field is relatively
sparse For example, Kupper (1984) shows that high correlations between the variables just
descnbed will result in "unreliable parameter estimates with large variances " Gail (1985)
considered the special case of omitting a balanced covanate from the analysis of a cohort
study and concluded that "In principle, the bias may be either toward of away from zero,
though in typical examples—the bias is toward zero In applications with additive or
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multiplicative regression, there is no bias " Neither paper provided information on how to
attempt to correct for the bias or on approaches for investigating the possible bias in a given
situation
Most studies of respiratory disease and NO2 exposures discussed here measured
important covanates such as age, socioeconomic level of the parents, gender, and parental
smoking habits The estimated effect (regression coefficient of disease on NO2 exposure)
will be overestimated if a missing covanate is positively (or negatively) correlated with both
exposure and outcome The estimated effect will be underestimated if positively correlated
with either exposure or outcome and negatively correlated with the other Ware et al
(1984), for example, found that parents with some college education were more likely to
report respiratory symptoms and were less likely to use a gas stove, leading to an
underestimate of the health effect if education were left out of the analysis
14.2.4 Selection Bias
The possibility of selection bias, although a concern of every study, seems very low for
the epidemiologic studies of NO2 Selection bias would require selection of participants
based on exposure (e g , use of gas stove) and also health outcome Because most
epidemiologic studies of these exposures are population based, there is little possibility of
selection based on health end points Nevertheless, the loss of subjects by attrition associated
with both exposure and health studies must be considered
14.2.5 Internal Consistency
Internal consistency is always a check on the validity of a study, but often the authors
do not report sufficient detail by which to check for such consistency For known risk
factors for respiratory effects, a study should provide evidence of expected associations
(e g , between passive smoking and increased respiratory illness among exposed coworkers
or children or more wheeze in exposed asthmatic children) Furthermore, certain patterns of
age or gender relationships to observed health outcomes should be expected On the other
hand, study results suggesting a significant beneficial effect of NO2 amid other deleterious
effects must be viewed with extreme caution in the absence of independent animal toxicologic
14-5
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or other types of evidence for plausible mechanisms to account for such effects Consistency
between studies provides a further indication of the overall strength of the total data base
14.2.6 Plausibility of Effects
Health outcomes measured should be ones for which there are plausible bases to suspect
that they could be affected by NO2 exposure Two health outcome measures have been most
extensively considered in the NO2 epidemiologic studies reviewed here lung function
measurements and respiratory illness occurrence Human clinical and animal toxicological
studies have not indicated a demonstrated effect on lung function at ambient levels in normal
subjects (see Chapters 13 and 15) However, in contrast, animal toxicological studies in
Chapter 13 have shown that NO2 exposure can impair components of the respiratory host
defense system, resulting in the host being more susceptible to respiratory infection Thus
observed increases in respiratory symptoms and disease among children in epidemiologic
studies of NO2 exposure may be more plausibly hypothesized to be the result of an increase
in respiratory infection
Special attention is accorded to considering all of the above factors in evaluating the
studies reviewed below Those studies that address these factors most appropnately provide
a stronger basis for accepting conclusions based on their results
14.3 STUDIES OF RESPIRATORY ILLNESS
Respiratory illness and the factors determining occurrence and seventy are important
public health concerns This section discusses epidemiological findings relating estimates of
NO2 exposure to respiratory illness This effect is of public health importance because of the
widespread potential for exposure to NO2 and because the occurrence of childhood
respiratory illness is common (Samet et al, 1983, Samet and Utell, 1990) This takes on
added importance because recurrent childhood respiratory illness (independent of NO2) may
be a risk factor for later susceptibility to lung damage (Glezen, 1989, Samet et al, 1983,
Goldetal., 1989)
The NO2 studies used standard respiratory questionnaires that evaluated respiratory
health by asking questions about each child's respiratory disease and symptom experience
14-6
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The reported symptoms and diseases (typically based on parental recall) characterize lower
respiratory morbidity in the cohorts studied A brief discussion of aspects of epidemiology
of lower respiratory morbidity in children provides a background for studies examining NC>2
exposure in relation to lower respiratory health Lower respiratory morbidity in children
typically includes asthma, bronchitis, croup, tracheobronchitis, bronchiolitis, and pneumonia
Asthma and bronchitis are briefly discussed individually below, and the latter four are
discussed together as part of lower respiratory illness syndromes
Asthma is characterized by reversible airway obsl ruction, airway inflammation, and
increased airway responsiveness to stimuli (National Institutes of Health, 1991) Schenker
et al (1983) report a prevalence of approximately 3 5/100 for M D -diagnosed asthma in
children 5 to 9 years of age The Centers for Disease Control (1990) indicate that, for those
less than 20 years of age, the prevalence of asthma increased from approximately 3 5/100
persons in 1980 to 5 0/100 persons in 1987 Asthma patients develop such clinical
symptoms as wheezing and dyspnea after exposure to allergens, environmental irritants, viral
infections, cold air, or exercise Exacerbations of asthma are acute or subacute episodes of
progressively worsening shortness of breath, cough, wheezing, chest tightness, or some
combination of these symptoms Although viral respiratory tract infections are common
asthma taggers, especially in young children (National Institutes of Health, 1991), symptoms
such as wheezing may occur without an infectious cause
Chronic bronchitis is defined in adults as a clinical disorder characterized by excessive
mucous secretion in the bronchial tube with an associated chronic productive cough on most
days for a minimum of 3 mo of the year for not less than 2 successive years (American
Thoracic Society, 1962) The diagnosis can only be made after excluding other disorders
with similar symptoms In contrast, Morgan and Taussig (1984) state that a clear definition
and etiology of chronic bronchitis in childhood have not yet been described They
characterize chronic bronchitis in children as a symptom complex consisting of a chronic or
recurrent "wet" cough, increased phlegm production, and wheezing that may be associated
with evidence of airway inflammation A rational appioach would be to view it as a clinical
presentation of chronic or recurrent airway disease
Symptoms and findings observed in children with physician-diagnosed chronic
bronchitis commonly include recurrent respiratory infections and wheezing, with chronic
14-7
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phlegm production and chronic cough being less prevalent (Burrows and Lebowitz, 1975)
Schenker et al (1983) report a prevalence of approximately 22/100 for M D -diagnosed
bronchitis in children 5 to 9 years of age Respiratory syncytial virus (RSV) and
parainfluenza virus are isolated in cases of bronchitis (Chanock and Parrott, 1965), but
symptoms of bronchitis may occur without an infectious cause
Lower respiratory illnesses are generally classified into one of four clinical syndromes
croup (laryngotracheobronchitis), tracheobronchitis, bronchiolitis, and pneumonia (Glezen
and Denny, 1973, Wright et al, 1989, McConnochie et al, 1988) In a study in Tucson,
the most common diagnosis during the first year of life was bronchiolitis, which accounts for
60% of all lower respiratory illness (Wright et al, 1989) The most common signs and
symptoms associated with lower respiratory illnesses were wet cough (85%), wheeze (77%),
tachypnea (48%), fever (54%), and croupy cough (38%) as reported by Wright et al (1989)
A few infectious agents are presumed to cause the majority of childhood lower respiratory
illness Bacteria are not thought to be common causes of lower respiratory illness in
nonhospitalized infants in the United States (Wright et al, 1989) Seventy-five percent of
the isolated microbes were one of four types RSV, parainfluenza virus types 1 and 3, and
Mycoplasma pneumomae (Glezen and Denny, 1973, McConnochie et al, 1988) Respiratory
syncytial virus is particularly likely to cause lower respiratory illness during the first 2 years
of life. More than half of all illnesses diagnosed as bronchiolitis, for which an agent was
identified, were positive for RSV (Wright et al, 1989) Wright et al (1989) noted that
studies that rely on parental reports of symptoms may underestimate illness Asking parents
about illnesses at the end of the first year of life revealed that one-third of them failed to
report illnesses diagnosed by pediatricians and evaluated by study nurses
Various studies of lower respiratory illness have reported rates based on visits to
physicians ranging from about 20 to 30 illnesses/100 children in the first year of life (Glezen
and Denny, 1973, Wright et al, 1989, Denny and Clyde, 1986, McConnochie et al, 1988)
Glezen and Denny (1973) reported that the rate for lower respiratory illnesses ranged from
24/100 person-years in infants under 1 year of age and decreased steadily each year through
the preschool years, tending to level off in school children (age 12 to 14 years) to about
7 5 illnesses/100 person-years Several factors affect the rate of lower respiratory illness in
children, including age, immunologic status, prior viral infections, level of health, SES
14-8
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(Chanock et al , 1989), day care attendance, home dampness and hurfiidity, environmental
tobacco smoke, NO2, participate matter, and other pollutants Rates also depend on method
of illness ascertainment Studies in the United States (Wright et al, 1989, Denny and Clyde,
1986, McConnochie et al , 1988) indicated that the overall pattern and incidence of lower
respiratory illness is consistent in different geographic legions during the two decades
covered by the studies, suggesting that diagnosis and infectious agents have changed little in
that time period In summary, lower respiratory illness remains one of the major causes of
childhood morbidity in the United States (McConnochie et al, 1988)
A large number of factors affect the susceptibility of children and, thus, the subsequent
occurrence of respiratory symptoms Special attention is directed at viral lower respiratory
morbidity in the first 2 years of life, because the highest incidence and rate of hospitahzation
for lower respiratory illnesses are found at this time and because of the risk of chronic
sequelae from lower respiratory morbidity in early childhood There is an immunologic
basis for increased susceptibility of the neonate to infection (Wilson, 1986) Full-term
infants are immune-deficient (as compared with older children and adults) in essentially all
measured immunologic parameters due to lack of prior exposure and subsequent development
of immunity, thus rendering them susceptible to serious infections (Bernbaum et al , 1984,
Kibler et al , 1986)
The occurrence of lower respiratory morbidity in early childhood may be associated
with unpaired lung function and growth that appears to persist through adolescence Early
insult from virus infection in the lower respiratory tract may be an essential element in the
development of chronic and persistent lung function impairment (Glezen, 1989, Gold et al ,
1989) Britten et al (1987) reported that the extension to age 36 of the earlier work of
BMRC's National Survey of Health and Development of the 1946 Great Britain Cohort
indicates that there can be little doubt in this cohort of the existence of an association
between childhood respiratory experience and adult respiratory morbidity They comment
that their study, coupled with evidence from Colley et al (1976), lends support to the model
of acquired lung damage predisposing individuals to increased respiratory diseases during
adulthood, with genetic susceptibility to respiratory disease being less of a factor Denny
and Clyde (1986) stated that it is now recognized that infections, reactive airways, and
inhaled pollutants (mostly cigarette smoke) are the mosl important nsk factors in the
14-9
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development of chronic lung disease Thus, factors such as the presence of NO2 (which
increases the risk for respiratory symptoms and related respiratory morbidity) are important
because of associated public health concern with regard to both the immediate symptoms
produced and the longer term potential for increases in the development of chronic lung
disease
The rest of this section examines epidemiological studies relating NO2 exposures to
respiratory illness The respiratory illness studies in this section are divided into indoor and
outdoor subsections
14.3.1 Indoor Studies
In this section, studies that meet criteria (see Section 14 6) for use in a quantitative
analysis are presented. Studies conducted by Melia and colleagues in Great Britain are
discussed first Next, two large studies conducted in six United States cities are examined
Then, other quantitative studies are presented that were conducted by different authors in
various locations These are followed by a quantitative study of infants in Albuquerque,
NM Finally, a discussion of selective studies that provide useful information concerning
NC>2 relationships to respiratory illness is presented
Many indoor studies report the results of their analyses as odds ratios The odds ratio
is defined as \pt (1 — pc)]/[pc (1 — Pt)L where pt is the probability of disease in the exposed
group, and pc is the probability of disease in the control group For small probabilities, the
odds ratio approaches the relative risk, pt/pc Odds ratios or relative risks greater than one
suggest an adverse effect of the exposure Although the odds ratio is more difficult to
interpret than the relative nsk, it is a natural measure resulting from many epidemiological
analyses
14.3.1.1 United Kingdom Studies
Results of several British studies have been reported by Melia et al (1977, 1978, 1979,
1980, 1982, 1985, 1988), Goldstein et al (1979, 1981), and Florey et al (1979, 1982)
Aspects of these studies were reviewed previously (U S Environmental Protection Agency,
1982a), but their importance requires a further, more complete discussion of them here
14-10
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The initial study, reported by Melia et al (1977), was based on a survey of 5,658
children (excludes asthmatics, thus 100 less than the number reported), aged 6 to 11 years,
with sufficient questionnaire information in 28 randomly selected areas of England and
Scotland The study included a self-administered questionnaire (completed by a parent) that
obtained information on the presence of morning cough day or night cough, colds going to
chest, chest sounds of wheezing or whistling, and attacks of bronchitis The questionnaire
was distributed in 1973 and asked about symptoms during the previous 12 mo Colds going
to the chest accounted for the majority of the symptoms reported Information about cooking
fuel (gas or electric), age, gender, and social class (manual versus nonmanual labor) was
obtained, but information on parental smoking was not Melia et al (1977) note that,
although they could not include family smoking habits in the analysis, the known relation
between smoking and social class (Tobacco Research Council, 1976) allowed them to avoid
at least some of the potential bias from this source It seemed unlikely to the authors that
within the social class groups there was a higher prevalence of smoking in homes where gas
was used for cooking No measurements of NO2, either indoors or outdoors, were given
The authors presented the results in the form of a contingency table for nonasthmatics
with complete covanate information Table 14-1 is a summary of that data for nonasthmatic
children The authors indicated that there was a trend for increased symptoms in homes with
gas stoves, but the increase was only significant foi girls m urban areas The authors gave
no measures of increased risk
Hasselblad et al (1992) reanalyzed the data in Table 14-1 using a multiple logistic
model, with the results as shown in Table 14-2 Because it had been suggested that gender
had an effect on the relationship with "gas cooker", interaction terms for gender were
included in the original model None of these proved to be significant, and they were
subsequently dropped from the model When separate terms for each gender were used for
the effect of "gas cooker", an estimated odds ratio of 1 25 was obtained for boys and an
odds ratio of 1 39 was obtained for girls The combined odds ratio for both genders was
1 31 (95% confidence limits of 1 16 and 1 48) and was statistically significant (p < 0 0001)
The other main effects of gender, SES, and age were all statistically significant This
reanalysis suggests that gas stove use m this study is associated with an estimated 31 %
increase in the odds for children of having respiratory illness symptoms
14-11
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TABLE 14-1. RESPIRATORY SYMPTOM RATES OF UNITED KINGDOM
CHILDREN BY GENDER, SOCIAL CLASS, AND COOKING TYPEa
Social Classes I-DI Social Classes m-V
(Nonmanual) (Manual)
Age < 8 years
Boys
Girls
Electric
256%
(203)
222%
(171)
Gas
261%
(88)
304%
(112)
Electric
299%
(375)
31 8%
(393)
Gas
375%
(309)
335%
(337)
Age ^ 8 years
Boys
Girls
208%
(365)
18 1%
(303)
233%
(189)
192%
(187)
250%
(675)
178%
(674)
290%
(654)
278%
(623)
aNumbers in parentheses refer to number of subjects
Source Mehaetal (1977)
TABLE 14-2. HASSELBLAD ET AL. (1992) MULTIPLE LOGISTIC ANALYSIS
OF DATA FROM THE MELIA ET AL. (1977) STUDY
Factora
SES and age
by gender interactions (2 d f )
Gas by gender interaction (1 d f )
Gas cooker
Gender (female)
SES (manual)
Age (<8 years)
Regression
Coefficient
02733
-0 1531
02730
03864
Standard Likelihood Ratio
Error Chi-Square p-Value
00616
00612
00702
00626
246
072
1978
629
1548
3777
02922
03953
< 00001
00121
00001
<0 0001
SES = Socioeconomic status
d f = Degrees of freedom
14-12
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Meha et al (1979) report further results of the national survey covering a new cohort
of 4,827 boys and girls, aged 5 to 10 years, from 27 randomly selected areas that were
examined in 1977 The 1977 study collected information on the number of smokers in the
home In the 1977 cross-sectional study, only the prevalence of day or night cough in boys
(p s± 0 02) and colds going to the chest in girls (p < 0 05) were found to be significantly
higher in children from homes where gas was used for cooking compared with children from
homes where electricity was used Grouping responses according to the six respiratory
questions into (a) none or (b) one or more symptoms or diseases yielded a prevalence higher
in children from homes where gas was used for cooking than in those from homes where
electricity was used (p = 0 01 in boys, p = 0 07 in giils) The results of this analysis are
presented in Table 14-3 The effects of gender, social class, use of pilot lights, and number
of smokers in the house were examined
The reanalysis of the data in Table 14-3 by Hasselblad et al (1992), applying a
multiple logistic model, is given in Table 14-4 This model contained the same terms as the
analysis in Table 14-2 As in the previous analysis, none of the interaction terms proved to
be significant, and they were subsequently dropped from the model When separate terms
for each gender were used for the effect of "gas cooker", an estimated odds ratio of 1 29 was
obtained for boys and an odds ratio of 1 19 was obtained for girls The combined odds ratio
for both genders was 1 24 (95 % confidence limits of 1 09 and 1 42) This effect was
statistically significant (p < 0 0002) The other mam effects of gender, SES, and age were
all statistically significant This reanalysis suggests that gas stove use in this study is
associated with an estimated 24% increase in the odds of having symptoms
This study was followed by a study in 1978 of 808 schoolchildren (Meha et al, 1980),
aged 6 to 7 years, in Middlesborough, an urban area of northern England Respiratory
illness was defined in the same manner as in the previous study Weekly indoor NO2
measurements were collected from 66% of the homes, with the remaining 34% refusing to
participate Nitrogen dioxide was measured weekly by tnethanolamme diffusion tubes
(Palmes tubes) attached to walls in the kitchen area and in the children's bedrooms
In homes with gas stoves, weekly levels of NO2 in kitchens ranged from 0 005 to 0 317 ppm
o 3
(10 to 596 jitg/m ) with a mean of 0 112 ppm (211 jug/in ), and levels in bedrooms ranged
from 0 004 to 0 169 ppm (8 to 318 j^g/m3) with a mean of 0 031 ppm (56 jwg/m3) In homes
14-13
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TABLE 14-3. UNADJUSTED RATES OF ONE OR MORE RESPIRATORY
SYMPTOMS AMONG UNITED KINGDOM CHILDREN BY GENDER,
SOCIAL CLASS, AND COOKING TYPE3
Social Classes I-m Social Classes ffl-V
(Nonmanual) (Manual)
Age < 8 years
Boys
Girls
Electee
274%
(277)
244%
(291)
Gas
317%
(145)
276%
(134)
Electee
328%
(485)
278%
(497)
Gas
367%
(313)
363%
(336)
Age S: 8 years
Boys
Girls
192%
(286)
148%
(243)
283%
(113)
186%
(118)
236%
(501)
21 5%
(437)
269%
(338)
185%
(313)
Numbers in parentheses refer to number of subjects
Source Meha et al (1979)
TABLE 14-4. HASSELBLAD ET AL. (1992) MULTIPLE LOGISTIC ANALYSIS OF
DATA FROM MELIA ET AL. (1979) STUDY
Factora
SES and Age
by gender interactions (2 d f )
Gas by gender interaction (1 d f )
Gas cooker
Gender (female)
SES (manual)
Age (<8 years)
Regression Standard Likelihood Ratio
Coefficient Error Chi-Square p-Value
0 2183 0 0674
-0 1970 0 0664
0 2225 0 0764
0 5253 0 0675
1 11
035
1043
881
860
6148
05749
05566
00012
00030
00034
< 00001
aSES = Socioeconomic status
d f = Degrees of freedom
14-14
-------
with electee stoves, weekly levels of NO2 in kitchens ranged from 0 006 to 0 188 ppm
(11 to 353 jug/m3) with a mean of 0 018 ppm (34 /tig/m3), and levels m bedrooms ranged
from 0 003 to 0 037 ppm (6 to 70 /*g/m3) with a mean of 0 014 ppm (26 /ttg/m3) Outdoor
levels of NO2 were determined using diffusion tubes systematically located throughout the
3
area, and the weekly average ranged from 0 014 to 0 024 ppm (26 to 45 jwg/m )
One analysis by the authors was restricted to those 103 children in homes where gas
stoves were present and where bedroom NO2 exposure was measured, the data are shown in
Table 14-5 A linear regression model was fit to the logistic transformation of the symptom
or illness rates Cooking fuel was found to be associated with respiratory illness,
independent of social class, age, gender, or presence of a smoker in the house (p = 0 06)
However, when social class was excluded from the regiession, the association was weaker
(p = 0 11) For the 6- to 7-year-old children living in gas stove homes, there appeared to
be an increase of respiratory illness with increasing levels of NO2 in their bedrooms
(p = 0 10), but no significant relationship was found between respiratory symptoms in those
children or their siblings or parents and levels of NO2 ui kitchens
TABLE 14-5. UNADJUSTED RATES OF ONE OR MORE RESPIRATORY
SYMPTOMS AMONG UNITED KINGDOM BOYS AND GIRLS BY
BEDROOM LEVELS OF NITROGEN DIOXIDE3
Bedroom Levels of NO2 (ppm)
Boys
Girls
TOTAL
<0020
435%
(23)
440%
(25)
437%
(48)
0 020-0 039
579%
(19)
600%
(15)
588%
(34)
>0039
692%
(13)
750%
(8)
71 4%
(21)
Total
545%
(55)
542%
(48)
544%
(103)
aNumbers in parentheses refer to number of subjects
Source Meha et al (1980)
14-15
-------
Because no concentration-response estimates were given by the authors, a multiple
logistic model was fitted by Hasselblad et al (1992) to the data in Table 14-5, using a linear
slope for NO2 and separate intercepts for boys and girls Nitrogen dioxide levels for the
groups were estimated by fitting a lognormal distribution to the grouped NO2 data, and the
average exposures within each interval were estimated (see Hasselblad et al, 1980) The
O
estimated logistic regression coefficient for NO2 (in jwg/m ) was 0 015 with a standard error
of 0 007 The likelihood ratio test for NO2 yielded a chi-square of 4 94 with one degree of
freedom, with a corresponding p-value of 0 03
The study was repeated in January to March of 1980 by Melia et al (1982a) This
tune, children aged 5 to 6 years were sampled from the same neighborhood as the previous
study, but only families with gas stoves were recruited Environmental measurements were
made and covanate data were collected in a manner similar to the previous study (Melia
et al, 1980) Measurements of NO2 were available for 54% of the homes The unadjusted
rates of one or more symptoms by gender and exposure level are shown in Table 14-6 The
authors concluded that" no relation was found between the prevalence of respiratory
illness and levels of NO2 " The reanalysis by Hasselblad et al (1992) of the data in
Table 14-6 was made using a multiple logistic model similar to the one used for the previous
study (Melia et al, 1980) The model included a linear slope for NO2 and separate
intercepts for boys and girls Nitrogen dioxide levels for the groups were estimated by
fitting a lognormal distribution to the grouped bedroom NO2 data The estimated logistic
regression coefficient for NO2 (in /*g/m3) was 0 0037 with a standard error of 0 0052 The
likelihood ratio test for the effect of NO2 gave a chi-square of 0 51 with one degree of
freedom (p = 0.48)
Melia et al (1982b) report an association between the prevalence of respiratory
symptoms in children and relative humidity in bedrooms Florey et al (1979) had
hypothesized that the respiratory health effects seen at the observed NO2 levels may be a
proxy for some other factor such as temperature or humidity After further study, these
researchers (Melia et al, 1982b) conclude that this study did not support the hypothesis that
high humidity or low temperature are associated with levels of NO2 within homes with a gas
cooker and state that these two environmental variables are thus unlikely to explain their
original observation of an association between respiratory illness among primary school
14-16
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TABLE 14-6. UNADJUSTED RATES OF ONE OR MORE RESPIRATORY
SYMPTOMS AMONG UNITED KINGDOM BOYS AND GIRLS BY
BEDROOM LEVELS OF NITROGEN DIOXIDE3
Bedroom Levels of NO2 (ppm)
Boys
Girls
<0020
564%
(39)
600%
(25)
0 020-0 039
676%
(37)
410%
(39)
>0039
720%
(25)
522%
(23)
Total
644%
(101)
494%
(87)
Numbers in parentheses refer to number of subjects
Source Meha et al (1982a)
children and NO2 Also, Melia et al (1982b) note that, contrary to their original hypothesis,
homes with an electric cooker tended to have slightly higher relative humidity than homes
with a gas cooker Arundel et al (1986) comment in general that the majority of health
effects related to relative humidity would be minimized by maintaining indoor levels between
40 and 60% and that this would require humidification during winter because indoor relative
humidities below 40% are widespread in winter
Melia et al (1983) investigated the association belween gas cooking in the home and
respiratory illness in a study of 390 infants born between 1975 and 1978 When the child
reached 1 year of age, the mother was interviewed by a trained field worker to complete a
questionnaire The mother was asked whether the child usually experienced morning cough,
day or night cough, wheeze, or colds going to the chest, and whether the child had
experienced bronchitis, asthma, or pneumonia during the past 12 mo No relationship was
found between type of fuel used for cooking at home and the prevalence of respiratory
symptoms and diseases recalled by the mother after allowing for the effects of gender, social
class, and parental smoking The authors reported prevalence rates for children having at
least one symptom by gas stove use and gender The combined odds ratio for presence of
symptoms by gas stove use was 0 63 with 95 % confidence interval of 0 36 to 1 10
Melia et al (1988) studied factors affecting respiratory morbidity in 1,964 primary
school children living in 20 inner city areas of England in 1983 as part of a national study of
health and growth Data on age, gender, respiratory illness, cooking fuels, mother's
14-17
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education, and size of family were obtained by questionnaire Smoking was not studied
The same respiratory questions were asked as in the previous studies Melia et al (1990)
reported indoor levels of NO2 associated with gas stoves m inner city areas of England m
1987. The mean weekly NO2 level measured in 22 bedrooms of homes with gas stoves was
0.0241 ± 0 013 ppm The mean weekly NO2 level measured in four bedrooms of homes
without gas stoves was 0 0207 + 0 0118 ppm Melia et al (1988) reported a relative risk of
1.06 (95% confidence interval of 0 94 to 1 17) for one or more respiratory conditions
relative to risk in white boys aged 8 years with mothers educated up to secondary school
level, one child in family, two-parent family, and no gas or kerosene fuel used in the home
14.3.1.2 United States Six Cities Studies
Several authors (Spengler et al, 1979, Sperzer et al , 1980, Ferns et al , 1983,
Spengler et al, 1986, Berkey et al, 1986, Ware et al, 1984, Quackenboss et al, 1986,
Dockery et al, 1989a, Neas et al, 1990, Neas et al, 1991) have reported on two cohorts of
children studied in six different U S cities (Watertown, MA, Kingston and Harnman, TN,
southeast St Louis, MO, Steubenville, OH, Portage, WI, and Topeka, KS) The six cities
were selected to represent a range of air quality based on their historic levels of outdoor
pollution In each community during the period 1974 through 1977, approximately
1,000 first- and second-grade schoolchildren were enrolled in the first year and an additional
500 first graders were enrolled during the following year (Ferns et al, 1979) Families
reported the number of persons living in the home and their smoking habits, parental
occupation and educational background, and the fuels used for cooking and heating Outdoor
pollution was measured at fixed sites in the communities as well as at selected households
Indoor pollution, including NO2, was measured in several rooms of selected households
Spengler et al (1979) show that a striking difference in NO2 levels exists between homes
with gas versus electnc cooking Later results of monitoring in Portage, WI, verify the fact
that the presence of a gas stove contributes to the indoor NO2 levels Table 14-7 is taken
from Quackenboss et al (1986) based on data collected in 1981 and 1982 These results
clearly show that gas stoves increase indoor concentrations and therefore also increase the
personal exposures of children
14-18
-------
TABLE 14-7. NITROGEN DIOXIDE CONCENTRATIONS (ppm) BY SEASON
AND STOVE TYPE IN PORTAGE, WISCONSIN
Indoor
Season
Summer
Winter
Stove
Gas
Electric
Gas
Electric
Mean
0016
0007
0027
0005
Std
Dev
0006
0003
0013
0003
Outdoor
Mean
0006
0008
0 008
0009
Std
Dev
0003
0003
0003
0003
Personal
Mean
0014
0009
0023
0008
Std
Dev
0004
0003
0009
0003
Source Quackenboss et al (1986)
Speizer et al (1980) first reported on results from the six cities studies, based on
evaluations of 8,120 children (aged 6 to 10 years) who had been followed for 1 to 3 years
Health end points were measured by a standard respiratory questionnaire, completed by the
parents of the children The authors used log-linear models to estimate the effect of current
gas stoves versus electric stoves on the rates of serious lespiratory illness before age 2 The
analysis gave an odds ratio of 1 12 (95 % confidence limits of 1 00 and 1 26) for gas stove
use The results were adjusted for the presence of adult smokers, presence of air
conditioning, and SES of the family
Ware et al (1984) later reported results from a larger cohort of 10,160 white children,
aged 6 to 9 years, in the same six communities over a longer period (1974 to 1979)
Directly standardized rates of reported illnesses and symptoms did not show any consistent
pattern of increased risk for children from homes with gas stoves Logistic regression
analyses controlling for age, gender, city, and maternal smoking level gave estimated odds
ratios for the effect of gas stoves ranging from 0 93 to 1 07 for bronchitis, chronic cough,
persistent wheeze, lower respiratory illness index, and illness for the last year The lower
respiratory illness index indicated the presence of bronchitis, restriction of activity due to
chest illness, or chronic cough during the past year None of these symptom-specific odds
ratios were statistically different from 1 Only two odds ratios approached statistical
significance (1) history of bronchitis (odds ratio = 0 86, 95 % confidence interval 0 74 to
1 00) and (2) respiratory illness before age 2 (odds ratio = 1 13, 95% confidence
interval 0 99 to 1 28) When the odds ratio for respiratory illness before age 2 was adjusted
14-19
-------
for parental education, the odds ratio was 111 with 95% confidence limits of 0 97 and 1 27
(p = 0 14). Thus, the study suggests an increase of about 11% in respiratory illness before
age 2 years, which is nearly the same as that reported by Speizer et al (1980), although the
increase was not statistically significant at the p < 0 05 level The end point in the Ware
et al (1984) study most similar to that of the Meka studies was the lower respiratory illness
index. The authors gave the unadjusted prevalence, and from those data, an estimated odds
ratio of 1 08 with 95 % confidence limits of 0 97 and 1 19 was calculated by Hasselblad
et al (1992) This rate was not adjusted for other covanates The analysis of Ware et al
(1984) on the other end points found that the effect of adjustment for covanates was
minimal
During the period 1983 through 1986, a new cohort of approximately 1,000 second-
through fifth-grade schoolchildren in each community were enrolled and given an initial
symptom questionnaire Dockery et al (1989a) evaluated reported respiratory symptoms on
a subsequent symptom questionnaire (second annual) for 5,338 white children who were aged
7 to 11 years at the time of enrollment The end points of chronic cough, bronchitis,
restriction of activity due to chest illness, and persistent wheeze were not found to be
associated with gas stove use in the home But the health end point of doctor-diagnosed
respiratory illness prior to age 2, yielded an odds ratio of 1 15 with 95% confidence limits of
0 96 and 1 37 The odds ratio for chronic cough was 1 15 with 95 % confidence limits of
0 89 and 1.91 and was adjusted for age, sex, parental education, city of residence, and use
of unvented kerosene heaters
Neas et al (1990, 1991) studied a stratified one-third random sample of the children
that were part of the Dockery et al (1989a) analysis The sample was restricted to
1,286 white children 7 to 11 years of age at enrollment having complete covanate
information and at least one valid indoor measurement of both NO2 and respirable particles
Methods for measuring indoor pollutants were described by Spengler et al (1986) Indoor
pollutants were measured in each child's home for 2 weeks during the heating season and
2 weeks during the cooling season Nitrogen dioxide was measured by Palmes passive
diffusion tubes at three locations kitchen, activity room, and the child's bedroom
Brunekreef et al (1989) examined children studied m the six cities studies and
concluded that home dampness is a strong predictor of respiratory symptoms among 8- to
14-20
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12-year-old children Dampness was determined by response to these three questions on a
questionnaire (1) Does water ever collect on the basement floor*? (2) Has there ever been
water damage to the building"? and (3) Has there ever been mold or mildew on any surface
inside the home9 Brunekreef et al (1989) comment that relative humidity of the indoor air
is less important for the growth of mites and fungi than the dampness of specific surfaces or
parts of the building structure Dampness did not confound the gas stove (Dockery et al ,
1989a) nor the NO2 association (Neas et al, 1991)
The analyses by Neas et al (1990, 1991) were based on the final symptom
questionnaire (third annual), which was completed by parents following the indoor
measurements The questionnaire reported symptoms during the previous year, including
attacks of shortness of breath with wheeze, persistent wheeze, chronic cough, chronic
phlegm, and bronchitis The authors used a multiple logistic model with separate city
intercepts, indicator variables for gender and age, parental history of chronic obstructive
pulmonary disease, parental history of asthma, parental education, and single parent family
3
status. The increases in symptoms were estimated for an additional 0 015 ppm (28 3 ^g/ni)
NO2 exposure Table 14-8 shows the odds ratios for the five separate symptoms associated
with the increase in NO2 exposure All of these odds ratios are consistent with the size of
effect seen in the other analyses of the Six City data and the analyses of the British studies
TABLE 14-8. ODDS RATIOS AND 95% CONFIDENCE INTERVALS FOR THE
EFFECT OF AN ADDITIONAL 0.015 ppm NITROGEN DIOXIDE ON THE
SYMPTOM PREVALENCE
Symptom
Shortness of breath
Persistent wheeze
Chronic cough
Chronic phlegm
Bronchitis
Odds Ratio
1 23
1 16
1 18
125
1 05
95 % Confidence Interval
0 93 to 1 61
0 89 to 1 52
0 87 to 1 60
0 94 to 1 66
0 75 to 1 47
Source Neas et al (1991)
14-21
-------
Neas et al (1990, 1991) defined a combined symptom measure, which was the
presence of any of the above-noted symptoms A multiple logistic regression of this
combined lower respiratory symptom measure, equivalent to the single response regressions,
gave an estimated odds ratio of 1 40 with a 95 % confidence interval of 1 14 to 1 72 The
odds ratio for the combined symptom score was slightly higher than in other studies, but is
not inconsistent with those results The reference category for the symptom-specific odds
ratios included some children with the other lower respiratory symptoms, whereas the
children in the reference category for combined lower respiratory symptoms were free of any
of these symptoms When split by gender, the odds ratio was higher in girls, and, when
split by smoking versus nonsmoking homes, the odds ratio was higher in smoking homes
When separate logistic analyses were performed for each community, the adjusted odds ratios
ranged from 1 26 for Topeka, KS, to 1 86 for Portage, WI When the cohort was restricted
to the 495 children in homes with a gas stove, the adjusted odds ratio was 1 37 with a 95 %
confidence interval of 1 02 to 1 84 Table 14-9 provides the adjusted odds ratios for
combined lower respiratory symptoms across ordered NO2 exposure categories The
association is statistically significant for the upper exposure category, and the lower exposure
categories are consistent with a linear dose-response relationship between NO2 and lower
respiratory symptoms in children
TABLE 14-9. ODDS RATIOS AND 95% CONFIDENCE INTERVALS FOR
THE EFFECT OF ORDERED NITROGEN DIOXIDE EXPOSURES
ON THE PREVALENCE OF LOWER RESPIRATORY SYMPTOMS
NO2 Level (ppm)
Range Mean
0 to 0 0049 0 0037
0.0050 to 0.0099 0 0073
0.0100 to 0 0199 0 0144
0 0200 to 0 0782 0 0310
Number of
Children
263
360
317
346
Odds Ratio
100
106
1 36
165
95%
Confidence Interval
0 71 to 1 58
0 89 to 2 08
1 03 to 2 63
Source Neas et al (1991)
14-22
-------
Neas et al (1992) reported that the estimated effect of exposure to an additional
0 015 ppm (28 3 jwg/m3) NO2 on lower respiratory symptoms was consistent across the
seasons and sampling locations Table 14-10 provides ihe odds ratios and 95% confidence
intervals for this association by season and sampler location The NO2 levels measured by
the activity room and bedroom sampler were more strongly associated with lower respiratory
symptoms than those in the kitchen The NO2 measurements in the kitchen were suggested
to be influenced more by the transient peak levels associated with meal preparation on gas
stoves, whereas the other sampling locations were more reflective of the child's long-term
average exposures to NO2 in the home Spengler et al (1992) indicated that children spend
relatively little tune (0 5 hours per day) in the kitchen when the range is operating
TABLE 14-10. ODDS RATIOS AND 95% CONFIDENCE INTERVALS FOR THE
EFFECT OF AN ADDITIONAL 0.015 ppm NITROGEN DIOXIDE
ON THE PREVALENCE OF LOWER RESPIRATORY
SYMPTOMS BY SAMPLING LOCATION AND SEASON
Mean Difference 95 % Confidence
Sampler Location and Season Gas vs Electric Odds Ratio Interval
Household annual average
Household winter average
Household summer average
Kitchen annual average
Activity room annual average
Bedroom annual average
0 016 ppm
0 018 ppm
0 014 ppm
0 022 ppm
0 014 ppm
0 013 ppm
140
1 16
146
1 23
150
147
1 14 to 1 72
1 04 to 1 29
1 13 to 1 89
1 05 to 1 44
1 20 to 1 87
1 17 to 1 85
Source Neas et al (1992)
14.3.1.3 Iowa Study
Ekwo et al (1983) surveyed 1,355 children 6 to 12 years of age for respiratory
symptoms and lung function in the Iowa City School District Parents of the school children
completed a questionnaire that was a modification of the questionnaire developed by the
American Thoracic Society The children were a random sample from those families whose
parents had completed the questionnaire Eight different measures of respiratory illness were
14-23
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reported by the authors, but only two of those were similar to the end points used in the
British studies and the Six City studies Parental smoking was also measured and used as a
covanate in the analyses The results of the analyses are presented in Table 14-11, and are
based on 1,138 children No measurements of NO2 exposure, either inside or outside the
homes, were reported
TABLE 14-11. ANALYSIS OF IOWA CITY SCHOOL CHILDREN RESPIRATORY
SYMPTOMS BY GAS STOVE TYPE AND PARENTAL SMOKING
Hospitahzation for Chest Chest Congestion and
Illness Before Age Two Phlegm with Colds
Factor
Gas stove use
Smoking effects
Father alone smokes
Mother alone smokes
Both smoke
Odds Ratio
24b
23b
29b
1 6
SEa
0684
0856
1 239
0859
Odds Ratio
1 1
10
1 3
12
SEa
0188
0213
0363
0383
SE = Standard error of the odds ratio
Indicates statistical significance at the 0 05 probability level
Source Ekwo et al (1983)
14.3.1.4 Dutch Studies
In the Netherlands, Houthuijs et al (1987), Brunekreef et al (1987), and Dijkstra et al
(1990) studied the effect of indoor factors on respiratory health in children The population
consisted of 6- to 9-year-old children from 10 primary schools in five nonindustnal
communities in the southeast region of the Netherlands Concentrations of NO2 in the home
and personal exposures to NO2 were measured An important NO2 emission/exposure source
in these homes were geysers, which are unvented, gas-fired, hot water sources at the water
tap. Exposure to tobacco smoke was assessed with a questionnaire that also reported
symptom information The study used Palmes diffusion tubes to measure a single weekly
average personal NO2 exposure In January and February of 1985, the homes of
593 children who had not moved in the last 4 years were measured for 1 week for NO2
14-24
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Personal exposure was also estimated from time budgets and room monitoring Estimated
and measured exposures to NO2 are given in Table 14-12
TABLE 14-12. DUTCH STUDY ESTIMATED AND MEASURED PERSONAL
NITROGEN DIOXIDE EXPOSURE (ppm) FOR
A SINGLE WEEKLY AVERAGE3
Estimated
NO2 Source
No geyser
Vented geyser
Unvented geyser
Number
370
112
111
Arith Mean
0012
0015
0021
SD
0004
0005
0005
Measured
Arith Mean
0012
0016
0022
SD
0005
0006
0006
Arith Mean = Arithmetic mean
S D = Standard deviation
Source Houthuijs et al (1987)
Three measures of health were obtained from the questionnaire, which was a modified
form of the World Health Organization questionnaire The different items were combined to
create three categories cough, wheeze, and asthma Asthma was defined as attacks of
shortness of breath with wheezing in the last year The presence of any of the three
symptoms was used as a combination variable The results are presented in Table 14-13
A logistic regression model was used to fit the combinalion variable by Hasselblad et al
(1992) Exposure was estimated by fitting a lognormal distribution to the grouped data and
the mean exposure values for each group were estimated by a maximum likelihood technique
(Hasselblad et al , 1980) The estimated logistic regression coefficient was —0 002,
corresponding to an odds ratio of 0 94 for an increase of 0 015 ppm (28 3 /ig/m ) in NO2,
with 95 % confidence interval of 0 70 to 1 27 Thus, the Dutch studies did not demonstrate
an increase in respiratory disease with increasing NO2 exposure, but the range of uncertainty
is quite large and the rates were not adjusted for covanates such as parental smoking and age
of the child
Of several potential explanations for the negative findings of the study with respect to
NO2 exposure offered by the authors, one consideration was that the power of the study to
14-25
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TABLE 14-13. FREQUENCY AND PREVALENCE OF REPORTED
RESPIRATORY SYMPTOMS FOR DIFFERENT CATEGORIES OF MEAN
INDOOR NITROGEN DIOXIDE CONCENTRATIONS IN A POPULATION
OF 775 DUTCH CHILDREN 6 TO 12 YEARS OLD
Frequency and Prevalence in Category of Indoor NO2
Symptom
Cough
Wheeze
Asthma
One or more
symptoms
0-0 Oil ppm
(n = 336)
16
30
22
36
48%
89%
66%
107%
0 011-0
(n =
12
18
12
24
021 ppm
= 267)
45%
67%
45%
90%
0 021-0 032 ppm
(n = 93)
7
3
2
8
75%
32%
22%
86%
> 0032 ppm
(n = 79)
3
7
3
8
3 8%
89%
3 8%
101%
Source Dykstraetal (1990)
detect health effects may have been reduced by the smaller sample size of the measured NO2
data compared to the categorical data (e g , geyser versus no geyser) They could not
estimate whether they gamed more precision by measured NO2 than was lost by the
reduction in the sample size Houthmjs et al (1987) report in an earlier analysis that the
presence of an unvented geyser in the kitchen is associated with a higher prevalence of
respiratory symptoms and that the difference between no geyser present and an unvented
geyser is about 0 010 ppm
14.3.1.5 Ohio Study
Keller et al (1979a) and Mitchell et al (1975) conducted a 12-mo study of respiratory
illness and pulmonary function in families in Columbus, OH, prior to 1978 The sample
included 441 families divided into two groups those using gas and those using electric
cooking Participating households were given diaries to record respiratory illnesses for
2-week periods Respiratory illnesses included colds, sore throat, hoarseness, earache,
phlegm, and cough Only the first incident of illness per person per 2-week period was
recorded
The study measured NO2 exposure by both Jacobs-Hochheiser and continuous
o
chemiluminescence methods The electric stove users averaged 0 020 ppm (38 /*g/m ) NO2
14-26
-------
3
exposure, whereas the gas stove users averaged 0 050 ppm (94 jtg/m ) The paper does not
report which rooms were measured in order to get this average
The analysis of incidence rates was done using the "Automatic Interaction Detector"
No differences were found in any of the illness rates for fathers, mothers, or children
No analyses were done using multiple logistic regression or Poisson regression (these
methods were relatively new at the time) No estimates were made that can be considered
comparable to the odds ratios reported in the other studies The authors did show a bar
graph of all respiratory illness for children under 12 The rates were 389 (per 100 person-
years) for electric stove use and 377 for gas stove use These rates were not significantly
different even after adjustment for covanates, including family size, age, gender, length of
residence, and father's education No mention was made of adjustments for smoking status
or smoking exposure for the children
In a second, related study (Keller et al, 1979b), 580 persons drawn from households
that participated in the earlier study were examined to confirm the reports and to determine
the frequency distribution of reported symptoms among parents and children in gas or
electric cooking homes A nurse-epidemiologist examined selected persons reported ill and
obtained throat cultures Unfortunately these rates were not adjusted for other covanates
The percent of children having respiratory illnesses in homes with a gas stove was 85 1 %
(n = 87) versus 88 8 % (n = 89) in homes with electric stoves Although the difference is
not statistically significant, these rates give an estimated odds ratio of 0 72 with 95 %
confidence interval of 0 30 to 1 74 Neas et al (1991) commented that Keller's model
controls for a series of variables that specify the child's prior illness history and that if
chronic exposure to NO2 is a risk factor for prior illnesses, controlling for the child's illness
history would substantially reduce the estimated effect of current NO2 exposure
14.3.1.6 Tayside Study
Ogston et al. (1985) studied infant mortality and morbidity in the Tayside region of
northern Scotland The subjects were 1,565 infants bom to mothers who were living in
Tayside in 1980 Episodes of respiratory illness were recorded during the first year of life
The information was supplemented by observations made by a health visitor and scrutinized
by a pediatrician who checked diagnostic criteria and validity One health end point assessed
14-27
-------
was defined as the presence of any respiratory disease during the year This end point was
analyzed using a multiple logistic regression model that included terms for parental smoking,
age of mother, and presence of a gas stove The results of this analysis are shown in
Table 14-14
TABLE 14-14. REGRESSION COEFFICIENTS FOR MULTIPLE LOGISTIC
ANALYSES OF RESPIRATORY ILLNESS IN TAYSIDE CHILDREN
Factor
Parental smoking
Age (in 5-year groups)
Presence of gas stove
Regres Coeff a
0429
-0094
0130
Odds Ratio
1 54
NAb
1 14
95 % Confidence Limits
0 86, 1 50
NA = Not available
Source Ogstonetal (1985)
Only the coefficient for parental smoking was statistically significant (p < 0 01)
A test for the significance for the coefficient for gas stove use gives a p-value of 0 14 The
study did not give measured NO2 exposure values, but referenced the other Melia studies
conducted elsewhere in the United Kingdom for exposure estimates
14.3.1.7 Albuquerque Study
Samet et al. (1993, 1992), Lambert et al (1993), and Samet and Spengler (1989)
reported preliminary results of a prospective cohort study of respiratory illness during the
first 18 mo of life in relationship to estimates of NO2 exposure in Albuquerque, MM
Exposure estimates were based on Palmes tube and activity data The study included
standardized ascertainment of illness and assessment of potential confounding factors
Samet et al (1993) conducted a prospective cohort study between January 1988 and
June 1990 to test the hypothesis that exposure to NO2 increases the incidence and seventy of
respiratory illness during the first 18 mo of life A total of 1,315 infants were enrolled into
the study at birth in Albuquerque, NM The subjects were healthy infants from homes
14-28
-------
without smokers and who spent less than 20 h per week in day care Illness experience was
monitored by a daily dairy of symptoms completed by the mother and a telephone interview
conducted every two weeks For a sample of the ill children, a nurse practitioner made a
home visit to conduct a standardized history and physical assessment Exposure to NO2 was
estimated by a two-week average concentration measured in the subjects' bedrooms with
passive samplers Estimates of exposure based on bedroom concentration were tightly
correlated with estimates of exposures calculated as time-weighted averages of the
concentrations in the kitchen, bedroom, and activity room Twenty-six percent of residences
had electric cooking ranges, 44% of homes had gas ranges with continuously burning pilot
lights, and 30% of homes had gas ranges with electronic ignition or burners that were lit
with matches In homes with gas stoves, the subjects' bedrooms were monitored every
2 weeks, year round In homes with electnc stoves, the child's bedroom was monitored
year-round during every other 2-week cycle Extensive internal and external quality
assurance and control procedures were implemented
Samet et al (1993) define illness events as the occurrence of at least two consecutive
days of any of the following runny or stuffy nose, wet cough, dry cough, wheezing, or
trouble with breathing Wheezing was defined as a high-pitched musical sound audible
during breathing, and trouble with breathing as the parent's perception of rapid or labored
breathing Illness events ended with two consecutive symptom-free days More specific
definitions were determined as follows "upper respiratory tract" was defined as at least
2 consecutive days of any combination of runny or stuffy nose, dry cough, and trouble
breathing, "lower respiratory tract" as at least 2 consecutive days of any of the upper
respiratory symptoms plus wet cough or wheezing or both being reported on at least 1 day,
"lower respiratory tract, wet cough" as any illness meeting the criteria for lower respiratory
tract but without wheezing at any tune, and "lower respiratory tract, wheezing" as any illness
meeting the criteria for lower respiratory tract with wheezing reported for at least 1 day
The analysis was limited to the 1,205 subjects completing at least 1 month of
observation, of these, 823 completed the full protocol Multivanate methods were used to
control for potential confounding factors and to test for effect modification In analyses of
determinants of incident illnesses, the outcome variable was the occurrence of illness during
2 week intervals of days at risk The independent variables considered in the multivanate
14-29
-------
analyses included the fixed factors of birth order (first born versus other), gender, ethnicity
(Hispanic versus non-Hispanic), parental asthma and atopic status (considered positive if hay
fever or desensitization shots were reported), household income (less than $10,000, $10,000
to $40,000, or greater than $40,000), and maternal education (12 years or less, 13 to
15 years, or 16 years or more) Other variables considered were the temporally varying
factors of age (6 mo or less, 7 to 12 mo, or 13 to 18 mo), calendar month, day-care
attendance (none, 1 to 4 h, or 5 or more hours per week), and breast feeding (none, partial,
or full). Potential confounding and effect modification by cigarette smoking were controlled
by excluding subjects from households with smokers
The overall distribution of tune at risk by level of bedroom NO2 level was skewed
toward lower contributions, with 22% of the total concentration above 0 02 ppm
(Figure 14-1) Lambert et al (1993) reports that during the summer, bedroom NO2
concentrations in homes with gas stoves averaged 0 014 ppm (standard deviation [SD] =
0.01 ppm) In the bedrooms of homes with electric stoves, the summer average
concentrations was 0 007 ppm (SD = 0 006 ppm) During the winter, bedroom
concentrations in homes with gas stoves averaged 0 021 ppm (SD = 0 022 ppm)
In bedrooms of homes with electric stoves, winter concentrations averaged 0 007 ppm
(SD = 0 006 ppm) The exposure estimates were stratified into three classes low (0 to
0 02 ppm), medium (0 02 to 0 04 ppm), and high (greater than 0 04 ppm) For these
exposure strata, personal exposures based on bedroom measurements were not substantially
different from those derived using the microenvironmental model Approximately 77% of
the bedroom NO2 observations were less than 0 02 ppm, only 5 % were greater than
0.04 ppm The 10th and 90th percentiles of the weekly measured concentrations were
0.005 and 0 050 ppm NO2, respectively, in bedrooms
Samet et al (1993) performed the analysis using the generalized estimated equations
described by Zeger and Liang (1986) This takes into account the correlation structure when
estimating regression coefficients and their standard errors The multivanate models
examined the effects of the unlagged NO2 exposures, lagged NO2 exposures, and stove type
(Table 14-15) None of the odds ratios was significantly different from unity, the value for
the reference category of 0 through 0 02 ppm Additionally, the odds ratios did not tend to
increase consistently from the middle category of exposure to the highest category Also,
14-30
-------
c
&
0-001 001-0.02 002-003 003-0.04 0.04-005 005-0.06 006-0.07 0.07-0.08 008+
Bedroom NO2 (ppm)
Figure 14-1. Distribution of time at risk by bedroom NO2 concentration.
Source Samet et al (1993)
exposure to NO2 and the durations of the four illness categones were not associated The
authors added NO2 exposure to the model as a continuous variable, while controlling for the
same covariates included in Table 14-15 For each of the five illness variables, the estimated
multiplier of the odds ratio per 0 001 ppm increment of NO2 was 0 999, with confidence
limits extending from approximately 0.995 to 1 002
Health Effects Institute Health Review Committee (1993) noted that although exposure
to NO2 levels in excess of those encountered in this study may be causally related to the
incidence or seventy of respiratory illness in children, other data indicate that an effect, if it
exists, is subtle and may be difficult to distinguish from other environmental risk factors,
especially environmental tobacco smoke
14-31
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TABLE 14-15. ODDS RATIOS3 FOR EFFECT OF NITROGEN DIOXIDE
EXPOSURE ON INCIDENCE OF RESPIRATORY ILLNESS
All Illnesses
NO2 Exposure
Unlagged6
0 02—0 04 ppm
>0 04 ppm
Lagged0
0.02-0 04 ppm
>0 04 ppm
Gas Stoved
Odd
Ratio
1 04
094
101
092
098
95%
096-1
081-1
093-1
077-1
090-1
CIb
12
08
10
10
07
All Lower
Odd
Ratio
098
093
097
091
091
95%
089-1
076-1
087-1
072-1
081-1
CIb
09
13
08
15
04
Lower,
Wet Cough
Odd
Ratio
1 00
094
097
089
094
95%
089-1
077-1
087-1
068-1
082-1
CIb
12
16
09
16
07
Lower,
Wheezing
Odd
Ratio
092
0 88
095
098
084
95% CIb
073-1 15
056-137
075-1 19
0 66-1 48
064-1 09
"Obtained by generalized estimating equation method Adjusted for season, age, gender, ethnicity, birth order,
day care, income, maternal education, breast feeding, parental atopy and asthma, and maternal history of
respiratory symptoms
CI = Confidence interval
°Reference category is 0-0 02 ppm NO2
Reference category is electric stove
Source Samet et al (1993)
Health Effects Institute Health Review Committee (1993) commented that performing
the multivanate Generalized Estimated Equation analyses without so many covanates being
considered simultaneously with an investigation of the separate and combined variables may
be informative In the present analyses, at least 11 variables were entered into the
multivariate analyses These include season, age, gender, ethnicity, birth order, day-care
status, income, maternal education, breast feeding, parental atopy and asthma, and a
maternal symptom report These analyses are unable to sort out the effects of the various
variables
The advantage of restrictions in the study population is that the effect of NO2 exposure
could be evaluated in a relatively homogeneous sample of infants The disadvantages are
that the results cannot be generalized to the potentially more susceptible portions of the
population, such as infants with parents or care givers who smoke, and infants who attend
day care (Health Effects Institute Health Review Committee, 1993)
The study design and implementation represent an effective reduction of
misclassification and potential bias The exposure estimates are a good representative
14-32
-------
estimate of NO2 exposure for the subjects over the tune period of the study The prospective
assessment of illness incidence limits potential bias of retrospective ascertainment of illness
The findings indicate that in a population of healthy infants (0 to 18 mo of age), no
significant associations between NO2 exposure estimates (in the range of 0 to 0 04 ppm) and
respiratory illness were found when precaution was taken to make an accurate assessment of
exposures, to validate the measurements of respiratory illness, to eliminate potentially
confounding variables, and to adjust for key variables
14.3.1.8 Chestnut Ridge Study
Schenker et al (1983) reported a large respiratory disease study of 4,071 children aged
5 to 14 years in the Chestnut Ridge region of western Pennsylvania The region is
predominately rural, and there are numerous underground coal mines and four large coal-
fired electricity-generating plants within the area The standardized ATS-DLD-78-C
children's questionnaire (Ferns, 1978) was sent to parents of all children in grades 1 through
6 in targeted schools An SES scale was derived from the parent's occupation and education
and divided into quintiles to provide SES strata V (lowest) through I (highest)
Important confounding factors were evaluated to include gender, SES, and maternal
smoking Persistent wheeze and chronic cough were the most commonly reported symptoms
No relationship was found between persistent wheeze, chronic phlegm, or chest illness in the
past year and SES Significant inverse trends with SES were present for chronic cough and
severe chest illness before 2 years of age, whereas physician-diagnosed bronchitis showed
significant trends of increased prevalence with higher SES
Significant linear associations were reported to be present only between the number of
parent smokers and the prevalence of chest illness in the past year, and of serious chest
illness before 2 years of age, but not with chronic respiratory symptoms Smoking
questionnaires were completed by 1,906 children in grades 4 through 6 Only 53 (2 7%)
said that they had ever smoked five or more cigarettes, and currently smoked
A significant inverse relationship was observed between gas cooking stove use and SES
When gas cooking stove use was tested in the multiple logistic model, a significant
association was not found between gas stove use and any of the respiratory or illness
variables after adjusting for SES No odds ratios or other numerical data were reported
14-33
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14.3.1.9 Swiss Study
Braun-Fahrlaender et al (1989, 1992) and Rutishauser et al (1990a,b) studied the
incidence and duration of common airway symptoms in children up to 5 years old The
study was conducted over a 1-year period in a rural, a suburban, and two urban areas of
Switzerland Parents were asked to record their child's respiratory symptoms (from a list)
on a diary form daily over a 6-week period Additionally, covanates including family size,
parental education, living conditions, health status of the child, parents' respiratory health,
and smoking habits of the family were assessed by questionnaire
Nitrogen dioxide was measured weekly during the same 6-week period using Palmes
tubes, both inside and outside the home of the participants Meteorological data were
obtained from local monitoring stations, but additional air quality data from fixed monitoring
sites were only available for the two urban study areas Figure 14-2 shows the average NO2
concentration outside and indoors and makes it clear that NO2 concentrations inside the home
were on average lower than the levels in the outside air Indoor NO2 levels for Basel,
Zurich, Wetzikon, and Rufzerfeld were 33 8, 28 4, 20 5, and 11 2 jug/m3 (0 0179, 0 0151,
0.0109, and 0 0059 ppm), respectively The indoor NO2 concentration depended to some
extent on the concentration of the outside air
0030
Figure 14-2. Nitrogen dioxide ambient and indoor concentrations in four Swiss regions
with 95% confidence range.
Source Braun-Fahrlaender et al (1989)
14-34
-------
The analysis was restricted to the 1,063 Swiss nationals (from a total of 1,225
participating families) For all four study areas, regional mean incidence rates of upper
respiratory illness, cough, breathing difficulties, and total respiratory illness, adjusted for
individual covanates and weather data, were regressed (using Poisson regression) against
regional differences in annual mean NO2 concentrations All the relative risks were
^
computed for a 0 0106-ppm (20-/*g/m ) increase in pollution concentration Nitrogen dioxide
by indoor passive sampler was predictive of the duration of any episode (relative duration of
1 16, 95% confidence interval of 1 12 to 1 21), upper respiratory episodes (relative duration
of 1 18, 95% confidence interval of 1 01 to 1 38), and coughing episodes (relative
duration of 1 15, 95% confidence interval of 1 03 to 1 29) A discussion of associations
with outdoor levels is presented in Section 14 3 2
14.3.1.10 Connecticut Study
Berwick (1987), Berwick et al (1984, 1987, 1989), and Leaderer et al (1986) reported
on a 12-week study (six 2-week tune periods) of lower and upper respiratory symptoms in
159 women and 121 children (aged 12 or less) living in Connecticut Nitrogen dioxide levels
were measured in 91 % of the homes, 57 of which had kerosene heaters and 62 of which did
not Ambient NO2 levels ranged from 9 to 19 /tg/m (0 005 to 0 01 ppm) for the six 2-week
tune periods Two-week average indoor NO2 levels in homes of monitored children were
highest for homes with kerosene heaters and gas stoves (91 jttg/m , 0 05 ppm, n = 8),
second highest for kerosene only (36 ptg/m3, 0 02 ppm, n = 45), third highest for gas stoves
3 3
only (32 jwg/m , 0 02 ppm, n = 13), and lowest for no sources (6 jtig/m , 0 003 ppm,
n = 43) Indoor levels did not fluctuate greatly over time, as indicated by the 2-week
averages A comparison of personal NO2 exposures, as measured by Palmes' diffusion
tubes, and NO2 exposures measured in residences had a correlation of 0 94 for a subsample
of 23 individuals Results of this comparison are depicted in Figure 14-3 and show an
excellent correlation between average household exposure and measured personal exposure
The study defined lower respiratory illness as the presence of at least two of the following
fever, chest pain, productive cough, wheeze, chest cold physician-diagnosed bronchitis,
physician-diagnosed pneumonia, or asthma Upper respiratory illness was defined as the
presence of two of the following fever, sore throat, nasal congestion, dry cough, croup, or
14-35
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0070
0060 -
0050 -
0040-
0030-
1
§ 0020-
S.
0010-
• Kerosene Heaters and Gas Stoves
• No Source
A Gas Stoves
V Kerosene Heaters
A
0010
0020
0030 0040
Average NO2/House (ppm)
)050
0060
0070
Figure 14-3. Total personal exposure to nitrogen dioxide versus nitrogen dioxide levels
in Connecticut residences.
Source Leaderer et al (1986)
head cold Although both upper and lower respiratory illness were investigated, the major
outcome of interest was lower respiratory symptoms The study obtained information on
many potential covanates, which included SES, age, gender, and exposure to environmental
tobacco smoke The covanates having the largest effect were age of the child, SES of the
family, and history of respiratory illness Multiple logistic analysis was used to allow for the
various factors
When controlling for SES and history of respiratory illness, children under the age of
*3
7 exposed to 30 jwg/m (0 016 ppm) NO2 or more were found to have an increased risk of
lower respiratory symptoms 2 25 times that of children who were not exposed
(95 % confidence limits of 1 69 and 4 79) They also had an increased risk of upper
respiratory symptoms of 1 33 (95% confidence limits of 1 19 and 1 49) Older children and
adults showed no increased risk
14-36
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Although the Berwick study had relatively extensive information on exposure, several
problems are evident The 3-year age-specific relative risks for lower respiratory disease are
very unstable, possibly due to the small sample sizes The rates do not appear to be
consistent with the rates for ages 0 to 6 and 7 and above, and it is not clear why a cut-off of
7 years of age was used The analyses may be sensitive to the adjustment for SES, which
can be correlated with exposure This is less of a problem in studies with larger sample
sizes (e g , Melia et al, 1977, 1979), but may be critical in the Berwick study Also, Neas
et al (1991) note that the Berwick study controls for prior illnesses, as did the Keller study,
which would reduce the estimated effect of current NO? exposure
14.3.1.11 Maryland Study
Helsing et al (1982) analyzed the records of 708 nonsmoking white adult residents of
Maryland to evaluate the effects of exposure to environmental tobacco smoke at home and
use of gas as a cooking fuel The frequency of cough and/or phlegm among these
nonsmokers showed a nonsignificant association with the presence of cigarette smokers in the
household Persons whose households had gas as a cooking fuel reported significantly more
chronic cough (relative risk of 2 1) and chronic cough amd phlegm (relative risk of 2 2) than
those in households using electricity for cooking The author noted that although gas
cooking has been considered by some as simply another indicator of poor social conditions,
the multiple adjustments for factors such as years of schooling and persons per room should
fully compensate for variations in socioeconomic level They also noted that all the
independent variables combined in the analysis accounted for only 5 to 10% of the variations
in symptomatology Respiratory ventilation function tests gave consistent results with
symptom reporting, with those using gas cooking showing impaired pulmonary function
14.3.1.12 German Study
Kuehr et al (1991) conducted a cross-sectional study on the prevalence of asthma in
childhood in relation to NO2 levels in the city of Freiburg and two Black Forest
communities A study group of 704 children aged 7 to 16 years took part in a standardized
interview and medical examination Indoor and outdooi exposure information was taken into
14-37
-------
account Passive smoking exposures were assessed Stoves used as heating devices carried a
4.8-fold relative risk for asthma compared to other types of heating
14.3.1.13 Canadian Studies
In a case-control study earned out in Montreal, Quebec, Canada, between 1988 and
1990, NO2 levels measured by passive NO2 monitoring badge were studied in relation to the
incidence of asthma among 3- and 4-year-old children (Infante-Rivard, 1993) Multivanate
unconditional logistic regression was earned out for the 140 subjects who had NO2
measurements, the analysis included NO2 and the vanables retained in the final conditional
model that includes SES and parental smoking The odds ratios for the NO2 categones
(defined as >0 0005 to 0 010, >0 010 to 0 015, and >0 015 ppm, in comparison with a
zero level) were 0 95 (95% confidence interval of 0 31 to 2 95), 3 85 (95% confidence
interval of 0 92 to 16 09), and 19 87 (95% confidence interval of 4 75 to 83 03),
respectively
Dekker et al (1991) studied asthma and wheezing syndromes as part of a questionnaire-
based study of 17,962 Canadian school children The questionnaire was developed from the
1978 American Thoracic Society questionnaire, which was the same one used in the Harvard
Six Cities Study For analysis, children were restncted to ages 5 through 8 years and those
with cystic fibrosis as well as those living in mobile homes, tents, vans, trailers, and boats
were excluded The authors calculated odds ratios adjusted for age, race, gender, parental
education, gender of the respondent, region of residence, crowding, dampness, and
environmental tobacco smoke The adjusted odds ratio of asthma as a function of gas
cooking was 1 95 with 95 % confidence limits of 1 41 and 2 68 The adjusted odds ratio of
wheezing as a function of gas cooking was 1 04 with 95 % confidence limits of 0 77 and
1 42 The authors note that this finding must be treated with caution, however, because of
the few subjects with asthma in the study who were exposed (n = 60)
14.3.1.14 North Carolina Study
Margoks et al (1992) studied the prevalence of persistent respiratory symptoms in
393 infants of different SES by analyzing data from a community-based cohort study of
respiratory illness in the first year of life in central North Carolina between 1986 and 1988
14-38
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Infants were limited to those weighing more than 2,000 g and who did not require neonatal
care outside the normal newborn nursery Of those eligible, 47% were enrolled, and of
these, 77% completed the study and were included in the analysis Compared with the
1,241 infants from families refusing enrollment, the 1,091 eligible study infants were more
likely to be of high SES and were more often black Study infants were less likely to have
mothers who smoked
The presence of persistent respiratory symptoms was measured at the 12-mo home
interview using an American Thoracic Society children questionnaire (modified for infants)
for studies of respiratory illness Infants who were reported to "usually cough" or
"occasionally wheeze" were classified as having persistent respiratory symptoms The
infant's SES was classified into three levels according to the highest level of education
achieved by the head of the household Each infant's exposure to tobacco smoke was
measured as the number of cigarettes smoked in the infants presence during the week prior to
the 12-mo home visit
Margolis et al (1992) used logistic regression to analyze to what extent the relationship
between SES and persistent respiratory symptoms could be accounted for by simultaneously
considering interactions between SES and other risk factors for lower respiratory illness and
confounding by other risk factors The relationship between the prevalence of persistent
respiratory symptoms and SES for infants in the study at low SES was 39 %, whereas 14 %
had persistent symptoms in the high-SES group Infants in the low-SES group were 2 9
(95 % confidence interval of 1 9 to 4 5) tunes more likely than infants of high SES to have
persistent respiratory symptoms Approximately 224 of the 393 infants in the study were
exposed to tobacco smokers Control for tobacco smoke exposure reduced the relative risk
of persistent symptoms among infants of low SES compared with high SES from 2 9 to 2 3
After accounting for all the risk factors, the effect of SES remained significant only for
infants not in day care
Of the 393 infants that Margolis et al (1992) included in their study, approximately
41 lived in homes with the environmental risk factor of gas cooking The relative risk of
persistent respiratory symptoms among infants exposed to gas cooking unadjusted for any
covanates was 1 12 (95 % confidence interval of 0 63 to 2 04)
14-39
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14.3.1.15 United States and Canadian Skating Rink Exposures
Hedberg et al (1989) reported that cough, shortness of breath, and other symptoms
among players and spectators of two high school hockey games played at an indoor ice arena
in Minnesota were related to emissions from a malfunctioning engine of the ice resurfacer
Although the exact levels of NO2 were not known at the tune of the hockey game, levels of
4 ppm (7,500 jig/m3) were detected 2 days later with the ventilation system working,
suggesting that levels during the games were higher Other pollutant levels such as PM10
may have also been elevated Hedberg et al (1989, 1990) reported that pulmonary function
testmg performed on members of one hockey team with a single exposure demonstrated no
decrease in lung function parameters at either 10 days or 2 mo after exposure Dewailly
et al (1988) reported another incident in a skating link in Quebec, Canada, in 1988
involving referees and employees reporting respiratory symptoms such as coughing, dyspnea,
and a suffocating feeling Five days after the incident, NO2 levels had come down to 3 ppm
(5,600 /ig/m3), suggesting much higher levels during the incident
In another skating rink study, Smith et al (1992) report the outcome of a questionnaire
administered to all students from two high schools on February 25, 1992, 3 days after
11 students participating in a Wisconsin indoor ice hockey tournament had been treated in
emergency rooms for acute respiratory symptoms (i e , cough, hemoptysis, chest pain, and
dyspnea) The game had been attended by 131 students, 57 of whom reported symptoms
A simulation test on February 24 provided levels of NO2 at 1 5 ppm (2,800 /xg/m3) in the air
over the rink after use of the ice resurfacing machine Higher levels may have been reached
the night of the game There are more than 800 ice arenas in the United States
14.3.2 Outdoor Studies
Several studies examined relationships between estimates of ambient NO2 levels and
respiratory health measures Those studies that provide a quantitative estimate of effect are
presented in Table 14-16 Health outcome measures include various respiratory
symptomologies
14-40
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TABLE 14-16. EFFECTS OF OUTDOOR NITROGEN DIOXIDE EXPOSURE
ON RESPIRATORY DISEASE
Odds Odds
Ratio or Ratio Conf
Study Health End Point NO2 Levels (ppm)/Penoda Estimate Interval
Dockery et al
(1989b)
Braun-Fahrlaender
etal 1992)
Bronchitis
Chronic cough
Chest illness
Wheeze
Asthma
Duration of
respiratory
episodes
Duration of
0 0065-0 0226/annual average
0 0065-0 0226/annual average
0 0065-0 0226/annual average
0 0065-0 0226/annual average
0 0065-0 0226/annual average
Change of 0 0106/6-week
average
Change of 0 0106/6-week
1 7
1 6
1 2
08
06
1 11
109
0 5 to 5 5
0 3 to 10 5
0 3 to 4 8
0 4 to 1 6
0 3 to 0 9
1 07 to 1 16
0 97 to 1 22
coughing episodes average
Schwartz et al Croup 0.0053-0 0371/daily average 1 28 1 07 to 1 54
(1991)
Schwartz and Phlegm 0 091/daily 1-h maximum 1 08 1 01 to 1 15
Zeger (1990) increase
Jaakkola et al Upper respiratory Contrasted polluted versus 16 1 1 to 2 1
(1991) infection less polluted areas by
comparison of annual levels
aMeasurement period over which stated nitrogen dioxide (NO2) level averaged
14.3.2.1 Six City Studies
As part of the Six City Studies, Dockery et al (1989b) obtained respiratory illness and
symptom data from questionnaires distributed from September 1980 to April 1981 Indoor
air aspects of this study (Dockery et al, 1989a) were described above, in the section on
indoor studies The questionnaires obtained information on bronchitis, cough, chest illness,
wheeze, and asthma A centrally located air monitoring station was established in 1974
where NO2, sulfur dioxide (SO2), ozone (O3), total suspended particulate (TSP), and
meteorological variables were measured The authors used multiple logistic regression
analysis in order to adjust for covanates of gender, age, maternal smoking, gas stove use,
and separate intercepts for each city Although the strongest associations were found
between respiratory symptoms and particulate matter, there were also increased odds ratios
for respiratory symptoms with ambient NO2 These were not statistically significant, but the
14-41
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direction for bronchitis, chronic cough, and chest illness was consistent with the studies of
indoor exposure The odds ratios for various health end points for an increase in NO2 from
the lowest exposure city to the highest exposure city (0 0065 to 0 0226 ppm, 12 to 43 i«g/m3)
are noted in Table 14-16
14.3.2.2 Swiss Study
Braun-Fahrlaender et al (1992) studied the incidence and duration of common airway
symptoms hi children up to 5 years old This study is also discussed in the earlier section on
indoor studies The study was conducted over a 1-year period in a rural, a suburban, and
two urban areas of Switzerland Parents were asked to record their child's respiratory
symptoms (from a list) on a diary form daily over a 6-week period Additionally, covanates
including family size, parental education, living conditions, health status of the child,
parents' respiratory health, and smoking habits of the family were assessed by questionnaire
Nitrogen dioxide was measured weekly during the same 6-week period using Palmes tubes,
both inside and outside the home of the participants Meteorological data were obtained
from local monitoring stations, but additional air quality data from fixed monitoring sites
were only available for the two urban study areas
The analysis was restricted to the 1,063 Swiss nationals (from a total of
1,225 participating families) For all four study areas, regional mean incidence rates of
upper respiratory illness, cough, breathing difficulties, and total respiratory illness, adjusted
for individual covanates and weather data, were regressed (using Poisson regression) against
regional differences in annual mean NO2 concentrations There was no association between
long-term differences in NO2 levels by region and mean annual rates of respiratory
incidence
The adjusted annual mean symptom duration (in days) by region and the corresponding
NO2 levels (measured by passive samplers to produce 6-week averages) are shown in
Table 14-17 A second-stage regression of the adjusted natural logarithm of regional mean
duration on NO2 levels yields significant associations between outdoor NO2 levels (6-week
averages) and the average duration (in days) of any respiratory episode (relative duration of
1.11, 95% confidence interval of 1 07 to 1 16) and upper respiratory episodes (relative
duration of 1 14, 95 % confidence interval of 1 03 to 1 25) A positive trend for the duration
14-42
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TABLE 14-17. ADJUSTED ANNUAL RESPIRATORY SYMPTOM DURATION
(DAYS) AND NITROGEN DIOXIDE LEVELS BY REGION (n = 1,063)
Region
Basel
Zurich
Wetzikon
Rafzerfeld
Any Symptom
Duration
450
421
400
3 88
URI
Duration
1 99
1 85
1 62
1 72
Cough Breathing Difficulty NO2 In
Duration Duration (ppm)
232
201
2 10
202
1 55
172
347
125
00166
00118
00103
00059
NO2 Out
(ppm)
00272
00248
00173
00133
aURI = Upper respiratory illness
Source Braun-Fahrlaender et al (1992)
of coughing episodes was also seen (relative duration of 1 09, 95% confidence interval of
0 97 to 1 22) No association was seen with the duration of breathing difficulties All the
't
relative risks are computed for a 0 0106-ppm (20-jwg/m ) increase in pollution concentration
In the suburban and rural areas, NO2 was the only air quality measure Correlation between
outdoor passive NO2 sampler and TSP measurements in the two urban study areas was quite
high (0 52) The high correlation between NO2 and TSP suggests that this NO2 association
may reflect confounding with TSP Unfortunately, the lack of TSP data for the other two
regions precludes eliminating TSP as a possible confourider in this analysis
Although the association with symptom duration in Zurich and Basel may well be due
to confounding with TSP, the cross-sectional association across the four regions supports a
possible contribution of NO2 The authors state that, thus, the association between medium
and long-term NO2 exposure and symptom duration deserves consideration as to a possible
causal relationship
14.3.2.3 German Studies
Schwartz et al (1991) studied respiratory illness in five German communities
Children's hospitals, pediatrics departments of general hospitals, and pediatricians reported
daily the numbers of cases of croup A diagnosis of croup was based on symptoms of
hoarseness and barking cough, inspiratory stridor, and dyspnea, and a sudden onset The
most important factors in croup etiology are parainfluenza viruses The croup counts were
modeled using Poisson regression with adjustments for weather, season, temperature,
14-43
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humidity, and autoregressive lag Statistically significant effects of both ambient participate
matter and NO2 were found on the counts of respiratory ilhiesses A relationship between
short-term fluctuations in air pollution and short-term fluctuations in medical visits for croup
symptoms was found in this study The estimated relative risk was 1 28 with 95 %
confidence limits of 1 07 and 1 54 for an increase from 0 0053 to 0 0371 ppm (10 to
*5
70 ^g/m ) of NO2 The NO2 results may be confounded with effects of particle levels
Rebmann et al (1991) studied 875 cases of croup in Baden-Wurttemberg in relation to
ambient NO2 levels over a 2-year period Statistical regression methods indicated weak but
statistically significant influences of the daily ambient NO2 mean on the occurrence of croup
Virologic testing was conducted on 205 cases that yielded positive results (including influenza
A and B, parainfluenza I-m, and RSV) in 34 cases
14.3.2.4 Los Angeles Student Nurses Data
Hammer et al. (1974) reported a daily diary study of morbidity symptoms in student
nurses in Los Angeles Diaries on morbidity symptoms were collected weekly from October
1961 to June 1964 Initially, 110 student nurses (over 90% of the class) agreed to
participate, but the class size decreased over the 3-year penod of the study so that by the end
of the third year, the cohort consisted of 30 student nurses Schwartz and Zeger (1990) and
Schwartz et al (1988) reported later analyses of the data They reexamined the original
diaries from the study, which contained smoking and allergy histories as well as symptom
reports. Ambient air pollution (NO2, SO2, carbon monoxide [CO], and photochemical
oxidants) was measured at a monitoring location approximately 2 5 miles from the
dormitory Pack-years of cigarettes were predictive of the number of episodes of coughing
o
and bringing up phlegm A daily 1-h maximum NO2 level of 0 091 ppm (170 jug/m ) was
associated with increased risk of phlegm (odds ratio of 1 08, 95% confidence interval of
1.015 to 1.15) and sore throat (odds ratio of 1 26, 95% confidence interval of 1 18 to 1 35)
Schwartz and Zeger (1990) note that although the use of only outdoor NO2 measurements
decreases sensitivity, the use of daily diaries should be more sensitive to detect effects of
NO2 than annual questionnaires Smoking, allergies, temperature, other pollutants, and
serial correlation were controlled for No paniculate measurements were available The
14-44
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mean of daily 1-h maximum NO2 levels over the study period was 0 13 ppm (245
with 25 and 75% levels of 0 06 and 0 17 ppm (113 and 320 /*g/m3), respectively
14.3.2.5 Chestnut Ridge Study
In the Fall of 1980, Vedal et al (1987) conducted a panel study on 351 children
selected from the 1979 Chestnut Ridge study Parents and children were instructed at the
beginning of the school year in completing daily diaries of respiratory symptoms, which were
used to define symptom outcomes Lower respiratory ilhiess was defined as wheeze, pain on
breathing, or phlegm production Of the 351 subjects selected for the 8 mo of follow-up,
128 participated in the completion of diaries Three subgroups were established one
without respiratory symptoms, one with symptoms of persistent wheeze, and one with cough
or phlegm production but without persistent wheeze Nitrogen dioxide was measured at a
single monitoring site in the study region Maximum hourly levels for each 24-h period
were used to reflect the daily pollutant level During the penod September 1980 to April
'j
1981, the mean NO2 maximum daily 1-h level was 40 5 /*g/m (0 021 ppm) with a range of
o
12 to 79 jwg/m (0 006 to 0 042 ppm) Regression models could not be fit for subjects who
never had symptoms, thus only 55 subjects were included in the analysis of lower respiratory
illness Nitrogen dioxide levels were not predictive of any symptom outcome
14.3.2.6 Finland Studies
Jaakkola et al (1991) studied the effects of low-level air pollution in three Finland
cities by comparing the frequency of upper respiratory infections over a 12-mo penod in
1982 as reported by parents of children ages 14 through 18 mo (n = 679) and 6 years
(n = 759) Pollutants studied included ambient levels of NO2 with an annual mean of
15 jiig/m (0 008 ppm) Other pollutants monitored weie SO2, hydrogen sulfide (H2S), and
particulate matter (measured as jwg/m3) Passive smoking and SES were taken into account
The authors report a significant association between the occurrence of upper respiratory
infections and living in an air-polluted area for both age groups studied, both between and
within cities The adjusted odds ratio was 1 6 (95 % confidence interval of 1 1 to 2 1) in the
6-year-old age group The authors conclude that the combined effect of SO2, particulate
matter, NO2, H2S, and other pollutants may be a contributing factor in the study results
14-45
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Ponka (1991) studied the effects of ambient air pollution and minimum temperature on
the number of patients who had asthma attacks and who were admitted to hospital in Helsinki
from 1987 to 1989 During the 3-year period, 4,209 hospitahzations for asthma occurred, an
average of 3 84 admissions a day The number of admissions increased during cold weather,
ranging from —37 0 to +26 4 °C, with a 3-year mean of 4 7 °C After standardization for
minimum temperature, the multiple-regression analysis indicated that NO2 and CO were
significantly related to asthma admission The annual NO2 levels averaged 38 6
(0.02 ppm) for the 3-year period During the period of high NO2 (daily mean 45 8 jug/m
[0.024 ppm]) levels, the mean number of all admissions was 29% greater than during the
o
period of lower pollution (28 1 j^g/m [0 015 ppm]) Indoor NO2 levels or cooking fuel use
were not discussed The observed association of incidence of asthma attacks with relatively
low levels of pollutants and cold weather accounted for an explanatory power of
approximately 14% in the regression analysis Other factors that may play a role in the
incidence of asthma attacks were not discussed
14.3.2.7 California Seventh-Day Adventist Study
In a California study, Euler et al (1988) assessed the risk of chronic respiratory disease
symptoms due to long-term exposure to ambient levels of TSP, oxidants, SO2, and NO2
Symptoms were ascertained using the National Heart, Lung, and Blood Institute questionnaire
on 8,572 Southern California Seventh-Day Adventists (nonsmokers—25 years and older) who
had lived 11 years or longer in their 1977 residential area Tobacco smoke (active and
passive) and occupational exposures were assessed by questionnaires, as were lifestyle
characteristics relative to pollution exposure, such as tune spent outside and residence
history For each of the 7,336 participants who responded and qualified for analysis,
cumulative exposures to each pollutant were estimated using monthly residence zip code
histories and interpolated exposures from state air monitoring stations
Multiple logistic regression analyses were conducted for pollutants individually and
together with eight covanables, including environmental tobacco smoke exposure at home
and at work, past smoking, occupational exposure, sex, age, race, and education
Statistically significant associations with chronic respiratory symptoms were seen for
(1) SO2 (p = 0 03), relative risk of 1 18 for 13% of the study population with 500 h/year of
14-46
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exposure above 0 04 ppm, (2) oxidants (p < 0 004) relative risk of 1 20 for 18% with
750 h/year above 0 1 ppm, and (3) TSP (p < 0 00001), relative risk of 1 22 for 25% with
750 h/year above 200 jtcg/m When these pollutant exposures were analyzed together, TSP
was the only one showing statistical significance (p < 0 01) Nitrogen dioxide exposure
levels in this population were not linked to chronic respiratory disease symptoms
Individuals working with smokers for 10 years had relative risks of 1 11 and those living
with a smoker for 10 years had relative risks of 1 07
14.3.2.8 Chattanooga Studies
Several studies were conducted in the greater Chattanooga area during the late 1960s
and early 1970s Although these studies were discussed in detail previously
(U S Environmental Protection Agency, 1982a), there are at least two additional points that
need to be made First, there were many measurements made in the area by methods other
than the Jacobs-Hochheiser method (e g , chemiluminescence) Reevaluation of the Jacobs-
Hochheiser method at a later tune questioned its accuracy for use in the studies to estimate
quantitative exposure-effect relationships Second, much of the pollution may have been in
the form of nitric acid (HNO3), and possible health effects may be related to HNO3 exposure
rather than NO2 itself The source of pollution was a large trinitrotoluene (TNT) plant,
located northeast of Chattanooga, which produced a substantial proportion of all TNT made
in the United States during World War n and the Korean War The plant was reopened in
April 1966 to supply munitions for use in Vietnam Annual averages of NO2 reached
-3
286 /*g/m (0 152 ppm) near the arsenal (as measured by the Saltzman method), and nitrate
3
fraction levels reached 4 1 fig/m at the downtown post office It is likely that the elevated
NO2 levels were accompanied by elevated HNO3 levels, although no direct measurements
were made The U S Environmental Protection Agency (1971) measured several factors
related to ambient air pollution including corrosion of zirtc, steel, and nylon The corrosion
levels in Chattanooga in 1967 and 1968 were among the highest in U S cities, and in the
case of nylon, were 10 to 100 times the levels of most other cities According to the report,
the arsenal was known to emit acid gases Additionally, Warner and Stevens (1973, 1975)
give other evidence suggesting the presence of sulfunc acid and HNO3 Thus, it is possible
that any adverse health effects seen in Chattanooga during this time period were associated
14-47
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with combined exposure to HNO3 and NO2 rather than with NO2 alone However, no
conclusion is possible because the health effects of HNO3 are poorly understood (see
Chapter 13)
Pearlman et al (1971) reported the results of a respiratory disease survey conducted in
the Chattanooga area in 1969 The study reported illness rates in children for the period
June 1966 to June 1969 Higher rates of bronchitis in school-aged children were found in
both the intermediate- and high-exposure areas, as compared with the low-exposure area
The results were not completely consistent with the exposure gradient because the rate of
bronchitis in the intermediate area was just as high as in the high-pollution area
Shy and co-workers (Shy, 1970, Shy et al , 1970a,b, 1973) studied the effects of
community exposure to NO2 in residential areas of Chattanooga on respiratory illness rates in
families. The incidence of acute respiratory disease was assessed at 2-week intervals during
the 1968-69 school year and the respiratory illness rates (adjusted for group differences in
family size and composition) were reported to be significantly higher for each family segment
(mothers, fathers, children) in the high-NO2 exposure neighborhood than in the intermediate-
and low-NO2 areas Although individual area pollution estimates are not available, one part
o
of the high pollution area had an annual average NO2 level of 286 /jg/m (0 152 ppm)
Areas more distant from the major NO2 source (TNT plant) had lower levels
Love et al (1982) studied acute respiratory disease in the same area during the years
1972-73 Fathers, mothers, school children, and preschool children all showed significantly
higher illness rates in the area designated as the high-pollution area during the beginning of
1972 There were almost no significant differences in illness rates during the periods
September to December 1972 and January to April 1973 During the period January to June
1972, NO2 levels (as measured by the continuous chemiluminescent method) ranged from
60 2 /tg/m3 (0 032 ppm) in the high area to 28 9 jug/m3 (0 015 ppm) in the low area
However, by the second half of 1972, the exposures in all areas were quite comparable
because of reduced emissions Thus, the results of the study tend to confirm the effect of
NO2 or its by-products on acute respiratory disease
14-48
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14.3.2.9 Glendora, California, Study
In another California study, Detels et al (1979, 1981a,b) and Rokaw et al (1980)
studied chronic respiratory disease symptoms and lung function in two areas of Los Angeles
County The low-exposure area was Lancaster, a city in the high desert country about
113 km from downtown Los Angeles, which was studied from November 1973 to October
1974 The high-exposure area was Glendora, an area in the Los Angeles basin, which was
studied from April 1977 to March 1978 The aerometnc exposures for Glendora were
estimated from a station in Azuza, about 5 km away Pollutants measured included total
oxidant (ultraviolet absorption method), NOX (Saltzman method), CO (nondispersive infrared
spectroscopy method), SO2 (conductimetnc method), hydrocarbons (flame lomzation
detection method), and particles (high-volume TSP method) The 5-year averages were
computed for those pollutants with sufficient data Comparing Lancaster to Glendora, the
NO2 levels were 0 033 versus 0 114 ppm, the total oxidant levels were 6 5 versus 116 ppm,
and the hydrocarbons were 2 9 versus 4 8 ppm Comparable differences existed for SO2,
particles, and sulfate fraction of particles, but the data were only complete for the year 1977
The authors evaluated symptom prevalence of cough, sputum production, wheezing, and
frequent chest illness All symptoms except frequent chest illness showed significantly
higher rates in Glendora for both sexes and all three smoking categories
The two primary weaknesses of the study are (1) the two areas (Lancaster and
Glendora) were measured at different tunes (1974 versus 1977), and (2) the areas are quite
different with respect to climate, commuting patterns, altitude, SES, season, and general
lifestyle No specific analyses related to NO2 levels were discussed The effects of smoking
habits were carefully controlled and should not be considered as a serious confounder The
authors also did attempt to control for variability in measurement methods and technicians,
and results of this are reported by Tashkin et al (1979)
14.4 STUDIES OF PULMONARY FUNCTION
Pulmonary function studies are part of a comprehensive investigation of the possible
effects of any air pollutant Measurements can be made in the field, they are nomnvasive,
and their reproducibility has been well documented Age, height, gender, and presence of
14-49
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respiratory symptoms are important determinants of lung function In addition, changes in
pulmonary function have been associated with environmental tobacco smoke
(Hasselblad et al, 1981), with paniculate matter in combination with SO2 (Dockery et al,
1982, Dassen et al, 1986), and with other factors
The rest of this section examines epidemiological studies relating NO2 exposure to
pulmonary function The pulmonary function studies in this section are divided into indoor
and outdoor subsections
14.4.1 Indoor Studies
Several of the studies discussed earlier with regard to respiratory disease symptoms
also included evaluations of pulmonary function Ware et al (1984), reporting on the Six
City Study, described analyses of lung function values using multiple linear regression on the
logarithm of the lung function measures Covanates included sex, height, age, weight,
smoking status of each parent, and educational attainment of the parents Forced expiratory
volume in 1 s (FEVj) values were significantly lower for children of current smokers than
for children of nonsmokers at both examinations and were highest for children of
ex-smokers Forced vital capacity (FVC) values were lower for children of nonsmokers than
for children of current smokers at both examinations, but the difference was statistically
significant only at the first examination Both the increase in mean FVC and the decrease in
mean FEVj among children of current smokers were linearly related to daily cigarette
consumption Exposure to gas stoves was associated with reductions of 0 7% in mean FEVj
and 0 6% in mean FVC at the first examination (p < 0 01), and reductions of 0 3% at the
second examination (not significant) The estimated effect of exposure to gas stoves was
reduced by approximately 30 % after adjustment for parental education The authors state
that the adjustment for parental education may be an over-adjustment, and may partially
represent gas stove use because of association between parental education and type of stove
Hasabelnaby et al (1989) provide estimation formulas for linear regression models that
incorporate errors in exposure variables using this data set as an example
Berkey et al (1986) used the Six City Study data from children seen at two to five
annual visits to evaluate factors affecting pulmonary function growth Children whose
mothers smoked one pack of cigarettes per day had levels of FEVX at age 8 years that were
14-50
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approximately 0 81 % lower than children of nonsmoking mothers (p < 0 0001) and had
FEV1 growth rates approximately 0 17% per year lower (p = 0 05) The same data
provided no evidence for an effect of gas stove exposure on growth rate
Neas et al (1991), discussed earlier, also reported that indoor NO2 levels were not
significantly associated with a decline in children's pulmonary function levels on either of
two examinations conducted prior to the indoor monitoring The ratio between FEVj and
FVC was actually increased in both boys and girls on both examinations
Goldstein et al (1988) reported preliminary data examining effects of acute exposure to
NO2 in inner-city apartments with gas cooking stoves on pulmonary function Eleven
asthmatic and 12 nonasthmatic women and children were monitored for 5 days with a
portable continuous NO2 monitoring instrument held at breathing level that provided 5-min
average NO2 levels The average levels observed over the 48-h sampling penod were over
100 ^g/m3 (0 053 ppm) NO2 in kitchens and over 70 pg/m3 (0 037 ppm) NO2 in bedrooms,
with peak levels significantly higher Pulmonary function (FEVl5 FVC, functional
expiratory volume at 25 to 75% of vital capacity [FEV25_75%], and peak flow), as well as
tracings of the entire flow curve, were monitored at several different points during the
exposure Although the data are limited, in this pilot study it seemed that at 5-min average
<3
NO2 exposures below 0 3 ppm (564 jwg/m ), FVC and peak expiratory flow (PEF) were as
likely to be increased as decreased, whereas at exposure above 0 3 ppm, FVC and PEF
mainly decreased for the adult asthmatic subjects
Ekwo et al (1983), discussed earlier, obtained pulmonary function measurements from
89 children whose parents did not smoke and 94 children whose parents smoked, and
reported no differences in lung function associated with gas stove use in a cohort of Iowa
children 6 to 12 years of age Dijkstra et al (1990) examined pulmonary functions in Dutch
children in a study discussed earlier Lung function was measured at the schools There
was a weak, negative association between maximal midexpiratory flow (MMEF) and
exposure to NO2 Forced expiratory volume in 1 s, PEF, and MMEF were all negatively
associated with exposure to tobacco smoke The authois concluded that the study failed to
document clear associations between indoor exposure to NO2 and lung function in Dutch
children 6 to 12 years of age
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Lebowitz et al (1985) studied a cluster sample of 117 middle-class households in
Tucson, AZ. Symptom diaries and peak flows were obtained over a 2-year period Outdoor
sampling of O3, TSP, CO, and NO2 was measured in or near the clusters Indoor sampling
of O3, TSP, respirable suspended particles, and CO was measured in a subsample of the
homes Additional information, such as the presence of a gas stove or smoking, also was
obtained The presence of a gas stove was used as a surrogate for indoor NOX exposure
because it was not measured directly The relationship of children's peak flow and gas stove
use was of borderline significance (p = 0 066) for an analysis excluding TSP, and was not
close to significance with TSP included in the analysis For asthmatic subjects, gas stove use
was significantly associated with peak flow decrements (p < 0 001) This was true across
smoking groups, but the difference was greatest for smokers Peak flow in adults was also
related to gas stove use, but the level of significance was not given
14.4.2 Outdoor Studies
Schwartz (1989) studied the effect of air pollution on lung function in U S children and
youths aged 6 to 24 years Forced expiratory volume, FEVl5 and peak flow measurements
taken as part of the National Health and Nutrition Examination Survey n Study were
examined after controlling for age, height, race, gender, body mass, cigarette smoking, and
respiratory symptoms Air pollution measurements were taken from all population-oriented
monitors in the U S Environmental Protection Agency's (EPA's) SAROADS data base
Each person was assigned the average value of each air pollutant for the 365 days preceding
the spkogram Highly significant negative regression coefficients were found for three
pollutants (TSP, NO2, and O3) with the three lung function measurements For an increase
3
of NO2 exposure of 0 015 ppm (28 3 jng/m ), an estimated decrease of about 0 045 L was
seen in both FVC and FEVj
Vedal et al (1987) conducted a panel study on 351 children selected from the 1979
Chestnut Ridge cross-sectional study of Pennsylvania elementary school-aged children (mean
age = 95 years) Peak expiratory flow rate (PEFR) was measured daily in 144 children for
9 consecutive weeks and was regressed against daily maximum hourly ambient concentrations
of NO2, O3, SO2, and coefficient of haze No air pollutant was strongly associated with
level of PEFR All pollutant levels were relatively low, daily maximum hourly NO2 levels
14-52
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o
ranged from 0 006 to 0 042 ppm (12 to 79 /ug/m ) No indoor measurements were made,
nor were any surrogates for indoor pollution included in the analysis
As part of the Six City Studies, Dockery et al (1989b) obtained pulmonary function
data during the 1980 to 1981 school year Respiratory illness and symptom data results were
discussed for this study in the earlier studies of respiratory illness section Only TSP
concentration was consistently associated with estimated lower levels of pulmonary function
There was little evidence for an association between lower pulmonary function level and the
annual mean concentration of NO2 or any other pollutant
In a study discussed earlier, Detels et al (1979, 1981a,b) and Rokaw et al (1980)
studied lung function in adults in two areas of Los Angeles County Lancaster, the low
NO2-exposure area, and Glendora, the high NO2-exposure area The authors found
significantly lower peak flow values in Glendora when compared with Lancaster, and these
differences were significant across all smoking categories and both sexes The percent of
subjects with FEVl or FVC below 50% of expected was also significantly higher in
Glendora Other lung function measurements showed less significant differences, but the
trend was always toward lower values in Glendora The primary weaknesses of the study
were discussed earlier
14.5 OUTCOMES RESULTING FROM OCCUPATIONAL EXPOSURES
Gamble et al (1987) studied 232 workers in four diesel bus garages for the effects of
NO2 on acute respiratory illness and pulmonary function Effects were assessed by an acute
respiratory questionnaire and before- and after-shift spirometry Measurements of NO2 over
the 6- to 7-h shift (using passive Palmes tube samplers) were made for each worker and were
collected on the same day as the pulmonary function tests and questionnaires Other irritant
gases were measured and were well below federal standards Mean NO2 levels over a shift
ranged from 0 56 (SD= 0 38) ppm NO2 in the highest garage to 0 13 (SD = 0 06) ppm
NO2 in the lowest garage Short-term NO2 measurements indicated levels above 1 ppm as
being common The authors report that the prevalence of acute respiratory symptoms were
elevated above expected in the high-exposure ( > 0 3 ppm) group only No reduction in
pulmonary function was associated with exposure
14-53
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Gamble et al (1983) examined chrome respiratory effects in 259 sodium chloride
miners. The Medical Research Council respiratory symptom questionnaire containing
smoking history was administered by trained interviewers A chest X ray and spirometry
were also conducted Personal samples of NO2 and respirable particles for jobs in each mine
were used to estimate cumulative exposure The cumulative mean exposure ranged from a
low of 0 2 (SD = 01) ppm NO2 to a high of 2 5 (SD = 13) ppm NO2 Diesel emissions
were the principal NO2 source The author reported that although cough was associated with
age and smoking and dyspnea was associated with age, neither symptom was associated with
indices of air pollution exposure (i e , years worked, estimated cumulative NO2 or
respiratory particle exposure) Reduced pulmonary function showed no association with NO2
exposure
Robertson et al (1984) reported on a 4-year study of lung function in 560 British coal
miners. Overall mean work shift average NO2 levels at nine coal mine sites ranged from
0.02 to 0 06 ppm (38 to 113 /*g/m3), and nitric oxide (NO) levels ranged from 0 13 to
1 19 ppm No relationship was found between exposure and decline in FEVj or respiratory
symptoms Jacobsen et al (1988) conducted a more extensive investigation on nearly
20,000 miners at the same nine British coal mmes to examine whether long-term exposure to
low concentration of NO2 and NO were associated with increased susceptibility to respiratory
infections The NOX source consisted of diesel emissions and blasting Work-shift median
levels were 0 2 ppm NO and 0 03 ppm NO2 This complete and intensive study had
problems with misclassification of exposure and outcome that are not uncommon when
existing data are used for purposes that were not foreseen when the data were collected The
authors concluded that the long-term exposure to the levels above do not detectably increase
the chance that miners will absent themselves from work because of a chest infection
Douglas et al (1989) report data between 1955 and 1987 on 17 patients examined at the
Mayo Clinic for silo-filler's disease shortly after exposure to silo gas (NO2 levels ranged
from 200 to 2,000 ppm) Health outcomes evaluated included hypoxemia, transient
obstruction of the airways, and death Meulenbelt and Sangster (1990) indicate that after a
symptom-free episode immediately following exposure to NO2, a severe respiratory failure
can develop several hours later The principal symptom, breathlessness, may become
manifest 6 or more hours following exposure as a result of adult respiratory distress
14-54
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syndrome Epler (1989) notes that prevention is essential for elimination of silo-filler's
disease
Other studies on high exposures (Lowry and Schuman, 1956, Grayson, 1956, Gregory
et al , 1969, Yockey et al, 1980) are reviewed in U S Environmental Protection Agency
(1982a) Levels above 300 ppm in occupational settings are likely to result in rapid death,
whereas levels of 150 to 200 ppm may lead to death 2 to 3 weeks after exposure Levels
between 25 and 100 ppm are usually, but not always, followed by essentially complete
recovery
14.6 SYNTHESIS OF THE EVIDENCE
The weight of the evidence does not indicate that NO2 exposure at levels reported in
the studies reviewed here has any consistent effect on pulmonary function of a biologically
significant magnitude Many of the indoor studies, however, do suggest an increase in
respiratory morbidity in children from exposure to NO2 levels measured in these studies,
although the effects reported did not reach statistical significance (p < 0 05) in the majority
of the studies The consistency of results across the indoor studies is examined and the
evidence is synthesized in a quantitative analysis presented below
In order to compare available studies on respiratory effects of NO2, a common end
point for a health outcome effect was defined, and then each indoor study was compared with
this standard end point The end point chosen was the presence of lower respiratory
symptoms and illness in children aged 5 to 12 years An attempt was made to include as
many indoor studies as possible The requirements foi inclusion were (1) the health end
point measured must be reasonably close to the standard end point, (2) significant exposure
differences between subjects must exist and some estimate of exposure must be available, and
(3) an odds ratio for a specified exposure estimate must have been calculated, or data must
be presented so that an odds ratio can be calculated
14.6.1 Health Outcome Measures
One major concern in attempting to interpret these studies is that the respiratory
morbidity variables measured in the various studies may represent differing health outcomes
14-55
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that may have different mechanisms of causation related to NO2 exposure estimates For
example, the origin of cough may be different than that of wheeze (i e , the agents that cause
them may be different) Various disease syndromes were evaluated in different studies
croup, bronchitis, bronchiolitis, asthma, and pneumonia The instruments measuring health
outcomes in the studies are also different If the similarity between the outcome measures
between and within the studies is not adequate, the potential interpretation of a quantitative
analysis may be limited Are the respiratory morbidity measures in these studies of children
a relatively similar outcome measure in both a statistical sense and as a biological end point
across the studies9 This discussion considers this question by evaluating outcome measures
used in the various NO2 studies, similarities in outcome measures in lower respiratory illness
studies in children, the use of questionnaires as instruments to measure lower respiratory
morbidity, and, in general, measures of lower respiratory morbidity
The studies in the quantitative analysis that follows used health outcome measures that
provide an indication of the state of respiratory health of various samples of children aged
5 to 12 years old The NO2 studies utilized standard questionnaires to evaluate lower
respiratory health in children Diagnoses of specific respiratory diseases such as
bronchiolitis or asthma were not made The factor of importance here is that an attempt was
made to measure some aspect of lower respiratory morbidity Table 14-18 lists the health
outcome measures for each study considered Whereas specific measures such as colds going
to chest (Melia et al, 1977), chest congestion, and phlegm with colds (Ekwo et al, 1983)
are used to provide measures of lower respiratory morbidity, other measures use indexes,
grouped responses, or combined indicators of lower respiratory morbidity, some of which
include measures such as colds going to chest
In the Melia et al (1977, 1979, 1980, 1982a) studies, a self-administered questionnaire
was completed by parents of children in the study Questions were asked about each child's
respiratory disease episodes and symptoms during the previous 12 mo The respiratory
symptoms and diseases surveyed included asthma in the last year, wheeze, bronchitis in the
last year, cough (night or day), and colds going to chest Irwig et al (1975) examined the
association of these reported questions with objectively measured reduced peak flow rates in
a sample of the children examined in 1973 Because the answers to the morbidity questions
were related to the reduced adjusted mean peak flow rates (an indicator of decreased
14-56
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TABLE 14-18. HEALTH OUTCOME AND NITROGEN DIOXIDE EXPOSURE MEASURES USED IN
SELECTED INDOOR NITROGEN DIOXIDE EPIDEMIOLOGY STUDffiS
Reference
Melia et al
Melia et al
Melia et al
Florey et al
Goldstem et
MeLa et al
Ware et al
(1977)
(1979)
(1980)
(1979)
al (1979)
(1982a)
(1984)
Health Outcome Used in Meta-Analysis
Colds going to chest showed a prevalence
of 26 8-19 8%
Responses to respiratory questions
grouped into (a) none or (b) one or more
symptoms or disease types Colds going
to chest (26 4-19 6%) showed the highest
prevalence, followed by wheeze
(10 1-6 2%), cough, and episodes of
asthma or bronchitis in last year
Group response to respiratory questions
as above
As above
Lower respiratory illness index (mdex of
Method*
Symptoms during past
12 mo recalled by
child's parent in
completing respiratory
symptoms
questionnaire
As above
As above
As above
Questionnaire (Ferns,
NO2 Exposure
Measure Used Age
in Analysis (years)
Gas stove vs 6-1 1
electric stove
Gas stove vs 5-10
electee stove
NO2 measured with 6-7
Palmes tubes Gas
stove homes only
NO2 measured with 5-6
Palmes tubes Gas
stove homes only
Gas vs electric 6-10
Sample
Size Where/When
5,658 28 Areas of
England and
Scotland (1973)
4,827 27 areas of
England and
Scotland (1977)
103 Middlesborough,
England (1978)
188 Middlesborough,
England (1980)
8,240 Six US cities
respiratory health) indicating during past
year the presence of (a) bronchitis,
(b) respiratory illness that kept the child
home 3 days or more, or (c) persistent
cough for 3 mo of the year
1978) completed by
parent for symptoms
during previous 12 mo
(1974-1979)
-------
TABLE 14-18 (cont'd). HEALTH OUTCOME AND NITROGEN DIOXIDE EXPOSURE MEASURES USED IN
SELECTED INDOOR NITROGEN DIOXIDE EPIDEMIOLOGY STUDDZS
Reference
Health Outcome Used in Meta-Analysis
Method*
N02 Exposure
Measure Used
in Analysis
Age Sample
(years) Size
Where/When
Neas et al (1990,1991) Combined indicator of one or more lower Symptom questionnaire NO2 measured with 7-11
respiratory symptoms as defined The
highest prevalences were for chronic
phlegm and wheeze The other
symptoms in the index are shortness of
breath, chronic cough, and bronchitis
Chest illness reflects a restriction of the
child's activities for 3 or more days
completed by parent for Palmes tubes Gas
the year during which and electric stoves
measurements of NO2
were taken
1,286 Six US cities
(1983-1986)
I— '
s
oo
Ekwo et al (1983)
Dykstraetal (1990)
Brunekreef et al (1987)
Keller et al (1979a,b)
Chest congestion and phlegm with colds
Respiratory illness combination variable
of presence of one or more of cough,
wheeze, or asthma
Respiratory illness
Questionnaire (ATS)
completed by parent
Questionnaire (WHO)
completed by parent
Telephone interview by
nurse epidemiologist
Gas stove vs 6-12
electric stove
NO2 measured with 6-12
Palmes tubes Gas
and electric
appliances
Gas stove vs < 12
electric stove
1,138 Iowa City, Iowa
775 Netherlands
(1986)
176 Columbus, Ohio
(1978)
ATS = American Thoracic Society
WHO = World Health Organization
NO2 = Nitrogen dioxide
-------
respiratory function), this suggested that the questions may be indicators of lower respiratory
morbidity (whereas others such as earache or hospitahzation for upper respiratory disease in
the last 12 mo may not) Thus, the more subjective morbidity measurements by the
questionnaires were supported to some extent by more direct objective lung function
measurements Melia et al (1977) indicated that the highest prevalence was for colds going
to the chest (approximately 25%), followed by wheeze (approximately 10%) Bronchitis and
asthma episodes in the past year had respective prevalences of less than 6 % and 3 % The
1977 studies showed very similar prevalences (Melia et al , 1979) to the 1973 data, with the
prevalence of asthma and bronchitis episodes both under 4% and colds going to chest at
approximately 25% in 1977
For these Melia studies, two indicators, colds going to chest and wheeze, provide the
major contnbution to the combined indicator for lower respiratory health Disease indicators
such as asthma and bronchitis episodes in the past year, although measures of lower
respiratory disease, may play a smaller role The Melia et al (1979) study provides data
that allow the development of graphs of the marginal likelihood functions (see Figure 14-4)
of the odds ratios for symptoms and diseases and any respiratory illness for the combined
indicator of boys and girls The odds ratio for colds to chest is 1 21 (SD of the log [OR] =
0 0675) and for any respiratory illness is 1 24 (SD of the log [OR] = 0 0703) This
demonstrates the similarity of these two outcome measures More specifically, it shows how
colds going to the chest represent an important component of Melia's respiratory index
In the quantitative analysis, the outcome measure used is colds going to chest for the Melia
et al (1977) study and the lower respiratory index for the other Melia studies
Other NO2 studies use different indexes or combinations that include symptoms such as
chronic phlegm and wheeze, persistent cough, respiratory illness, and asthma and bronchitis
(Ware et al, 1984, Neas et al , 1990, 1991, Dijkstra et al, 1990, Keller et al, 1979b)
These symptoms in the indexes are all indicators of involvement of the lower respiratory
tract and, as such, form a measure of lower respiratory health In most cases, chest colds
and wheeze, or similar indicators, are the prominent symptoms in the index Use of the
same mdex in several of the NO2 studies and similar indexes in the other NO2 studies
provides consistency in the outcome measurements of the studies
14-59
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Colds to
Chest
Wheeze
Asthma
05
1
Odds Ratio
Combined Indicators
Lower Respiratory
Illness
Morning
Cough
15
Figure 14-4. For the Melia et al. (1979) study, a graph of the marginal likelihood
function of the odds ratios for combined gender (boys and girls) of the
respiratory illness outcome measures developed by EPA.
How do the outcome measures in the NO2 studies compare with indicators of lower
respiratory health status done in other studies9 The studies of lower respiratory illness
(unrelated to NO2 exposures) discussed in Section 14 3 were based on visits to physicians
The signs and symptoms that were most common in those studies, wet cough and wheeze,
are similar to the most common end points in the NO2 studies, that is, colds going to chest
and wheeze Thus, to an extent, the NO2 respiratory studies are providing a measure of
lower respiratory health somewhat equivalent to these other studies Specific pediatnc
diseases and agents such as bronchiohtis and RSV are investigated in these other studies and
not in the NO2 respiratory studies The overall pattern and incidence of lower respiratory
14-60
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illness are considered to be consistent in different geographic regions over tune (Wright
et al, 1989, Denny and Clyde, 1986, McConnochie et al, 1988)
Lower respiratory illness is an entity commonly defined by criteria for clinical
diagnosis (Wright et al, 1989) Key studies (Monto et al, 1971, Taussig et al, 1989,
Glezen et al , 1971, Gardner et al, 1984, and Maletzky et al., 1971) consider the
classification of lower respiratory disease and surveillance methods Standardized and
uniformly accepted clinical criteria have not been developed for the illnesses considered to
make up lower respiratory illness, that is, for croup, tracheobroncmtis, bronchiolitis, and
pneumonia The extent to which the diagnostic criteria for lower respiratory illness were
documented vanes in these studies The characteristic signs and symptoms are wheeze,
phlegm from chest, and deep cough These are basic respiratory symptoms indicative of
lower respiratory involvement Samet et al (1992) classified illnesses as lower respiratory
tract illness if wet cough or wheeze occurred at any tune during the illness exposure Bates
et al (1990) note that differentiation among "acute bronchitis", "acute bronchiohtis", and
acute exacerbation of "asthma" is clinically difficult, therefore, it is unrealistic to expect
finite and noncontroversial differentiation among these conditions
For the purpose of analysis of illness rates, childien with lower respiratory morbidity
may be considered to belong to a single population with a similar illness. Although different
diseases are grouped together as lower respiratory illness (such as croup, bronchiolitis, and
pneumonia), the lower respiratory illness syndrome designation (Monto et al, 1971) is a
means of grouping these illnesses of similar pattern for analytic purposes It is relatively
easy to classify illnesses on the basis of anatomic area of involvement Graham (1990) states
that classification by anatomic site remains the preferred system for most physicians and is
compatible with the International Classification of Diseases system Such a classification is
often a good indication of the seventy of illness
The respiratory questionnaires used in the NO2 studies determined the state of lower
respiratory health in the subjects by tabulating responses to questions on respiratory
symptoms such as cough and wheeze and on respiratory illnesses such as asthma and
bronchitis Symptom reporting provides subjective evidence of respiratory infection and
diseases such as bronchitis Diseases are characterized by groups of symptoms Disease end
points such as asthma and bronchiolitis may have similar defining symptoms Lower
14-61
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respiratory morbidity reflects a broader grouping, within which the ability to differentiate
between the wheeze of asthma and wheeze related to other causes or infections in the
pediatnc age group may not be readily possible Wright et al (1989) note that wheeze is
recorded as occurring for several diagnoses to include croup, bronchiolitis, bronchitis, and
pneumonia Questions of asthma in the past year may not represent a disease-defining
measure as much as a measure of wheeze Additionally, asthma may be related to the
occurrence of infection as a precipitating cause, thus further confusing differences between
symptoms and disease
The NO2 studies of lower respiratory health are implemented by use of standardized
questionnaires filled out by the parent for illness and symptoms during the past 12 mo The
purpose of a respiratory symptom questionnaire is to compare prevalence of symptoms
(Fairbairn et al., 1959) and to follow their rate of progression in populations This facilitates
the quantitative comparison between groups This, however, contrasts fundamentally with
the usual objective of clinical medicine, that is, a decision about an individual providing an
accurate diagnosis of the patient's condition to determine the correct treatment or prognosis
In surveys of prevalence, valid comparisons can be made with less accurate data, providing
that a sufficient population is studied and that inaccuracies of reporting are randomly
distributed between the groups being compared Standardized questionnaires permit
meaningful comparisons of the results of prevalence surveys (Samet, 1978) Mitchell et al
(1976) note that the information obtained from the subjects in a respiratory health study that
used a questionnaire are probably an accurate reflection of the lung disease experienced by
the total population of these communities
Childhood lower respiratory morbidity is characterized by a grouping of similar
symptoms and diseases that reflect changes located anatomically in the lower respiratory
tract. This characterization represents an indication of seventy of the respiratory morbidity
status of the children and is a multifaceted approach to respiratory health in a population
living under natural conditions Lower respiratory morbidity is the combination of different
respiratory effects that have in common an evaluation of the morbidity status of the lower
respiratory tract. The measure of effect on the lower respiratory tract varied among the
studies; the indicators, however, are conventional symptom and illness outcomes The
symptoms are tabulated from similar standardized questionnaires (Ferns, 1978) and are
14-62
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directed at eliciting the same basic data—an indication of the presence of illness or infection
in the lower respiratory tract
Although the use of identical health outcome measures would be most desirable, the
level of similarity and common elements between the outcome measures in the NO2 studies
provide some confidence in their use in the quantitative analysis However, the symptoms
and illnesses combined are to some extent different and could indeed reflect different
underlying processes Thus, caution is necessary in interpreting the analysis This concern
is addressed further later in this section as part of the statistical aspects of the random-effects
model
14.6.2 Biologically Plausible Hypothesis
The human clinical and animal toxicological studies in Chapters 13 and 15 that
examined NO2 effects on aspects of the respiratory host defense system provide a
biologically plausible hypothesis compatible with the relationship seen between respiratory
symptoms and morbidity and NO2 exposure in epidemiologic studies However, research
gaps in both animal toxicological and clinical studies exist, indicating the need for further
research efforts A brief discussion is presented here.
The lung is one of the sites of microbial infection Although many types of
microorganisms are implicated in respiratory infection, viruses represent a major cause of
respiratory disease, particularly for infants and children In a viral respiratory infection,
viral replication produces injury and, thus, the signs and symptoms of respiratory illness
(Douglas, 1986) The respiratory system has several defense mechanisms against inhaled
infectious and chemical agents Host defense mechanisms consist of nonspecific and specific
components The nonspecific aspects include mucociliary clearance and alveolar
macrophages, whereas humoral and cellular immunity offer specific defenses The immune
system functions through a sequence of events, starting with the nonspecific components
followed by responses by the specific components The immune system is a principal factor
in the host's interaction with infectious agents such as viruses and in the host's ability to
contain and/or eradicate the establishment of infection To some extent, an increase of
reported respiratory symptoms in some epidemiology studies may be an indication of the
ability of the respiratory host-defense mechanism to either overcome an infection or to limit
14-63
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its severity. Nitrogen dioxide may affect the immune system in such a way that one or
several aspects of the immune system do not function at a level sufficient to limit the extent
or occurrence of infection Nitrogen dioxide may to some degree influence respiratory
symptom rates by direct toxic mechanisms Meulenbelt and Sangster (1990) note that NO2
may cause direct epithelial damage that could increase susceptibility to infection
The evidence from animal toxicological and human clinical studies of host defenses
provide a rationale for investigating the relation between exposure to NO2 and an increase in
frequency and seventy of respiratory symptoms and/or infections in humans When
microorganisms attack a lung that has been exposed to NO2, host defense mechanisms altered
by the NO2 exposure may result in increased seventy or rate of respiratory illness Although
the host defense system reacts both very specifically and generally to the challenge, the
overall response in humans is expressed as a generalized demonstration of signs and
symptoms that may be associated with a site such as the lower respiratory tract and also may
be reported or objectively discerned as a general outcome such as a chest cold, cough, or an
incident of asthma or bronchitis
14.6.3 Publication Bias
Publication bias, also known as the "file drawer problem" (Rosenthal, 1979), is the
result of the increased likelihood of publication of studies that have positive results, leading
to a bias in the literature reviewed towards positive results There are two factors that make
this bias less likely for epidemiological studies First, the studies are expensive, well
publicized, and the results are usually published in order to give credit to the researchers
involved. Second, many of the studies included in this section did not produce statistically
significant findings, indicating that there was not a problem in publishing negative studies
However, some studies are necessarily excluded because they provide insufficient
information. Although this can lead to bias, there is little that can be done to correct for this
problem This problem is not normally referred to as publication bias, but it is a similar
problem.
14-64
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14.6.4 Quantitative Analysis
In order to compare available indoor studies on respiratory effects of NO2, a common
end point for a health outcome effect was defined, and then each study was compared with
this standard end point The end point chosen was the presence of lower respiratory
symptoms and illness in children aged 5 to 12 years An assumption has been made that the
relative odds of developing this lower respiratory morbidity outcome is similar across this
age range as a function of NO2 exposure, even though Ihe actual rates may not be (This is
a common assumption in many analyses )
An attempt was made to include as many indoor studies as possible The requirements
for inclusion were (1) the health end point measured must be reasonably close to the standard
end point, (2) significant exposure differences between subjects must exist and some estimate
of exposure must be available, and (3) an odds ratio foi a specified exposure estimate must
have been calculated, or data must be presented so that an odds ratio can be calculated All
studies that met the criteria for inclusion were included Quality scores were not assigned to
the studies There is no evidence that the use of quality scores improves estimates (see
Emerson et al, 1990) Separate analyses for studies that had specific features that might be
considered in quality scores, such as measured NO2 instead of surrogate estimates, are
examined later as part of the analysis
The term "exposure" is used to denote the pattern of concentrations of a pollutant in air
through which an individual passes during a fixed period of tune The term "exposure
estimate" will be used for any measure that estimates some function of that pattern, such as
the arithmetic average Exposure estimates include both personal monitors used for some
fraction of the total exposure time as well as fixed monitors located in rooms known to be
occupied by individuals for some fraction of the tune Some estimates may be based on
averages from sites with similar exposure characteristics, such as presence of a gas stove
Thus, exposure estimates are defined as an estimate based on some data of the exposure of
the group being studied Such an estimate cannot perfectly characterize true exposure See
Section 7 3 for a more detailed discussion on exposure to NO2
The goal was to estimate the odds ratio corresponding to an increase in concentration
level of 0 015 ppm (28 3 /*g/m3) as an estimate of NO2 exposure Studies with NO2
exposure measures used 1- to 2-week integrated indoor measurements by Palmes passive
14-65
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diffusion tubes that provide an estimate for chronic exposure Studies that characterized NO2
exposure by differences between gas stove and electric stove use estimated a value for this
difference Four studies measured NO2 levels Five studies estimated exposure to NO2
based on the presence or absence of a gas stove as a surrogate for NO2 measurements
Exposure measurement error related to use of a surrogate was discussed earlier To use
these studies in the meta-analysis, numerical values of exposure estimates must be
determined Three of the studies were conducted in the United States (Ware et al, 1984,
Ekwo et al, 1983, and Keller et al, 1979a,b) and two in Great Britain (Melia et al , 1977,
1979).
Limited data are available from which to estimate NO2 exposure values Appropriate
estimates ideally would be country specific, current with the studies in location and tune, and
derived from a representative sample that appropriately characterizes the exposure The
Neas et al. (1991) study may be an appropriate source to estimate a value for the three
United States studies This study had a large sample size, measured levels during two
seasons, and was conducted in the same United States cities as Ware et al (1984) was Neas
et al. (1991) reported a housewide average difference of 0 0173 ppm (32 5 /ig/m ) NO2
between homes with electric stoves and homes with gas stoves with pilot lights In two
British studies (Melia et al, 1980, 1982a,b) conducted by the same authors that conducted
Melia et al (1977, 1979), data on the difference in levels between homes with electric stoves
and homes with gas stoves is provided Melia et al (1980, 1982a,b) provide data that
indicate 0.0165 ppm (311 /ig/m ) as an estimate of this difference in average NO2
concentrations in bedrooms of homes in Britain
The effects studied may be related to peak exposures, average exposures, or a
combination of the two To the extent that health effects depend on peak exposures rather
than average exposures, the above exposure estimates introduce exposure measurement error
These studies cannot distinguish between the relative contributions of peak and average
exposures and their relationship with the observed health effects Additionally, a by-product
of NO2, HONO, may be a factor in observed effects, however, limited health and aerometnc
data are available that examine such possibilities
The above factors are used when evaluating each study The British studies provide
several estimates of the subject odds ratio Meka et al (1977) studied children aged 6 to
14-66
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11 years and developed an indicator of the presence of at least one of a group of symptoms
including cough, colds going to the chest, and bronchitis The symptom reported most often
was "colds going to chest", which was used as an indicator of lower respiratory morbidity
This study did not measure NO2 exposure, and so the assumption was made that the increase
in NO2 exposure from gas stove use in England was reasonably similar to that in the other
^
British studies that measured NO2, that is, an increase of 0 0165 ppm (311 /*g/m ) The
estimated odds ratio was 1 31, with 95% confidence limits of 1 16 and 1 48 After adjusting
O
to a standard increase of 0 015 ppm (28 3 /*g/m ), the odds ratio became 1 28 with
95% confidence limits of 1 14 and 1 43 No adjustment was made for parental smoking in
this study
The cross-sectional data reported by Meha et al (1979) on children aged 5 to 10 years
also was employed to estimate an odds ratio, although no exposure estimates were made
The presence or absence of a gas stove was used as a surrogate as in the Meha et al (1977)
study The estimated odds ratio was 1 24, with 95 % confidence limits of 1 09 and 1 42
After adjusting to a standard NO2 increase of 0 015 ppm (28 3 jwg/m3), the odds ratio
became 1 22 with 95% confidence limits of 1 08 and 1.37
Meha et al (1980) studied children aged 6 to 7 years and measured bedroom NO2
levels for the exposure estimate This study applied the same combined health end point as
the previous study The estimated odds ratio for an NO2 increase of 0 015 ppm
(28 3 /*g/m3) was 1 49 with 95% confidence limits of 1 04 and 2 14
Melia et al (1982a) studied children aged 5 to 6 years, measured NO2 exposure in the
bedroom, and also applied the same combined health end point The 10th and 90th
percentiles of the weekly measured concentrations were 0 009 and 0 065 ppm NC^,
respectively, in bedrooms (Melia et al, 1982b) The estimated odds ratio for an NO2
increase of 0 015 ppm was 1 11, with 95% confidence limits of 0 84 and 1 46
In the first Six City study cohort, Ware et al (1984) reported an index of respiratory
illness Exposure to NO2 was based on the presence or absence of a gas stove (0 0173 ppm
[32 5 /ig/m3]) The estimated odds ratio was 1 08 with 95 % confidence limits of 0 97 and
*5
1.19 After adjusting to a standard NO2 increase of 0 015 ppm (28 3 jwg/m ), the odds ratios
became 1 07 with 95% confidence limits of 0 98 and 1 17
14-67
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A second cohort of subjects in the Six City study was initially reported on by Dockery
et al. (1989a). This cohort of children aged 7 to 11 years was then reinterviewed after
indoor NO2 measurements were made, and the results of this analysis were reported by Neas
et al. (1990, 1991). The 10th and 90th percentiles of the weekly measured concentrations
were 0.008 and 0 033 ppm NO2, respectively in bedrooms (Neas et al, 1991) The
estimated odds ratio for an increase in the presence of any respiratory symptom resulting
3
from an increase in NO2 exposure of 0 015 ppm (28 3 ftg/m ) was 1 40, with 95 %
confidence limits of 1 14 and 1 72
Ekwo et al (1983) studied several respiratory illness end points from children surveyed
at ages 6 to 12 years No exposure measurements were obtained, and the exposure was
based on the presence or absence of a gas stove (0.0173 ppm [32 5 /tg/m3]) None of the
end points matched the end pomt of interest closely The two most similar end points were
hospitahzation for chest illness before age 2 and chest congestion and phlegm with colds
The estimated odds ratio for hospitalization was 2 40. The estimated confidence limits for
cough and phlegm with colds was 1 09, with 95% confidence limits of 0 82 and 1 45 This
last symptom appears to be most similar to the end point of interest, and so it was included
in the synthesis
The data presented by Dijkstra et al (1990) on the study in the Netherlands were
analyzed and gave an estimated odds ratio of 0 94 for an increase of 0 015 ppm (28 3 /tg/m3)
in NO2 exposure The 95% confidence limits were 0 70 and 1.27. The study had measured
NO2 exposure data, but the EPA analysis did not adjust for covanates because the covanates
were not included in the tables that included NO2 exposure
Keller et al (1979b) did not find any statistically significant changes in respiratory
disease associated with gas stove use, but the unadjusted estimated odds ratio for lower
respiratory illness was 0 72, with 95 % confidence limits of 0 30 and 1 74. Assuming that
3
the NO2 exposure increase was 0.0173 ppm (32 5 /xg/m ), the odds ratio was adjusted to an
exposure of 0.015 ppm (28.3 jtg/m3) This resulted in an odds ratio of 0 75 with 95%
confidence limits of 0 35 and 1 62
Three studies with sufficient information for analysis were excluded from the synthesis
The Berwick et al (1989) analysis has been criticized for its lack of consistency across age
groups, which may have resulted from the very small sample sizes The Swiss study
14-68
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(Braun-Fahrlaender et al, 1989, 1992) examined end points that might not be considered as
similar to the other studies, such as upper respiratory disease, breathing difficulties, and
duration of various respiratory measures The Melia et al (1988, 1990) study did not
demonstrate significant estimated NO2 exposure differences between the two groups
contrasted (0 0034 ppm [6 4 jitg/m3]) These differences estimated in exposure were much
smaller than those seen for any other study of gas stove exposure This may reflect use of
gas ranges without pilot lights and changes in cooking practices such as increased use of
microwave ranges (see Section 7-3) If the relative risk were adjusted for an increase of
^
0 015 ppm (28 3 jttg/m ), the relative risk would be about 1 29, which is comparable to the
odds ratios seen in the other studies Because the difference in exposure groups was so
small, requiring a very large adjustment, it was decided not to combine this study with the
other studies For these reasons, the above studies were not included in the synthesis
These studies, however, qualitatively support the results of the synthesis
Graphs of the odds ratio from each indoor study included in the quantitative analysis
are depicted in Figure 14-5 Each curve can be given one of three interpretations (1) the
normal approximation to the marginal likelihood of the logarithm of the odds ratio,
(2) a distribution such that the 2 5 percentile and the 97 5 percentile points of the distribution
are the 95 % confidence limits of the estimated odds ratio, and (3) the posterior for the odds
ratio for a particular study given a flat prior on the log-odds ratio The basic information for
each curve is provided in Table 14-19
Synthesizing evidence (often referred to as meta-amalysis) is not new, having been used
as early as 1904 (Pearson, 1904) Sacks et al (1987) defines meta-analysis as a discipline
that critically reviews and statistically combines the results of previous research Meta-
analyses are being used much more frequently now, as indicated by Mann (1990) For
example, the National Research Council (1986) combined evidence on the effect of
environmental tobacco smoke on lung cancer using Peto's method as described by Yusuf
et al (1985) Also, several methods for combining clinical trials were discussed by Laird
and Mosteller (1990) The evidence to be combined in this section comes from
epidemiological studies and, as a result, some of the methods used for clinical trials are not
appropriate for this section
14-69
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Meliaetal (1977)
Meliaetal (1982a)
/Neasetal (1991)
Ekwoetal (1983
Keller etal (1979b)
Odds Ratio
Figure 14-5. U.S. Environmental Protection Agency meta-analysis of indoor
epidemiologic studies of nitrogen dioxide exposure effects on respiratory
disease in children 5 to 12 years old. Each curve can be treated as a
likelihood function or posterior probability distribution. If treated as a
likelihood function, the 95% confidence limits for the odds ratio can be
calculated as those two points on the horizontal axis for which 95% of the
area under the curve is contained between the two points. If treated as a
posterior probability distribution, then the area under the curve between
any two points is the probability that the odds ratio lies between those two
points.
Two basic models are employed in order to combine evidence (Hasselblad et al, 1992)
The first model assumes that each study estimates the same fixed, but unknown, parameter
Most methods of combining evidence make this assumption One of the earliest attempts to
combine data using a fixed-effects model was given by Birge (1932) His method weights
the estimates inversely by their variances and produces a combined estimate and associated
confidence limits Other methods include the Mantel-Haenszel method (Mantel and
Haenszel, 1959), which is used to combine contingency tables Recently, Bayesian methods
14-70
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TABLE 14-19. SUMMARY OF ODDS RATIOS FROM INDOOR STUDIES OF THE
EFFECTS OF NITROGEN DIOXIDE INCREASED BY 0.015 ppm
Authors
Mehaetal (1977)
Mehaetal (1979)
Mehaetal (1980)
Mehaetal (1982a)
Wareetal (1984)
Neas et al (1991)
Ekwo et al (1983)
Dykstraetal (1990)
Keller et al (1979b)
Estimated Odds Ratio
1 28
1 22
1 49
1 11
107
1 40
109
094
075
2 5 and 97 5 Percentiles
(Confidence Interval)
1 14 to 1 43
1 08 to 1 37
1 04 to 2 14
0 84 to 1 46
0 98 to 1 17
1 14 to 1 72
0 82 to 1 45
0 70 to 1 27
0 35 to 1 62
have been used to combine evidence, and methods particularly appropnate to these lands of
studies were described by Eddy (1989) and Eddy et al (1990a,b) Bayesian analyses require
the choice of a prior distribution for the parameter of interest, which is often a
nomnformative prior A nomnformative prior is one that, prior to seeing the evidence,
favors no value of the parameter over any other The interesting fact about use of these
methods is that, for the data sets considered in Table 14-19, the results of the computations
were nearly identical This is because the (marginal) likelihood for the odds ratio is closely
approximated by a lognormal curve The interpretations of these curves are different, as
described earlier
The second basic model assumes that the parameter of interest is not fixed, but is itself
a random variable from a distribution The value of this random variable is different for
each study, but each study gives some information aboul the mean of the distribution These
models go by several names, including random-effects models, mixed models, two-stage
models, or hierarchical models The purpose of a random-effects model is to relax the
assumption that each study is estimating exactly the same parameter This idea is not new,
having been discussed by Cochran (1937) For a discussion of the interpretation of random-
effects models in clinical trials and several methods of estimating the parameters of these
models, see DerSimoman and Laird (1986) If the studies being combined tend to estimate
the same parameter, then the results using a random-effects model will approach the results
using a fixed-effects model On the other hand, if the studies are estimating very different
14-71
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parameters, then the confidence limits will tend to be much broader than those obtained from
a fixed-effects model
The nine indoor studies described earlier (Tables 14-18 and 14-19) were combined
using both kinds of models The results using a fixed-effects model are labeled "fixed", and
results of the random-effects model are labeled "random" (see Figure 14-5) Methods for
estimating the parameters of a random-effects model were descnbed by DerSimonian and
Laird (1986) and Eddy et al (1992) The results of the analyses are provided in
Table 14-20
TABLE 14-20. U.S. ENVIRONMENTAL PROTECTION AGENCY COMBINED
ANALYSES OF INDOOR STUDIES ON RESPIRATORY ILLNESS EFFECTS OF
NITROGEN DIOXIDE INCREASED BY 0.015 ppm
Fixed-Effects Model
Random-Effects Model
Number of
Groupa Studies
All
United States
British
Measured NO2
Gas stove surrogate
SES adjusted
SES not adjusted
Smoking adjusted
Smoking not adjusted
Gender adjusted
Gender not adjusted
9
4
4
4
5
3
6
2
7
5
4
Odds
Ratio
1 17
1 11
125
123
1 15
1 27
107
1 28
1 15
126
1 05
Confidence
Interval
1 11 to 1 23
1 02 to 1 20
1 15 to 1 35
1 08 to 1 41
1 09 to 1 23
1 17 to 1 37
1 00 to 1 16
1 09 to 1 52
1 09 to 1 22
1 18 to 1 36
0 97 to 1 14
Odds
Ratio
1 18
1 13
125
1 22
1 16
1 27
108
1 25
1 16
127
1 05
Confidence
Interval
1 08 to 1 28
0 97 to 1 32
1 13 to 1 37
0 99 to 1 50
1 03 to 1 30
1 15 to 1 41
0 97 to 1 21
0 92 to 1 71
1 04 to 1 28
1 16 to 1 39
0 96 to 1 15
NO2 = Nitrogen dioxide
SES = Socioeconomic status
The first line of Table 14-20 shows the results of combining all nine indoor studies
using a fixed model The estimated odds ratio is 1 17 and the 95 % confidence limits are
1.11 and 1 23 The analysis was made assuming that the parameters were the same
(homogeneous), and this can be tested The chi-square test for homogeneity for the nine
studies was 12 32 for 8 degrees of freedom, which has a p-value of 0 1375 Thus, there is
some evidence that the parameters from each study are not identical The estimates for the
random-effects model (also shown on the first line of Table 14-20) are similar to the
estimates for the fixed model, but the confidence limits are slightly broader The conclusion
14-72
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from both models is the same, namely that the odds ratio is estimated to be about 1 2 with
95 % confidence intervals ranging from about 1 1 to 1 3 (Hasselblad et al , 1992) Many
researchers have suggested that the random-effects model is the more appropnate model
because it does not assume that all studies estimate the same parameter Furthermore, the
random-effects estimates will approach the fixed-effects estimates when studies give similar
estimates
These studies include results from North America and Europe Meta-analyses of
studies from different countries are common For example, Canner (1987), Littenberg
(1988), and Jaeschke et al (1990) all combined studies done in both North America and
Europe and did not adjust for geographic differences The indoor NO2 studies were
compared by country Four of them were done in Great Britain (Melia studies), and four in
the United States (Ware et al, Neas et al, Ekwo et al , Keller et al) The British studies
provide the highest estimated odds ratio (random-effects model), 1 25, the U S studies give
a combined estimate of 1 13
Four of the nine indoor studies used measured NO2 values, whereas the other five did
not The use of a surrogate for exposure should tend to reduce the estimate of the effect (see
Samet and Utell, 1990) The measured NO2 studies gave an estimated odds ratio (random-
effects model) of 1 22, whereas the others gave an estimate of 1 16, which is consistent with
a measurement error effect
Table 14-21 lists the important covanates considered in these nine studies and shows if
the covanate was used m the study and the meta-analysis Study design and exposure
measurement source are also presented The effect of having adjusted for various covanates
can be seen m Table 14-20 In general, those studies that adjusted for a particular covanate
found larger odds ratios as compared with those that did not
Although there may be reasons to weight certain studies or groups of studies more
heavily than others, the final conclusion has to be that there is an increase m the odds of
respiratory disease of children exposed to NO2, especially those of elementary school age
Subject to assumptions made for the combined analysis, the main conclusion from that
analysis was that an increased nsk of about 20% for respiratory symptoms and disease
•>
conesponded to each increase of 0 015 ppm (28 3 jwg/m ) in estimated 2-week average NO2
exposure, where mean weekly concentrations in bedrooms m studies reporting NO2 levels
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TABLE 14-21. COVARIATE TREATMENT AND OTHER FACTORS IN SELECTED
NITROGEN DIOXIDE EPIDEMIOLOGY STUDIES IN META-ANALYSIS
Covanatesa
Reference
Meliaetal (1977)
Meliaetal (1979)
Meliaetal (1980)
Mehaetal (1982a)
Wareetal (1984)
Neas et al (1991)
Ekwo et al (1983)
Dykstraetal (1990)
KeUeretal (1979b)
Parental
SES Smoking
A
A
M
M
M
A
NM
M
M
NM
M
M
M
M
A
A
M
NM
Gender Design
A
A
A
A
M
A
M
M
M
Cross-
sectional
Cross-
sectional
Cross-
sectional
Cross-
sectional
Cross-
sectional
Cross-
sectional
Cross-
sectional
Cross-
sectional
Prospective
Exposure Measurement
Source
Gas stove vs electric
stove
Gas stove vs electee
stove
NO2 measured with Palmes
tubes Gas stove homes
only
NO2 measured with Palmes
tubes Gas stove homes
only
Gas stove vs electee
stove °
NO2 measured with Palmes
tubes Gas and electee
stove homes
Gas stove vs electee
stove c
NO2 measured with Palmes
tubes NO2 emissions
sources in homes
Gas stove vs electee
stove c
SES = Socioeconomic status
A = Covanate included in study and meta-analysis
NM = Not measured in study
M = Measured in study but data not available for meta-analysis
Estimate of exposure derived from assumption of gas stove versus electric stove levels in bedrooms in
England from data in Melia et al (1980, 1982a,b) of approximately 0 0165 ppm
cEstimate of exposure derived from assumption of gas stove with, pilot light versus electric stove
levels averaged in the home in the Unites States in Neas et al (1991) of approximately 0 0173 ppm
were predominately between 0 008 and 0 065 ppm NO2 (Hasselblad et al, 1992) The
studies using measured NO2 give a slightly higher estimate of the odds ratio The estimates
are not sensitive to the assumption that each study is estimating the same parameter as
indicated by the random-effects model In fact, the finding of increased nsk across a wide
14-74
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variety of study conditions suggests that the effects seen are not an artifact of any one
particular study
These results are not sensitive to the inclusion or exclusion of any one study It would
have been possible to include the hospitalrzation results of Ekwo et al. (1983), the analysis of
the Swiss study, or the Berwick et al (1989) study None of these studies would have made
any real change in the estimated odds ratios or therr 95% confidence limits
Various researchers have conducted studies of infants less than 2 years of age (see
Table 14-22) A major difference for this group of studies is that the health outcome
measures are less uniform than the studies of older children For purposes of comparability,
a meta-analysis similar to the one for older children was calculated
The seven studies of infants shown in Table 14-22 show mixed results A test of
homogeneity of the odds ratios gives a chi-squared value of 22 66 for 6 degrees of freedom,
which has a p-value of 0 0009 that implies that the studies are not homogenous The
variation in results could be due to several factors, including different health outcome
measures and other factors Dockery et al (1989a) note that the associations discussed in
Ware et al (1984) and Dockery et al (1989a) must be viewed with caution because they
compare recalled respiratory events early in the child's life Because of the heterogeneity,
the studies were combined using a random effects model Subject to the assumptions made
for the meta-analysis, the combined odds ratio for the increase in respiratory disease per
increase of 0 015 ppm NO2 was 1 09 with a 95 % confidence interval of 0 95 to 1 26, where
mean weekly concentrations in bedrooms were predominantly between 0 005 and 0 050 ppm
NO2 in studies reporting levels Thus, although the overall combined estimate is positive, it
clearly contains the no-effect value of 1 0 (i e , is not statistically significant), and so we
cannot conclude that the evidence suggests an effect in infants comparable to that seen in
older children
There is always the concern that the studies described in this document are not the
complete list of studies, but contain primarily the positive studies because these are the
studies most likely to get published Alternatively, nonsignificant results may not be
reported with sufficient quantitative detail to permit then inclusion Both of these effects can
be considered as "publication bias" There are two reasons to be less concerned with
14-75
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TABLE 14-22. SUMMARY OF ODDS RATIOS OF THE EFFECTS OF NITROGEN DIOXIDE, HEALTH OUTCOME,
AND EXPOSURE ESTIMATES FOR INFANT (<2 years) EPIDEMIOLOGY STUDIES
Reference
Meha et al. (1983)
Ekwoetal (1983)
Wareetal (1984)
Ogstonetal (1985)
Dockeryetal (1989a)
Margolisetal (1992)
Samet et al (1993)
Estimated
Odds Ratio
063
24
1 11
1 14
1 15
1 12
09856
2 5 and 97 5 Percentiles
(Confidence Interval)
0 36-1 10
1 06-3 74
0 97-1 27
0 86-1 50
0 96-1 37
0 63-2 04
0 935-1 0386
Health Outcome
Respiratory illness
incidence
Hospitakzation for chest
illness before age 2
Respiratory illness before
age 2
Respiratory illness
incidence
Respiratory illness before
age 2
Persistent lower
respiratory symptoms
Lower respiratory illness
incidence
NO2 Exposure
Estimate (ppm)
0 0165tt
0 0173b
0 0173b
0 0165a
0015C
0 0105d
0015f
Age
<1 year
<2 years
<2 years
<1 years
< 2 years
<1 year
<18mo
Where/When
England (1978)
Iowa
Six US cities
(1974-1979)
Scotland (1980)
Six US Cities
(1983-1986)
North Carolina
(1986-1988)
Albuquerque
(1988-1990)
^Estimate of exposure derived from assumption of gas stove versus electric stove levels m bedrooms in England from data in Melia et al (1980, 1982) of
approximately 0 0165 ppm
Estimate of exposure derived from assumption of gas stove with pilot light versus electee stove levels averaged in the home in the Unites States in Neas et al
(1991) of approximately 0 0173 ppm
cEstimate of exposure derived from assumption of gas stove versus electric stove levels averaged in the home in United States in Neas et al (1991) of
approximately 0 015 ppm
Estimate of exposure derived from assumption of gas stove versus electric stove levels averaged in the home in the Albuquerque study (Samet et al, 1993) of
approximately 0 0105 ppm
6Computed from logistic regression coefficient derived from Samet et al (1993)
Exposure level used to convert logistic regression to an odds ratio
-------
publication bias in this particular situation First, epidemiological studies are very expensive
and require the work of many individuals The designs of studies are usually described to
the scientific community before the results are even known. Second, most of the studies
cited in this section were reported as negative studies by the authors themselves, indicating
that there was no difficulty publishing negative results However, some studies are
necessarily excluded because they provide insufficient information Although this can lead to
bias, there is little that can be done to correct for this problem This problem is not
normally referred to as publication bias, but it is a similar problem
14.6.5 Summary of Synthesis of Evidence
The evidence from individual studies of the effect of NO2 on lower respiratory
symptoms and disease is somewhat mixed Most of the indoor studies used in the synthesis
showed increased respiratory disease rates associated with increased exposure A few of the
individual studies were statistically significant Combining the indoor studies giving
quantitative estimates of effects tend to show increases of lower respiratory morbidity in
children associated with long-term exposure to NO2 Combining the indoor studies as if the
end points were similar gives an estimated odds ratio oi 1 2 (95 % confidence limits of
1 1 and 1 3) for the effect per 0 015 ppm increase of NO2 on lower respiratory morbidity
(Hasselblad et al , 1992) This suggests that subject to assumptions made for the combined
analysis, the main conclusion from that analysis was that an increase of about 20% in the
odds of lower respiratory symptoms and disease corresponded to each increase of 0 015 ppm
3
(28 3 jwg/m ) in estimated 2-week average NO2 exposuie, where mean weekly concentrations
in bedrooms in studies reporting NO2 levels were predominately between 0 008 and
0 065 ppm NO2 (Hasselblad et al, 1992) Thus, the combined evidence is supportive for the
effects of estimated exposure to NO2 on lower respiratory symptoms and disease in children
aged 5 to 12 years
In the individual indoor studies of infants 2 years of age and younger, no consistent
relationship was found between estimates of NO2 exposure and the prevalence of respiratory
symptoms and disease Based on a meta-analyses of these indoor infant studies, subject to
the assumptions made for the meta-analysis, the combined odds ratio for the increase in
respiratory disease per increase of 0 015 ppm NO2 was 1 09 with a 95 % confidence interval
14-77
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of 0.95 to 1 26, where mean weekly concentrations in bedrooms were predominately between
0.005 and 0 050 ppm NO2 in studies reporting levels Thus, although the overall combined
estimate is positive, it clearly contains the no-effect value of 1 0, (i e , is not statistically
significant), and so we cannot conclude that the evidence suggests an effect in infants
comparable to that seen in older children
Several uncertainties need to be considered in interpreting the above studies and results
of the EPA meta-analysis Measurement error in exposure is potentially one of the most
important methodological problems in epidemiological studies of NO2 Thus, measured NO2
concentrations are not exposure values per se, rather, estimating actual exposure requires
knowledge of both pollutant levels and related human activity patterns The effects studied
may be related to peak exposures, average exposures, or a combination of the two To the
extent that health effects depend on peak exposures rather than average exposures, the
exposure estimates used ML the above studies and meta-analyses introduce exposure
measurement error These studies cannot distinguish between the relative contributions of
peak and average exposures and their relationship with the observed health effects
Additionally, a by-product of NO2, HONO, may be a factor in observed effects However,
only very limited health and aerometnc data are available that examine such possibilities
Also, although the level of similarity and common elements between the outcome measures
in the NO2 studies provide some confidence in their use in the quantitative analysis, the
symptoms and illnesses combined are to some extent different and could indeed reflect
different underlying processes Thus, caution is necessary in interpreting the meta-analysis
results
14.7 CONCLUSIONS
Although there is evidence that suggests that increased estimated NO2 exposure is
associated with increased respiratory symptoms in children aged 5 to 12 years, the exposure
estimated may be inadequate to determine a quantitative relationship between estimates of
exposure and symptoms The studies with measured NO2 exposure did so only for periods
of 1 to 2 weeks and reported the values as averages None of the studies attempted to relate
14-78
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the effects seen to the pattern of exposure, such as short-term peaks Furthermore, the
extrapolation to possible patterns of ambient exposure is difficult
Several researchers studied a different population group that consisted of infants 2 years
of age and younger In the individual studies of infants 2 years of age and younger, no
consistent relationship was found between estimates of NO2 exposure and the prevalence of
respiratory symptoms and disease Based on a meta-analyses of these infant studies, the
overall combined estimate is positive, however, it clearly contains the no-effect value of 1 0,
(i e , is not statistically significant); and so we cannot conclude that the evidence suggests an
effect in infants.
Several studies attempted to relate some measure of indoor and outdoor NO2 exposure
to long-term changes in pulmonary function These changes were marginally significant
No short-term studies had indoor exposures Most studies did not find any effects, which is
consistent with results from controlled human exposure studies (see Chapter 15) However,
the basic conclusion is that there is insufficient epidemiological evidence to make any
conclusion about the long- or short-term effects of NO2 on pulmonary function
14.8 SUMMARY
This chapter discusses the epidemiological evidence for the effects of NOX on human
health The major emphasis is on the effects of NO2 because it is the NOX compound
studied in most epidemiological studies and because it is the NOX compound currently of
greatest concern from a public health perspective The results from the various
epidemiological studies of NO2 exposure effects on human health outcomes are summarized
in Appendix 14A
The studies considered in this chapter were evaluated for several key factors, including
(1) measurement error in exposure, (2) misclassification of the health outcome,
(3) adjustment for covanates, (4) selection bias, (5) internal consistency, and (6) plausibility
of the effect based on other evidence The health outcome should be an outcome for which
there is good reason to suspect that NO2 exposure has an effect Two health outcome
measures are generally considered lung function measurements and respiratory illness
Each study is reviewed with special attention given to the factors just discussed Those
14-79
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studies that address these factors more appropriately provide a stronger basis for the
conclusions that they draw
Respiratory illness and factors that affect its rate and/or seventy are important public
health concerns This chapter discussed epidemiological findings relating NO2 exposure to
respiratory illness This effect is of public health importance because of the potential for
exposure to NO2 and because childhood respiratory illness is common (Samet et al, 1983,
Samet and Utell, 1990) This takes on added importance because recurrent childhood
respiratory illness may be a risk factor for later increased susceptibility to lung damage
(Glezen, 1989)
Animal toxicological studies in Chapter 13 suggest that NO2 exposure can impair
components of the respiratory host defense system The observed increase in respiratory
symptoms and disease among children in epidemiologic studies of NO2 exposure may be the
result of an impaired respiratory host defense system The biological plausibility of this
hypothesis is supported by the animal toxicology data, but the hypothesis requires further
testing.
Several of the indoor epidemiological studies gave some evidence that repeated NO2
exposure increases respiratory illness in children, although many were not statistically
significant Melia et al (1977) first reported on a survey of children in randomly selected
areas of England and Scotland using the presence of a gas stove as a measure of NO2
exposure A reanalysis of those data yields an estimated odds ratio of 1 31 for the presence
of respiratory symptoms The cross-sectional study of Melia et al (1979) also found that the
presence of a gas stove was associated with increased risk of respiratory disease The odds
ratio was 1 24 with 95% confidence limits of 1 09 and 1 42 Melia et al (1980) described
the results of a third study of respiratory symptoms in children aged 6 to 7 years in northern
England. Multiple logistic regression analysis of the data presented by Melia et al (1980)
showed a significant increase in symptoms as a function of bedroom NO2 levels
Melia et al (1982a) reported on a fourth study of children in England Multiple logistic
regression analysis of these data was not statistically significant, although the symptoms were
positively related to NO2 exposure The analysis by Hasselblad et al (1992) suggests that an
increase of 0 015 ppm (28 3 pig/m3) in bedroom NO2 levels yields an 11 % increase in the
odds of respiratory illness
14-80
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The analysis of the Six City studies by Ware et al (1984) estimated an unadjusted odds
ratio of 1 08 (95 % confidence limits of 0 97 and 1 19) for a lower respiratory illness index
associated with gas stove use Other indicators such as bronchitis, cough, and wheeze did
not show any increased incidence Neas et al (1990, 1991) analyzed a different Six City
Study cohort enrolled later, used a different symptom questionnaire, and made indoor NO2
measurements for all subjects They found increased respiratory disease and gave an
estimated odds ratio of 1 40 (95 % confidence limits of 1 14 and 1 72) at an exposure of
0 015 ppm (28 3 jwg/m3)
Ekwo et al (1983) studied respiratory symptoms in relation to gas stove use in Iowa
City, IA Gas stove use provided in an odds ratio of 2 4 for hospitalization for chest illness
before age 2, and 1 1 for chest congestion and phlegm with colds Dijkstra et al (1990)
studied the effect of indoor factors on respiratory health in children in the Netherlands
A logistic regression analysis (Hasselblad et al, 1992) yielded an odds ratio of 0 94 with
95% confidence limits of 0 66 and 1 33, thus showing no evidence of an increase in
respiratory disease with increasing NO2 exposure Keller et al (1979b) did not find any
statistically significant changes in respiratory disease associated with gas stove use,the
unadjusted estimated odds ratio for respiratory illness was 0 72, with 95 % confidence limits
of 0 30 and 1 74
Samet et al (1993) report preliminary results of a prospective cohort study of
respiratory illness during the first 18 mo of life in relationship to estimates of NO2 exposure
in Albuquerque, NM The findings indicated that in a population of healthy infants, no
significant association between NO2 exposure estimate aind respiratory illness were found
Other studies did not provide sufficient information to derive any quantitative estimates
of the effect of NO2 or gas stove use on respiratory disease Several other studies contain
information about the effects of NO2 on respiratory illness, but most of the studies either
used very different health end points or did not provide quantitative estimates of the effects
In Melia et al (1983), infants under 1 year of age were examined No relation was found
between type of fuel used for cooking and the prevalence of respiratory symptoms Ogston
et al (1985) studied respiratory disease in 1-year-olds in the Tayside region of northern
Scotland The presence of a gas stove yielded an increase in upper respiratory illness
Schenker et al (1983) studied children in Chestnut Ridge, PA, but did not report any
14-81
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quantitative data of any relationship between daily respiratory symptoms and NO2 levels
Braun-Fahrlaender et al (1992) found indoor NO2 levels predictive of duration of respiratory
disease episodes. The study of Berwick (1987) showed increased relative risk of respiratory
disease in some age groups, but not in others Dekker et al (1991) reported an increase in
asthma in children aged 5 to 8 years (n = 60) in relation to the presence of a gas stove in
Canadian homes Hedberg et al (1989) and Smith et al (1992) reported respiratory
symptoms in relation to NO2 exposures greater than 1 5 ppm (2,800 /jg/m3) in skating rinks
Several studies examined the relationship between estimates of ambient NO2 levels and
respiratory health measures Dockery et al (1989b) examined relationships between various
respiratory symptoms and ambient NO2 levels Braun-Fahrlaender et al (1992) reported
associations between outdoor long-term measures of NO2 and duration of respiratory
episodes Schwartz et al (1991) showed a relationship between short-term fluctuations in air
pollution and short-term fluctuations in medical visits for croup symptoms Rebmann et al
(1991) reported a relationship between croup with positive virologic testing and NO2 levels
Several of the indoor studies suggest an increase in respiratory symptoms ui children
aged 5 to 12 years from estimated exposure to NO2 The associations in the majority of the
studies do not reach statistical significance The consistency of these studies was examined
and the evidence was synthesized in a quantitative analysis The studies described used
different indicators to study health end points In order to compare these indoor studies, a
standard end point was defined, and then each study was compared with this standard end
point. The end point chosen was the presence of lower respiratory symptoms and disease in
children aged 5 to 12 years It was assumed that the relative odds of developing lower
respiratory symptoms and disease are similar across this age range as a function of NO2
exposure, even though the actual rates may not be (This is a common assumption in many
analyses ) The goal was to estimate the odds ratio corresponding to each increase of
0.015 ppm (28 3 /tg/m3) in NO2 exposure (Hasselblad et al, 1992)
An attempt was made to include as many indoor studies as possible The requirements
for inclusion were (1) the health end point measured must be reasonably close to the standard
end point, (2) exposure differences between subjects must exist and some estimate of
exposure must be available, and (3) an odds ratio for a specified exposure estimate must have
14-82
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been calculated, or data must be presented so that it can be calculated (Hasselblad et al ,
1992)
Two models for combining evidence were employed (Hasselblad et al , 1992) The
first was a fixed-effects model, which assumed that every study was estimating the same
parameter The second basic model assumed that the parameter of interest was not fixed, but
was itself a random variable from a distribution This kind of model is designated by several
names, including random-effects models or hierarchical models The purpose of a random-
effects model is to relax the assumption that each study is estimating exactly the same
parameter DerSimonian and Laird (1986) discuss the random-effects model Many
researchers have suggested that the random-effects model is the more appropriate model
because it does not assume that all studies estimate the same parameter Furthermore, the
random-effects estimates will approach the fixed-effects estimates when studies give similar
estimates
Subject to assumptions made for the combined analysis, the main conclusion from that
analysis was that an increased risk of about 20% for respiratory symptoms and disease
o
corresponded to each increase of 0 015 ppm (28 3 jwg/m ) in estimated 2-week average NO2
exposure, where mean weekly concentrations in bedrooms in studies reporting NO2 levels
were predominately between 0 008 and 0 065 ppm NO? (Hasselblad et al, 1992) The
measured NO2 studies gave an estimated odds ratio (random-effects model) of 1 22, whereas
the others yield an estimate of 1 16, which is consistent with a measurement error effect
The effect of having adjusted for covanates such as SES, smoking, and gender was that those
studies that adjusted for a particular covanate found larger odds ratios as compared with
those that did not
Several uncertainties need to be considered in interpreting the above studies and results
of the EPA meta-analysis Measurement error in exposure is potentially one of the most
important methodological problems in epidemiological studies of NO2 Thus measured NO2
concentrations are not exposure values per se, rather, estimating actual exposure requires
knowledge of both pollutant levels and related human activity patterns The effects studied
may be related to peak exposures, average exposures, or a combination of the two To the
extent that health effects depend on peak exposures rather than average exposures, the
exposure estimates used in the above studies and meta-analyses introduce exposure
14-83
-------
measurement error These studies cannot distinguish between the relative contributions of
peak and average exposures and their relationship with the observed health effects
Additionally, a by-product of NO2, HONO, may be a factor in observed effects However,
only very limited health and aerometnc data are available that examine such possibilities
Also, although the level of similarity and common elements between the outcome measures
in the NO2 studies provide some confidence in then* use in the quantitative analysis, the
symptoms and illnesses combined are to some extent different and could indeed reflect
different underlying processes Thus, caution is necessary in interpreting the meta-analysis
results.
Although there is evidence that suggests that increased estimated NO2 exposure is
associated with increased respiratory symptoms in children aged 5 to 12 years, the exposure
estimated may be inadequate to determine a quantitative relationship between estimates of
exposure and symptoms The studies with measured NO2 exposure did so only for periods
of 1 to 2 weeks and reported the values as averages None of the studies attempted to relate
the effects seen to the pattern of exposure such as short-term peaks Furthermore, the
extrapolation to possible patterns of ambient exposure is difficult
Several researchers studied a different population group that consisted of infants 2 years
of age and younger In the individual studies of infants 2 years of age and younger, no
consistent relationship was found between estimates of NO2 exposure and the prevalence of
respiratory symptoms and disease Based on a meta-analyses of these infant studies, the
overall combined estimate is positive, however, it clearly contains the no-effect value of 1 0,
(i.e., is not statistically significant), and so we cannot conclude that the evidence suggests an
effect in infants
The Harvard Six City study (Ware et al, 1984, Berkey et al , 1986, Neas et al , 1991)
attempted to relate some measure of indoor and outdoor NO2 exposure to long-term changes
in pulmonary function These changes were marginally significant Most studies did not
find any effects, which is consistent with controlled human exposure study data (see
Chapter 15) However, the basic conclusion is that there is insufficient epidemiological
evidence to make any conclusion about the long- or short-term effects of NO2 on pulmonary
function
14-84
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14-100
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APPENDIX 14A.
SUMMARY OF EPIDEMIOLOGICAL STUDIES OF
NITROGEN DIOXIDE HEALTH EFFECTS
14A-1
-------
TABLE 14A-1. SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
NO2 Exposure
Effects Seen
Discussion
References
to
Children aged 6-11 years
with and without gas stoves
in the house in 28 areas of
England and Scotland
4,827 Children aged
5-10 years in a second
British Cohort
808 Children aged 6-7 years
in Middlesborough,
England
Children aged 5-6 years in
Middlesborough, England
Six City study of
9,000 grade-school children
in the United States
Six City study of 6,273
children aged 7-11 years in
the United States
Respiratory illness study of
1,565 infants in the Tayside
region of Scotland
Gas stove use
Gas stove use
N02, 7-318 fLgfia (0 004-
0 169 ppm) in bedroom
with gas stoves, 6-70 /tg/m
(0 004-0 0371 ppm) without
gas stoves
NO2 levels ranged from
16-530 ftg/m3 (0 009-
0 281 ppm) Gas stoves
only
7-49 /tg/m3
(0 004-0 26 ppm) estimate
of total personal exposure
gas stove use used as a
surrogate
Measured indoor exposure
average 17 4 ppb higher in
homes with gas stoves and
pilot lights
Gas stove use
Respiratory symptoms
(bronchitis, cough, wheeze)
higher with gas stoves
present
Respiratory symptoms
higher in children with gas
stoves Smoking in home,
and use of pilot lights
examined
Incidence of respiratory
illness higher m homes with
gas stoves (p = 0 10)
Slight trend for increase in
respiratory disease rates
Marginally significant
association of gas stove use
with respiratory illness rate
in children under 2 years
Increases in individual
symptoms of 5-29%,
combined symptom odds
ratio of 1 47
Increase of 14% in
respiratory disease in homes
with gas stove use
Indoor N02 not measured
at fame of study
Result significant for boys
but not girls
Marginally significant
results Subset data with
gas stove only
Results not even marginally
significant
Differences marginally
significant Incomplete
indoor exposure
Larger increase in
combined symptoms than in
individual symptoms
Indoor NO2 not measured
at time of study Results
not statistically significant
Mehaetal (1977)
Mehaetal (1979)
Mehaetal (1980)
Goldstein et al (1979)
and Florey et al (1979)
Mehaetal (1982)
Speizer et al (1980)
Wareetal (1984)
Dockeryetal (1989a)
Neas et al (1990, 1991)
Ogstonetal (1985)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
NO2 Exposure
Effects Seen
Discussion
References
Respiratory symptom study
of 1,355 children aged
6-12 years in the Iowa City
Schools
Respiratory symptom study
of 6- to 9-year-old children
in the southeastern region
of the Netherlands
Respiratory illness study of
children under age 12 years
in Columbus, OH
Prospective cohort study of
respiratory infection during
the first 18 mo of life in
relationship to NO2
exposure in Albuquerque,
NM
Respiratory symptom study
of children aged 0-5 years
in four areas of
Switzerland
Twelve-week study of
lower and upper respiratory
symptoms in women and
children
Gas stove use
Weekly average NO2
ranged from 22-42 ftg/m
(0 012-0 022 ppm) Gas
appliance
Annual average NO2
ranged from 38-94 jiig/m
(0 020-0 050 ppm)
Personal exposure estimate
based on Palmes tube and
activity data
Outdoor mean NO2 levels
ranged from 25-52 fig/m
Indoor levels ranged from
11-34 /tg/m3
Outdoor NO2 levels ranged
from 9-19 jiig/m Indoor
NO2 ranged from
6-91 jttg/rn3
(0 003-0 048 ppm)
Increase in chest congestion
of 10% and increase of
hospitahzation of more than
100%
No evidence of any increase
m respiratory disease
No difference found in
children's respiratory
illness
Careful standardized
ascertainment of illness,
assessment of potential
confounding factors
Increase symptom scores in
children exposed to outdoor
N02 levels of 30 jttg/m3
(0 0159 ppm)
Children under age 7 years
with exposure to more than
30 itig/m3 (0 0159 ppm) had
2 17 times the lower
respiratory illness
Only hospitalization was
statistically significant
End points different from
other studies No indoor
air measurements
Wide confidence intervals
for effects measured
Adjustments for previous
illness may have lost any
effect Analysis model
different from other studies
No significant associations
found between NO2
exposure estimates and
respiratory illness in infants
End points different from
other studies Effect of
indoor NO2 marginally
significant
Inconsistent results by age
group, possibly due to very
small sample sizes
available
Ekwoetal (1983)
Dykstraetal (1990)
Houthuys et al (1987)
Brunekreef et al (1987)
Keller et al (1979a,b)
Mitchell etal (1975)
Samet et al (1993, 1992),
Samet and Spengler (1989)
Braun-Fahrlaender et al
(1989)
Berwick et al (1984, 1987,
1989)
Berwick (1987)
Leaderer et al (1986)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
N02 Exposure
Effects Seen
Discussion
References
Study of 5,561 adults aged
25-39 years living in
Lancaster and Glendora,
CA
Respiratory disease study of
approximately 2,800 adults
and children for three
different 6-mo periods in
Chattanooga, TN
Study of respiratory
symptoms in four French
cities
Respiratory symptom and
pulmonary function study of
4th-6th graders in Akron,
OH
Study of asthma emergency
room visits and
hospitalizations for children
at the Children's Hospital
of Los Angeles
Study of acute illnesses in
elderly patients in long-term
care
Annual means over the
5-year penod in Lancaster
and in Glendora (Azuza
station) averaged 3 2 and
11 4pphm, respectively
Annual averages of daily
NO2 levels ranged from
43-91 jtg/m3 in 1972, and
from 22-40 fig/m3 in 1973
NO2, 12-16 ng/m
(0 006-0 008 ppm) daily
means, NO, 7-140 /tg/m3
daily means
2
NO2 averaged 54 jtig/m
(0 029 ppm) in polluted
area, 377 jug/m
(0 019 ppm) in cleaner
area
Monthly average NO2
levels ranged from
12-18 pphm, and NO levels
ranged from 13-59 pphm
Mean value of nitrogen
oxides indoors was
0 062 ppm and outdoors
was 0 055 ppm
Decreased FE^ and FVC
and increased cough,
phlegm, and wheezing
found in Glendora when
compared to Lancaster
Increased respiratory illness
in all age and sex groups
found in 1972, but not
found in 1973
No relationship of
respiratory symptoms with
N02
Higher rates of acute
respiratory disease in
polluted area
Increased asthma
emergency room visits and
hospitalizations correlated
significantly with NO and
N02, as well as coefficients
of haze and hydrocarbons
Respiratory diseases
correlated with NO2
Areas measured at different
times, other differences
impossible to adjust for
No indoor measurements
NO2 measured by Jacobs-
Hochhesiser method for
some periods
No indoor measurements
Low NO2 exposure
No indoor measurements
Small differences seen
Relatively low NO2
exposure
No indoor measurements
Pollutants could not be
separated
No adjustment for
covanates Details of
pollution monitoring not
given
Detels et al (1981a,b)
Loveetal (1982)
PAARC (1982a,b)
Mostardietal (1981a)
Richards et al (1981)
Loewenstem et al (1985)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY DLLNESS3
Study
NO2 Exposure
Effects Seen
Discussion
References
Study of COPD rates in
COPD and normal subjects
in Helsinki, Finland
Los Angeles Student Nurse
Data originally collected daily
from 1962-64, reanalyzed by
Schwartz Symptoms
included eye irritation, cough,
phlegm, sore throat,
headache, chest discomfort
Respiratory disease study of
4,071 children aged
5-14 years m Pennsylvania
Results based on parents
questionnaire
The data reported by Love
et al 1982 were analyzed for
short-term effects
Study of influenza cases
over a 2-mo period in
Sofia, Bulgaria
Acute symptom study of
35 asthmatics in Barcelona,
Spain
NO2, 5-70 fig/m (0 003-
0 0371 ppm)
COPD correlated with NO2
Daily outdoor NO2 exposure
averaged 0 13 ppm over a
3-year period Sites located irritation
within 2 5 mi of the subject
NO2 exposure related to
phlegm, sore throat, and eye
Presence of gas stove used as
a surrogate for NO2
exposure
Daily NO2 levels were split
into three categories using
the cut-offs of 75 and
150 jug/m3 (0 04-0 08 ppm)
NOX had means of
21 and 37 pg/m3 (0 014-
0 45 ppm) for the
two epidemics
Mean daily maximum hourly
N02 levels ranged from 271-
846 /tg/m3 (0 14-0 45 ppm)
for two weeks in December
1985
No significant association
between use of gas stove and
any symptom or illness
variable
No consistent short-term
effect of N02 on acute
respiratory disease was
found
NOX related to NO2
Two-day lag in illnesses
Symptoms in asthmatics not
related to NO2 levels
Second study relates
symptoms to soybean dust
No indoor measurements Pershagen et al (1984)
No adjustments for covanates
other than temperature
Small sample sizes
No indoor NO2 exposure
measurements, and only one
outdoor station
Schwartz et al (1988),
Schwartz and Zeger (1990)
No indoor NO2 exposure
No significant results
No indoor measurements
No biological explanation for
results found
No indoor measurements
No measurement method for
NOX given
Effects related to soybean
dust in definitive study
Schenkeretal (1983)
Harrington and Krupnick
(1985)
Kalpazanov et al (1976)
Anto et al
Anto et al
(1986)
(1989)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
NC>2 Exposure
Effects Seen
Discussion
References
o\
Study of asthma in
20 children seen in two
Japanese hospitals
Ecological study attempting
to relate nitrates and oxides
of nitrogen to mortality
Ecological analysis of
mortality rates and air
pollution
Study of variations in daily
mortality in relation to daily
air pollution in several U S
cities from 1962-65
A study of the prevalence
of respiratory symptoms in
school children in two
Israeli cities with different
NO2 levels
Quarterly mean NO2 levels
ranged from 3 0-5 7 pphm
County-wide NOX
estimates
NO2 data from the National
Air Sampling Network
Nonspecific NOX levels
Maximum N(X levels in the
3
cities of 62 and 23 jtig/m ,
respectively
No relationship found
between frequency of
attacks and N02 levels
Two million death
certificates examined
Mortality rate associated
with various disease end
points
Multiple regression analysis
showed significant negative
association between winter
mortality in New York City
and daily NOX
concentration but no
association in summer In
contrast, the data in Los
Angeles were positively
related in the winter but not
in the summer
Various respiratory
symptom rates were higher
in Ashdad, the more heavily
polluted city Confounding
factors controlled
No indoor measurements
Attack rates are generally
longest when NO2 is
highest
Both positive and negative
coefficients found in the
multiple regression, which
were almost always not
statistically significant
Quality and applicability of
both the mortality and
monitoring data preclude
considering the results as
anything but speculative
The results suggest that
other factors are causing the
relationship
Watanabeetal (1984)
Mendelsohn and Orcutt
(1979)
Hickeyetal (1970)
Lebowitz (1971)
Effects related to both SO2 Goren and Hellmann (1988)
andN
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
N02 Exposure
Effects Seen
Discussion
References
Case-control study of
231 children in the
Netherlands for respiratory
symptoms vs NO2 levels
with different NO2 levels
Study of changes in
respiratory symptom rates
in relation to NO2 levels m
a rural district of Japan
Study of respiratory illness
in 2,900 school children vs
levels of NO2 in Oska
Prefecture, Japan
Study of incidence of
mediastinal and
subcutaneous emphysema m
young bronchial asthma
patients in relation to NO2
levels m Japan
Study of respiratory
symptoms in over 90,000
school children in Japan in
relation to NO2 levels
Indoor NO2 levels
NO2 measurements from
1974-79
Annual outdoor NO2 levels
Daily average outdoor NO2
levels
Outdoor mean NO2 value
3 years prior to the survey
Respiratory symptoms
determined by questionnaire
and school health survey
data
Significant correlation
between daily maximum
NO2 and subacute phlegm,
cough, and wheezing for
pnck skin test (house dust
extract)—positive children
Children attending schools
in the highest polluted area
had the highest rates of
bronchial asthma and
recurring respiratory
infeciions
Report correlation between
NO2 levels and occurrence
at mediastinal and
subcutaneous emphysema
Asthma-like symptoms was
higher in district with the
highest NO2 concentration
No difference in levels
between case and controls
Similar results for SO2
during this period The fact
that SO2 and NO2 were
correlated and that no
covanates were included in
the analysis means the study
can only be considered
suggestive
No indoor NO2 data NO2
levels analyzed in relation
to distance between
children's homes and
distance from highways
No indoor N02 data
Other factors that cause
mediastinal and
subcutaneous emphysema
not evaluated
No indoor NO2 data,
confounding with other
pollutants Confoundmg
due to smoking and
socioeconomic factors
Hoeketal (1984)
Kagamimon et al (1986)
Nagira et al (1981)
Odajima and Baba (1987)
Tsunetoshi et al (1987)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
N02 Exposure
Effects Seen
Discussion
References
oo
Study of respiratory
symptoms in the district of
Pisa, Italy, using Italian
Natural Research Council
Questionnaire on 3,729
adult subjects
Study respiratory infection
in children and adults and
absenteeism in Helsinki,
Finland
Study of respiratory
illnesses in children and
mothers in Hong Kong
Study of croup incidence
and NO2 levels in Bochum,
Germany
Study of croup incidence
and NO2 levels in
Mannheim and Darmstadt,
Germany
Gas stove use
Annual average of N02 was
47 fig/m3 (0 025 ppm)
Mean levels of NO2
estimated by personal
samplers
Outdoor NO2 levels, daily
averages
Outdoor NO2 levels, annual
and monthly average
Higher prevalence rate for
respiratory symptoms for
those using tank gas for
cooking
The level of NO2 has little
effect on the frequency of
disease The effect of low
temperature is highly
significant
Among 312 mothers,
statistically significant
allergic rhinitis for NO2
levels of 22 6 vs 19 0 ppb,
whereas among the children
there was no statistically
significant difference in
presence vs absence of the
respiratory symptoms by
NO2 levels
Positive correlation between
NO2 levels and incidence of
croup as determined by
hospital admissions
Monthly NO2 average in
Mannheim but not in
Darmstadt showed linear
correlation with monthly
cases of croup
No NO2 measurements
No indoor NO2
measurements Possible
confounding with
temperature
Difference in mean levels
of NO2 for children for
different symptoms were
very small, ranging from
0 00-1 79 ppb
(0-3 4 jtig/m )
No indoor NO2 data No
discussion of confounding
factors
No indoor NO2 data No
discussion of confounding
factors
Viegi et al (1992, 1990)
Ponka (1990)
Kooetal (1990)
Sevenen and
Mietens (1987)
Wemmer (1984)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
NO2 Exposure
Effects Seen
Discussion
References
Study of croup incidence and
NO2 levels in Mannheim
and Darmstadt, Germany
Study on the influence of
various factors to include
N02 on the incidence of
croup in Innsbruck, Austria
A pilot study of 130 third
graders of NO2 levels and
hematological, clinical and
immunological end points in
Rostock, Germany
Study of NO2 exposure and
respiratory disease function
and symptoms in Japan
Study of acute illness
symptoms and pulmonary
function in New York in
relation to NO2 exposure
Outdoor NO2 levels, annual
and monthly average
NO2 levels were measured
by a chemolummescence
detector at a measuring
station in the inner city area
Indoor monitors for NO2
and other pollutants
Indoor NO2 measurements
urine hydroxprolme
measurements
Measures of personal
exposure to NO2
Monthly N02 average in
Mannheim, but not it
Darmstadt, showed linear
correlation with monthly
cases of croup
Pseudocroup has a
multifactor pathogenesis
Rapid changes in air
pollutants (especially NO
and NO2) are followed by
increased occurrence of
croup
Selected results of
questionnaires and smoking
history
Respiratory disease
questionnaires (American
Thoracic Society),
pulmonary function tests in
1,000 3-year-old children
Asthmatic and nonasthmatic
women and children are
evaluated for PFT and
respiratory symptoms
No indoor NO2 data No
discussion of confounding
factors
No indoor NO2 data
Weather conditions cannot
by this statistical evaluation
be eliminated as an
important factor whether
directly or indirectly
Limited findings presented in
the preliminary study
Prevalence rate of
respiratory symptoms higher
near roadways with heavy
traffic and higher pollutant
levels
Preliminary data
Wemmer (1984)
Guggenbichler et al (1990)
Thielebeule and Huelsse
(1989)
Ono et al (1990), Tominaga
and Ono (1985)
Goldstein et al (1987)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPJDEMJOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
NO2 Exposure
Effects Seen
Discussion
References
Study of chronic respiratory
disease symptoms in 8,572
Southern California Seventh-
Day Adventists
Study of 875 cases of croup
in Baden-Wurttemberg in
relation to NC>2 exposure in
a 2-year prospective study
Study of asthma attack
incidence over a 3-year
period in Finland in relation
to pollutant levels
Study of upper respiratory
infections in children in
Finland in relation to NO2
and other pollutants
Hours of S02 exposure
above 4 pphm, oxidants
above 10 pphm, and TSP
above 200 /ig/m3 N02
exposure levels were not
linked to health effects
Daily ambient levels
Ambient NO9 levels
3
averaged 38 6 jttg/m over
the 3-year period
Ambient NO9 levels
3
averaged 15 fig/m
Relative risks of about
1 2 for the pollutants listed
Statistical regression
methods indicate weak but
statistically significant
influences of the daily NO2
mean on the occurrence of
croup
During the high NO2 (mean
45 8 /tg/rn3) levels, the
mean number of all
admissions was 29% greater
than during lower levels
(28 1 jug/m3)
A significant association of
upper respiratory infections
and air pollution in the two
age groups studied
No relationship with
measured NO2.
Euleretal (1988)
Air pollution considered a
weak factor, whereas
essential conditions for
croup are individual and
familiar disposition and virus
infection
Indoor NO2 level and
cooking fuel used were not
considered Minimum
temperature was associated
with asthma admissions
Passive smoking and SES
evaluated Indoor NO2
levels and cooking fuels not
considered
Rebmannetal (1991)
Ponka (1991)
Jaakkolaetal (1991)
Study of prevalence of
asthma in children in
relation to NO2 levels in
Germany
Indoor and outdoor NO2
data taken into account.
Stoves used as a heatmg
device had a 4 8-fold
relative risk for asthma
compared to other heating
704 children, 7-16 years old,
took part in a standardized
interview and exam
Passive smoking was
examined
Kuehretal (1991)
-------
TABLE 14A-1 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
RESPIRATORY ILLNESS3
Study
NO2 Exposure
Effects Seen
Discussion
References
Panel study of 128 children
whose parents completed a
daily diary of respiratory
symptoms in Chestnut
Ridge region of western
Pennsylvania in relation to
NO2 levels
Study of 708 nonsmoking
white adult residents of
Maryland evaluating the
effect of gas cooking on
respiratory symptoms
Longitudinal study of
exacerbation of asthma
measured by wheezing
occurrence in relation to
NO2 exposure in
approximately 24 asthmatic
school children
Study of the prevalence of
persistent respiratory
symptoms in 393 infants in
North Carolina
Maximum hourly levels for
each 24-h period averaged
40 5 /ig/m with a range of
12-79 g/m3
Gas stove cooking or
electric stove use
Maximum daily ambient
levels with average levels of
75 and 169 jitg/m in the
"medium" and "high"
exposure categories
Yearly average levels were
2 0 and 0 4 jiig/m3,
respectively
Gas stove cooking or
electric stove use
Ambient NO2 levels were
not predictive of any
symptom outcome
measures
Increased chronic cough
and phlegm in households
using gas stoves
NO2 levels were not
associated with the
occurrence of symptoms in
this group of asthmatics for
the exposures that occurred
Relative risk of 1 12 (95%
confidence interval of 0 63
to 2 04)
The subgroup analysis had
55 subjects No indoor
levels or use of gas stoves
were noted
Adjustments for SES and
controlled for smoking
exposures
No indoor exposure data
The model adjusted for the
effect of the presence of
symptom on the previous
day
Approximately 41 infants
lived in homes with the
environmental risk factor of
gas cooking
Vedeletal (1987)
Helsingetal (1982)
Comstock et al (1981)
Henry et al (1991)
Margohs et al (1992)
-------
TABLE 14A-2. SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
PULMONARY FUNCTION
Study
N02 Exposure
Effects Seen
Discussion
References
h—^
to
Six City study of 9,000
grade school children
Six City study analyzed for
effects on pulmonary
function growth
Lung function panel study
of 351 children at the
Chestnut Ridge Elementary
School
Lung function study of
117 middle-class households
in Tucson, AZ
Study of traffic policemen
in urban Boston with nearby
suburban areas
Study of FEV0 75 in school
children in Chattanooga,
TN
Study of nonsmoking adults
in Los Angeles and San
Diego
7-49 ng/m (0 004-
0 026 ppm) estimate of total
personal exposure gas stove
use used as a surrogate
7-49 /ig/m3 (0 006-
0 04 ppm) estimate of total
personal exposure gas stove
use used as a surrogate
NO2 ranged from
12-79 /tg/m3 (0 006-
0 04 ppm)
Gas stove use
75-103 /ig/rn (0 04-
0 055 ppm) annual mean
daily concentrations
Ambient annual NO?
3
levels as high as 286 jitg/m
(0 15 ppm) (Saltzman
method)
43-96 jtig/m3 (0 023-
0 051 ppm) annual mean
daily concentrations 113-
118j«.g/m 90th percentile
FEVj, and FVC decreases
ranged from 0 2-0 6%,
depending on examination
and adjustment
No effect of gas stove use
on growth of pulmonary
Peak flow not affected by
pollutant levels
Peak flow was marginally
related to gas stove use
(p = 0 066)
No differences in various
pulmonary function tests
Suggestion of lower
FEV0 75 means in high
NC«2 area
No difference in several
ventilatory measurements
including spirometry and
flow volume curves
Differences marginally
significant, incomplete
indoor exposure
Differences marginally
significant, incomplete
indoor exposure
No indoor pollutant
measurements made,
outdoor levels were low
No indoor NO2
measurements made
Speizeretal (1980)
Berkey et al (1986)
Vedaletal (1987)
Lebowitzetal (1985)
No indoor measurements, Speizer and Ferns
small sample sizes (1973a,b)
No indoor measurements, Shy et al (1970a,b)
much of the NO2 measured
by Jacob-Hochheiser
method, no strong
differences found
No indoor exposures,
complicated outdoor
exposures
Cohen et al (1972)
-------
TABLE 14A-2 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
PULMONARY FUNCTION
Study
NO2 Exposure
Effects Seen
Discussion
References
h-»
OJ
Lung function study of
12 asthmatic children living
in the Sunair Home in
California
Office workers in Los
Angeles and San Francisco
Respiratory symptom and
pulmonary function study of
4th through 6th graders in
Akron, OH
Study of pulmonary
function in French cities
Maximum hourly value of
22pphm
65-130 jtig/m (0 034-
0 07 ppm) median NO2,
110-250 90th percentile
NO2 averaged 54 /ig/m
(0 029 ppm) in polluted
area
NO2, 12-16 fjLg/m (0 006-
0 008 ppm) daily means
NO, 7-140 nig/m3 daily
means
Study of 5,561 adults aged Annual means over the
25-39 years living in
Lancaster and Glendora,
CA
Japanese study of school
children in Japan, aged
11 years
5-year period in Lancaster
and Glendora (Aryza
station) averaged 3 2 and
114 pphm, respectively
40-360 /ig/m3 (0 02-
0 19 ppm) 1-h values at
time of testing
Morning peak flow was
reduced, but afternoon peak
flow was not
No difference in most
pulmonary tests
Small decrease seen in ratio
to FVC
No decrease in pulmonary
function related to NO2
exposure
Decreased FEV{ and FVC
and increased cough,
phlegm, and wheezing
found in Glendora when
compared to Lancaster
Correlation of peak flow
and 25% and 50% FVC
with N02, NO, SO2, and
TSP
Effects of other pollutants
could not be separated
Seasonal factors not
adjusted for No indoor
pollutant measurements
No indoor measurements
Small sample size
No rndoor measurements
Small differences seen
Relatively low NO2
exposure
No indoor measurements
Low NO2 exposure
Areas measured at different
times Other differences
between areas impossible to
adjust for
Correlations not adjusted
for other pollutants
Richards et al (1981)
Lmnetal (1976)
Mostardietal (1981a,b)
PAARC (1982a,b)
Detelsetal (1981a,b)
Kagawa and Toyama (1975)
-------
TABLE 14A-2 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
PULMONARY FUNCTION
Study
N02 Exposure
Effects Seen
Discussion
References
Case control study of
213 nonsmoking women in
the Tecumseh Community
Health study evaluated FEV
values vs various cooking
fuels
Study of 561 nonsmoking
white adult residents of
Maryland evaluating the
effects of gas cooking on
pulmonary function
National Health and
Nutrition Examination
Survey n lung function data
coupled with EPA's
SAROAD aerometnc data
Lung function measurements
included FVC, FEV^ and
PEF
In a sample of over
16,000 children, PFT were
compared to gas stove use
Cooking fuels, duration of
exposure to cooking fuels,
and exhaust fan use
Gas stove exposure
NO2 exposures ranged from
less than 0 01-0 08 ppm
Individual estimates based on
average of all stations within
10 mi of census tract
Gas stove use
Association of low FEV and
gas cooking was marginally
significant (p = 0 07)
The use of gas cooking
associated with a
significantly greater
percentage with impaired
ventilatory function as
measured both by
FEVj < 80% predicted and
by FEVj/FVC < 70%
Highly significant regression
coefficients showing a
decrease in FVC, FEV1( and
PEF with increasing NO2
exposure
Marginally significant
decrease (p = 0 052) in lung
function in girls 9-13 years
of age PFT numbers were
adjusted for parental
smoking habits
No indoor NO2 data Small
sample size reduces power,
especially the limited
number of gas stoves in
sample
Adjustments for SES and
control for smoking
Jones etal (1983)
Helsingetal (1982)
No indoor NO2 exposure
measurements Outdoor
estimates over a wide area
Schwartz (1989)
No indoor measurements Hasselblad et al (1981)
-------
TABLE 14A-2 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
PULMONARY FUNCTION
Study
NO2 Exposure
Effects Seen
Discussion
References
A study of PFT in women
in relation to NO2 levels in
the Netherlands
A study of the effect of
domestic air pollution on
respiratory function in a
group of housewives in
Lebanon, CT
Study of lung function and
respiratory function in
relation to homes with coal
or gas cooking fuels
Study of children's
pulmonary function in
relation to NO2 exposure in
the Netherlands
Study of NO2 exposure on
pulmonary function and
symptoms in Japan
Weekly personal exposure
estimates ranged from
15-300 fig/m (0 008-
0 159 ppm)
Gas stove use
NO2 levels of over
400 jtig/m3 (0 21 ppm) in
kitchens
N"U2 levels measured by
Palmes tubes in homes,
mean concentration ranged
from 23-72 /ig/m3 (0 012-
0 038 ppm) NO2
Indoor NO2 measurements,
urine hydroxyprolme
measurements
Statistically significant
negative associations
between various pulmonary
function measures and NO2
exposure in nonsmoking
women
Some decreases observed in
lung function in homes with
gas stoves
Lung function measures in
women were reduced by
elevated levels of pollutants
No respiratory symptoms
were related to gas
PFTs were evaluated in
over 800 children aged
6-12 years No relationship
was found The power of
the study to detect small
effects on lung function was
considered adequate
Respiratory disease
questionnaires, PFTs in
1,000 3-year-old children
No association with
longitudinal pulmonary
function decline
Sample sizes too small to
give meaningful
information
Method of analysis and
adjustments for covariate
were not given
Exposure measure may not
have captured short-term
peak concentrations
Ongoing study expected
completion in 1989-90 with
analysis at a later date
Fischer et al (1985, 1986)
Remijnetal (1985)
Hosein and Corey (1986)
Liu and Wang (1987)
Brunejcreef et ai (,1990)
Tommaga and Ono (1985)
-------
TABLE 14A-2 (cont'd). SUMMARY OF EPIDEMIOLOGICAL STUDIES OF NITROGEN DIOXIDE EFFECTS ON
PULMONARY FUNCTION
Study
NOj Exposure
Effects Seen
Discussion
References
O\
Study of children's
bronchial reactivity in
relation to N02 exposure in
Switzerland
NO2 levels measured by
passive samplers
Study of oxides of sulfur,
NO2, hydrocarbons, and
particulate exposure on
pulmonary function in two
cities in Southern
California
Outdoor daily peak hourly
levels for NO2 range from
003-0 llppm
Bronchial reaction to
carbachol measured in
312 school children in two
exposure groups
Chronic exposure to the
pollutant mix results in less
rapid growth of lung
function in children and a
greater rate of deterioration
in adulthood
Urban NO2 levels of
36 2 /tg/m3 vs 26 2 /tg/m3
in rural area A statistical
difference seen in bronchial
reactivity in healthy
children but not in
asthmatics Effects of anti-
asthmatic therapy in atopic
children could not be
determined
The proportion of
participants retested in the
study was low (45-47%)
due to migration out of the
study area The impact of
this may have been minimal
through Differences
between communities in age
and height could have
biased the results
Gschwend-Eigenmann et al
(1989)
Detels et al (1991)
Abbreviations NOj = Nitrogen dioxide, SC>2 = Sulfur dioxide, TSP = Total suspended particulate, FEV = Forced expiratory volume, FVC = Forced vital capacity, NO = Nitric oxide,
COPD = Chronic obstructive pulmonary disease, NOX = Nitrogen oxides, PFT = Pulmonary function test, SES = Socioeconomic status, PEF = Peak expiratory flow
-------
15. CONTROLLED HUMAN EXPOSURE STUDIES
OF NITROGEN OXIDES
15.1 INTRODUCTION
This chapter discusses the effects of nitrogen oxides (NOX) on human volunteers
exposed under controlled conditions The NOX species of primary concern is nitrogen
dioxide (NO2) Nitric oxide (NO) and nitrates have also been evaluated in controlled human
exposures, and nitric acid (HNO3) effects have only recently been studied The 1982 Air
Quality Criteria for Oxides of Nitrogen Document (U S Environmental Protection Agency,
1982a) presents a comprehensive review of studies conducted up to about 1980 The present
chapter focuses mainly on summaries and critiques of studies conducted since then, but also
includes some discussion of the earlier literature as well
Controlled human exposure studies of NO2 deal with relatively brief, experimental
exposures to higher concentrations compared to the annual arithmetic mean standard
(0 053 ppm) One of the purposes in reviewing these studies is to evaluate the data base for
short-term (typically < 4 h) NO2-related health effects, thus, consideration of the time course
of responses and the pattern of exposure in controlled human exposure studies is important
Because of the widespread occurrence of NO2 in both outdoor and indoor
environments, there are major concerns regarding potential impacts of NO2 exposure on
human health, particularly with regard to effects on the lung Dosimetry modeling and
animal histological studies indicate that NO2's impact should be primarily seen in the small
airways and gas exchange regions of the lung, thus, tests that specifically evaluate responses
in this region are of particular interest in evaluating the effects of NO2 In assessing NO2
lung effects for criteria development purposes, a number of important questions need to be
addressed, as posed in the following list of critical questions Some of these questions are of
a "generic" nature, applying to many ambient air pollutants, and others are specific to NO2
inhalation Several of the questions may be answered only partially by controlled human
exposure studies and are addressed further by animal toxicological studies and/or
epidemiological studies discussed in Chapters 13 and 14, respectively
15-1
-------
Nitrogen Dioxide Exposure and Human Health Critical Questions
1. Does short-term NO2 exposure cause acute changes in lung function, increased
respiratory symptoms, or increased airways responsiveness in normal, healthy
subjects at levels that may be expected in the ambient (or indoor) environment'?
Are these effects reproducible*? If so, what is the possibility that such acute
responses may contribute to chronic changes in lung function, promote the
development of respiratory disease, cause acceleration of normal age-related
declines in lung function, or aggravate existing respiratory disease9
2 Are there groups within the population at special risk for NO2 health effects
(i e , groups compnsed of persons who exhibit greater responses to NO2
exposure than the average healthy subject)*? Groups hypothesized as likely to be
at special risk include young children, adolescents, elderly subjects, and patients
of all ages with asthma, chronic obstructive lung disease, or other lung diseases
If there are subject groups who are more responsive, can they be identified
prospectively (i e , without exposing them to NO2 first)1?
3. Does NO2 cause an inflammatory response in the lungs of healthy individuals or
patients with lung disease1? Specifically, does NO2 exposure cause increased
capillary permeability, increased local blood flow, extravasation of fluid or influx
of leukocytes—especially neutrophils and eosinophils—into the interstitmm and
the airways, secretion of pro-inflammatory mediators, mast cell degranulation, or
epithelial desquamation*?
4. Does NO2 exposure cause increased responses of the lung (including airways
responsiveness, lung function, inflammation, cell damage, etc ) to (a) other
pollutants such as ozone (O3), sulfur dioxide (SO2), or acid aerosols,
(b) bronchoconstnctors such as histamine or methacholine, (c) other agents such
as cold-dry air or exercise, or (d) specific antigemc substances'?
5 Does NO2 exposure alter (a) respiratory tract host defenses, (b) airway epithelial
permeability or mucociliary clearance, or (c) local or systemic immune response
to infection*? As a consequence of NO2 impacts on host defense system
components, is the killing or removal of microorganisms unpaired by NO2
exposure*? Also, are inflammatory responses or tissue injury caused by
microorganisms worsened by coincident NO2 exposure*?
6 What is the time course of response to acute exposures'? Are there both
immediate and delayed responses'? Do responses increase or decrease with
mcreased exposure duration*? What is the tune course of response to repeated
exposures'? Do responses increase or decrease with mcreased frequency of
exposure*?
15-2
-------
Controlled human exposure studies serve as an important source of inhalation
toxicology data, particularly for the criteria pollutants such as NOX Methodological and
experimental design considerations for controlled human exposure studies have recently been
reviewed (Folinsbee, 1988) These studies are typically conducted on volunteers who have
been informed of the possible risks of such studies and who have given their "informed
consent" to participate The subject group that most often participates in such studies are
young adult males with no history of respiratory disease, allergies, smoking, and no
contraindication to exercise In addition to young men, participants of either gender and of
different racial groups have included children, adolescents, elderly persons, and adults
Other subject groups that have been specifically studied include healthy subjects with
allergies, asthmatics, smokers, patients with chronic obstructive pulmonary disease (COPD),
or otherwise healthy persons with upper respiratory infections These latter subject groups
may be considered potentially "sensitive subjects", especially asthmatics, COPD patients,
children, and the elderly For individuals with existing lung disease and/or hyperresponsive
airways, special consideration of the potential impact of pollutant exposure is required
These individuals, including the healthy elderly population, often have limited pulmonary
reserves and, therefore, a given insult has a greater physiological/pathological consequence
(increased airways resistance, restriction of lung volume) Children are of special concern
because their lungs are still growing and developing and, hence, the possibility of a long-
term impact on lung health may be greater than for the mature adult lung
Controlled exposures, by definition, occur in a laboratory setting The most "natural"
mode of exposure is unencumbered breathing within an exposure chamber Other modes of
exposure include facemask, hood, or mouthpiece exposures A controlled exposure implies
that the environmental variables such as the concentration of the pollutant, temperature, and
humidity are monitored and maintained at some specified level In addition, the duration of
the exposure and amount of activity during the exposure are closely regulated The activity
level is closely correlated with the volume of air breathed into the lung In order to simulate
an outdoor exposure where the subject is active, many exposure studies include some form of
controlled exercise However, exercise alone may have some important confounding effects,
particularly in the case of exercise-induced bronchoconstnction in asthmatics Exercise alone
may induce significant decrements in spirometric variables or significant increments in
15-3
-------
airway resistance Exercise-induced bronchoconstnction is followed by a refractory period
of several hours during which asthmatics are less susceptible to bronchoconstnction
(Edmunds et al, 1978) This period of refractoriness could alter the subject's responsiveness
to NO2 or other inhaled substances The major external determinants of the exposure "dose"
of a pollutant are the concentration of pollutant, the duration of the exposure, and the volume
of air breathed (specifically, the route, depth, and frequency of breathing) during the
exposure Further information is, of course, necessary to determine the actual dose delivered
to the various "target" regions of the respiratory tract (i e , total respiratory uptake), as is
presented in Chapter 13 Many of these considerations have been discussed in greater detail
by Folinsbee (1988)
In human exposure studies, the methods used for assessment of effects primarily
involve "noninvasive" procedures Various pulmonary function tests such as spirometnc
measures of lung volumes, measures of resistance of lung or nasal airways, ventilation
volume (volume of air inhaled into the lung per minute), breathing pattern (frequency and
depth of breathing), and numerous other "breathing" tests have been utilized (Bouhuys,
1974). These tests provide useful information about some of the basic physiological
functions of the lung Certain tests provide information primarily about large airway
function, these include (a) dynamic spirometry tests (e g , forced expiratory tests such as
forced expiratory volume mis [FEVj], maximal and partial flow-volume curves [including
those using gases of different densities such as helium], peak flow measurements, etc ), and
(b) specific airway resistance/conductance measurements (SRaw, SGaw) The reader should
refer to the glossary for more specific descriptions of various tests These "standard
pulmonary function" tests are relatively simple to administer, provide a good overall index of
lung function, and have a relatively low coefficient of variation (CV), the CV is about 3%
for FEVj and about 10 to 20% for SRaw However, because NO2 deposits primarily in
peripheral airways, many of the above tests may not provide the necessary information to
fully evaluate the effects of NO2 Other tests purported to provide evidence of small airway
function include multiple breath nitrogen washout tests, closing volume tests, aerosol
deposition/distribution tests, density dependence of flow-volume curves (using gases of
different densities such as helium), and frequency dependence of dynamic compliance, but
15-4
-------
none are used routinely and use of these procedures to assess "small airways function" is not
widely accepted
Somewhat more invasive procedures have also been more utilized in recent years to
determine human responses to air pollutant exposures, including pharmacologic airway
inhalation challenge tests, measurements of pulmonary clearance of inhaled aerosols,
bronchoalveolar lavage, nasal lavage, and arterial blood gas measurements
Airway inhalation challenge tests are used to evaluate the "responsiveness" of a
subject's airways to inhaled materials Airway responsiveness may change as a result of
alterations in a disease state, such as inflammation associated with asthma or viral respiratory
infection, or as a result of damage to the airway caused by disease or insult from inhaled
toxic or allergenic materials Thus, one of the problems in evaluating changes in airway
responsiveness with respect to inhalation of air pollutants is that the baseline responsiveness
can be changed by other factors not associated with pollutant exposure In order to test for
the degree of airway responsiveness, a chemical that causes constriction of the airways (such
as histamine, carbachol, or methacholine) is typically used Other challenge tests involve the
use of allergenic substances, exercise, hypertonic saline, or cold-dry air Responses are
usually measured by evaluating changes in airway resistance or spirometry after each dose of
the challenge is administered Usually, the test will proceed until some target effect level is
achieved (e g , doubling of airway resistance) and the airway responsiveness is then
characterized by the dose required to achieve that level The procedures for administering
and interpreting inhalation challenges are discussed in detail elsewhere (Cropp et al , 1980,
Cropp, 1979, Chai et al , 1975, Fish and Kelly, 1979, O'Byrne et al , 1982)
Asthmatics as a group are significantly more responsive than healthy normal subjects to
a variety of airway challenges The differences in airway responsiveness may span several
orders of magnitude (at least 100-fold) between normals and asthmatics (O'Connor et al,
1987) Nevertheless, there is considerable overlap between the more responsive healthy
subjects and the less responsive (histamine) asthmatics (Pattemore et al , 1990) Airway
responsiveness to methacholine appears to be somewhat better than airway responsiveness to
histamine at differentiating normals and asthmatics, although responses to these two
bronchoconstnctors are well correlated (r = 0 70) (Chatham et al, 1982) Unfortunately,
because of the number of different provocative agents used in the airway challenges and the
15-5
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variety of methodologies used to administer challenges, it is difficult to compare responses
between laboratories in a quantitative manner Thus, it is not useful to suggest standard
ranges of responsiveness for normals and asthmatics
Tests of pulmonary clearance of inhaled aerosols are used to assess the efficiency of the
mucociUary clearance mechanism and to estimate pulmonary epithelial permeability
Typically, a radioactively labeled test aerosol, of a specific size range that will deposit in the
lung region of interest, is deposited in the lung by inhalation External detectors are then
used to assess the amount of remaining test aerosol at various times after the initial
deposition of the aerosol This methodology is discussed in Clarke and Pavia (1980) and
Raabe (1982) and in Section 13 22 1 A particular application of clearance of radiolabeled
aerosols is for the estimation of epithelial permeability, typically using technetium-labeled
diethylene triamine penta-acetate or pentetate This methodology is discussed in Nolop et al
(1987)
In the past several years, bronchoalveolar lavage techniques have been used in clinical
exposure studies of several different pollutants In this procedure, a fiberoptic bronchoscope
is passed into the airways and wedged in a subsegmental bronchus, where sterile buffered
saline is used to wash free cells and airway secretions from the segment (Reynolds, 1987)
The resulting lavage fluid may be analyzed for various chemical mediators or reaction
products, numbers and types of cells, and the functions of some lung cell types Another
less invasive procedure, known as nasal lavage, may be used to obtain nasal secretions and
cells (Graham et al, 1988)
There are a number of limitations of controlled human exposure or "clinical" studies
Many experimental animal models are derived from genetically pure strains, thus reducing
the expected variability in biological response Because of their heterogeneity, humans are
expected to display a wider range of response to a variety of physiological and pathological
stimuli This variability and the small sample numbers limit the extent to which the data can
be generalized to the population as a whole or to certain defined segments of the population
(e.g , asthmatics) The small sample size may limit the interpretation of the study, especially
when the results are negative (i e , the null hypothesis is not rejected) One cannot have
great confidence in a study that finds no effect of a treatment (in this case NO2 exposure) if
the sample size (i e., the number of subjects) is too small to statistically detect an effect, if
15-6
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present This must be kept in mind in interpreting the results of human exposure studies
with a small number of subjects Investigators have reported a wide variation in
responsiveness of asthmatics to NO2, which may be partially attributed to intrinsic variation
in response as well as variation in exposure variables In addition, place of residence, season
of the year, and indoor home environment may all be determinants of the asthmatic's
response to NO2 Controlled human exposure studies are ethically limited to acute or
subchromc fully reversible functional and/or symptomatic responses This may in many
cases limit the magnitude of expected responses and, hence, the statistical significance of
responses in studies with small numbers of subjects Exposures seldom last longer than 1 to
2 weeks for up to 8 h per day These data, therefore, are pnmanly useful in evaluation of
short-term, NO2-induced health effects
True simulation of ambient conditions, given the number of potential pollutants and the
variety of possible combinations, is not a realistic goal for controlled human exposure
studies For example, the typical temporal pattern of ambient concentrations is seldom
duplicated in controlled exposure studies However, simple mixtures of two or three
pollutants can be evaluated to determine the potential for either additive or synergistic
effects Further discussion of the design considerations for human clinical studies are
presented by Bates et al (1970), Hackney et al (1975a), and Folinsbee (1988) and were the
subject of a symposium proceedings (Frank et al, 1985). Because controlled exposure
studies of humans deal exclusively with acute or subchromc exposures, the applicability of
these data is limited to short-term exposure effects and of limited usefulness in the evaluation
of the effects of chronic NO2 exposure
More than 25 additional studies on the effects of NO2 on healthy, normal subjects have
become available since the 1982 Air Quality Criteria for Oxides of Nitrogen Document (U S
Environmental Protection Agency, 1982a) Several new studies of the effects of NO2 on
individuals with pulmonary disease (asthma and COPD) have been published, helping to
alleviate a critical information deficit of the earlier review (U S Environmental Protection
Agency, 1982a,b) Although more than 10 new reports have been published, the data base
concerning NO2 effects in sensitive subjects still requires concentration-response studies in
moderately sensitive asthmatics, information concerning the inflammatory response to NO2
inhalation, further examination of the effects of NO2 on infectivity in humans, and further
15-7
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evaluation of patients with COPD Only one study is available on pulmonary epithelial
permeability or mucociliary clearance effects of NO2 in humans
One of the more important observations in studies of NO2-exposed animals is that NO2
exposure is associated with increased susceptibility to viral and bacterial infections due to
impairment of host-defense mechanisms (see Section 13 22 I) Also, epidemiology studies
clearly suggest a link between NO2 exposure and increased rates of respiratory illness,
especially in children (Section 14 3 1) These studies have provided a basis for several
recent investigations of human immune host defenses after NO2 exposure Studies have
utilized both in vitro exposure of cultured human cells (e g , macrophages) and in vivo
exposures of human subjects
This chapter opens with discussion of effects on healthy adults of controlled human
exposures to NO2 Recently published reports generally support prior conclusions regarding
the effects of NO2 exposure on healthy young adults Several of the newer studies examined
the specific effect of NO2 on cardiopulmonary function in normal adults (see Section 15 2)
The NO2 concentrations ranged from 0 2 to 4 0 ppm In another group of studies, the
effects of pollutant mixtures or ambient air, of which NO2 was a component, were examined
These studies are summarized in Section 15 2 3
Studies examining the specific effects of NO2 in normal subjects support conclusions
earlier reached in the Air Quality Criteria for Oxides of Nitrogen Document (U S
Environmental Protection Agency, 1982a), in that they consistently demonstrated the absence
of effect of NO2 on lung function at concentrations between 0 3 and 0 6 ppm (Adams et al ,
1987; Drechsler-Parks et al, 1987, Drechsler-Parks, 1987, Kagawa, 1986)
Four studies (Avol et al, 1983, 1985a, 1987, Linn et al, 1980a) have also been
published in which NO2 was a component of an ambient oxidant air mixture The effects of
ambient air exposures, if any, were attributed to O3 There was no apparent influence of the
very low (0.04 to 0 07 ppm) concentrations of NO2 present in the ambient air In addition,
several controlled exposure studies used pollutant mixtures containing NO2 in concentrations
from 0.16 to 5 0 ppm (Kagawa, 1983a,b, Folinsbee et al, 1981, Islam and Ulmer, 1979a,b,
Kagawa and Tsuru, 1979, Kagawa, 1986, Kleinman et al , 1985, Linn et al, 1980b, Toyama
et al., 1981, Von Nieding et al, 1979, Stacy et al, 1983, Drechsler-Parks et al , 1987)
15-8
-------
At concentrations less than 1 0 ppm, these studies demonstrated no obvious effects of NO2 in
pollutant mixtures, which contained O3, SO2, and/or pairticles in addition to NO2
After the discussion of healthy subjects, NO2 effects on sensitive subjects, including
asthmatics and patients with COPD, are presented (Section 15 3) Several studies examining
the effects of NO2 concentrations in the range of 0 1 to 0 5 ppm on spirometry in asthmatic
subjects suggest possible small changes in lung function (Bauer et al , 1986b, Roger et al ,
1985, Koemg et al , 1987a,b, Avol et al., 1986) However, these changes were absent at
higher NO2 concentrations (Avol et al, 1986, Bylin et al, 1985, Linn et al, 1985b, 1986),
thus failing to suggest a concentration-response relationship Studies examining patients with
COPD indicated pulmonary function changes with brief exposure to high concentrations (5 to
8 ppm for 5 min) or with more prolonged exposure to lower concentrations (0 3 ppm for
3 75 h).
Since a change in airway responsiveness appears to be one of the most sensitive
indicators of response to NO2 exposure, Section 15 4 discusses in more detail the effects of
NO2 exposure on airways responsiveness in both healthy and asthmatic subjects Airway
responsiveness has been shown to increase in healthy subjects after exposure to NO2
concentrations in excess of 1 0 ppm Evaluation in Section 15 4 of studies of NO2 exposure
effects on airways responsiveness in asthmatics indicates in some cases increased airway
responsiveness to a variety of provocative mediators at exposure levels of 0 2 to 0 3 ppm
NO2, however, the occurrence of these responses appears to be influenced by the exposure
protocol, particularly whether or not the exposure includes exercise
Section 15 5 next reviews the effects of NO2 and HNO3 exposure on blood, urine, and
bronchoalveolar lavage fluid (BAL) biochemistry This is followed by discussion of an
important array of studies examining the effects of exposure to NO2 or HNO3 exposure on
human pulmonary host defense responses These studies (Section 15 6) examine the roles
that NO2 exposure may play in potentiating susceptibility to respiratory infections Next,
Section 15 7 examines the effects of nitrates on human lung function Finally, Section 15 8
presents conclusions and a summary of the chapter
15-9
-------
15.2 EFFECTS OF NITROGEN OXIDES IN HEALTHY SUBJECTS
Early studies indicated that the effect of NO2 on airway resistance was noted at
concentrations above 1 5 ppm in healthy volunteers (Abe, 1967, Von Nieding et al, 1970,
1973a) Other studies have indicated no significant lung function effects of NO2 in healthy
normal subjects at concentrations below 1 0 ppm (Folinsbee et al, 1978, Hackney et al,
1978, Beil and Ulmer, 1976, Kerr et al, 1979) This section presents a discussion on these
and other studies, as well as studies on NO and NO2 mixtures
15.2.1 Lung Function Effects of Nitrogen Dioxide
This section is divided according to the concentrations of NO2 used m the study The
first subsection deals with the effects of exposure to greater than 1 0 ppm, the second with
the effects of exposure to less than 1 0 ppm Studies dealing with NO2 exposure m healthy
subjects are summarized in Table 15-1 (The details of the exposure conditions, number of
subjects, ventilation levels, temperature, relative humidity, and other experimental
information are presented in Table 15-1 Occasional reference to this information is made in
the text when necessary, but the reader should refer to the table for these details )
15.2.1.1 Concentrations Above 1.0 ppm
The effects of NO2 levels greater than 1 0 ppm have been examined in several
laboratories In two studies, Von Nieding and Wagner (1977) and Von Nieding et al (1979)
studied 11 males exposed to 5 0 ppm NO2 for 2 h while performing light, intermittent
exercise Airway resistance increased from 1 51 to 2 41 cm H2O/L/s after 2 h of exposure
There was also an apparent decrease of arterial oxygen partial pressure (PaO^) from 90 to
82 torr (These samples were taken from "artenahzed venous" blood drawn from the ear
lobe ) The statistical analysis of this data is impacted slightly due to an adjustment in the
PaO2 data prior to testing for significance Von Nieding and Wagner (1977) state
"To increase the power of the tests PaO2-differences <5 mm Hg and RT-increases ^0 5 cm
H2O/L/s were regarded as zero " This transformation would increase the likelihood of
finding a significant effect
In a subsequent synopsis of several studies, Von Nieding et al (1980) discussed the
results of two experimental exposures that were previously published (in German) In the
15-10
-------
TABLE 15-1. RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE8
Reference
Abe (1967)
Adams et al
(1987)
Beil and Ulmer
(1976)
Bousheyetal (1988)
(part 2)
Exposure Exercise Exercise
NO2 Duration Duration Vent Temp
(ppm) (mm) (nun) (L/min) (°C)
40-50 10
06 60 60 70 21-25
50
1 0 120
25 120
50 120
75 120
50 840
06 120/day 60 -30-40 210
for 4 days
Relative Number and
Humidity Gender of
(Percent) Subjects
—
45-60 20 M
20 F
16
16
16
16
8
56 4 M/l F
Subject
Characteristics
Normal
Healthy
11 S
5NS
8S
Healthy, NS,
21-36 years,
FEVi/FVC%
range 73-83%
"normal"
methacholine
responsiveness
Notes on Effects
Bag exposure technique
Airway resistance increased
30 mm after end of exposure
No change in spirometry
No effect of NO2 on
spirometry or airway
resistance
Responsiveness to
acetylcholrne challenge
increased after 7 5 ppm
(120 mm) and 5 0 ppm (14 h)
Resistance increased after all
but the 1 0-ppm exposure
No effects of repeated NO2
exposure on respiratory
function (SRaw, FVC, FEVj)
or symptoms Slight increase
in circulating (venous)
lymphocytes 1,792 ±
544 mm3 (post-NO2) vs
1,598 ± 549/mm3 (baseline)
No change in BAL lympho-
cytes except an increase in
natural killer cells 7 2 ± 3 1 %
(post-NO2) vs 4 2 ± 2 4%
(baseline) No change
observed m IL-1 or TNF
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE3
Reference
Bylinetal (1985)
Chaney et al (1981)
Devlin etal (1992)
Drechsler-Parks et al
(1987)
Drechsler-Parks
(1987)
Folinsbee et al
(1978)
N02
(ppm)
00
012
025
05
02
20
060
060
062
Exposure
Duration
(nun)
20
120
240
120
120
120
Exercise
Duration
(nun)
—
0
120
60
60
15
30
Exercise
Vent Temp
(L/min) (°C)
22
22
50 22
25 24
25 24
33 25
33
Relative
Humidity
(Percent)
35
40
40
54
55
45
Number and
Gender of
Subjects
5M/4F
19 M
(15 controls)
10
8M/8F
8M/8F
5M
5M
Subject
Characteristics
20-36 years,
NS
Young adult,
normal
Healthy NS
51-76 years
18-26 years, NS
Healthy
Notes on Effects
Suggestion of change in SR.^ in
normals SRaw tended to
increase at 0 25 ppm. and tended
to decrease at 0 50 ppm
Analysis of variance indicates
no significance No effects on
bronchial reactivity Median
odor threshold 0 04 ppm
Increase in blood glutathione
levels after NO2 exposure
Increased bronchial PMNs and
decreased macrophage
phagocytosis
No statistically significant
changes in lung function due to
NO2 exposure
No significant changes in
spirometry attributable to NO2
No significant pulmonary
function responses attributed to
NO2 exposure
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE3
Reference
Frampton et al
(1989a)
Frampton et al
(1989b)
NO2
(ppm)
06
005
with 2 0 spikes
(1)06
(2) VAR (0 05
background
with 3 X 15
mm at
2 0 ppm)
(3)06
(4)15
Exposure
Duration
(mm)
180
135
(3 X 15)
180
180
180
180
Exercise Exercise
Duration Vent Temp
(mm) (L/mrn) (°C)
60 «40 22
(6 X 10)
60
(6 X 10)
60 39 22 0
60 43 22 0
60 «40 22 0
60 39 22 0
Relative Number and
Humidity Gender of Subject
(Percent) Subjects Characteristics
30 7 M/2 F Healthy, NS
11 M/4F Nonreactive
(carbachol),
no recent upper
respiratory
infection
30 6 M/2 F 30 3 + 1 4 years
30 11M/4F 253 ± 1 2 years
30 5 M/3 F 326 ±16 years
30 12 M/3 F 23 5 ± 0 7 years
Healthy, NS
Notes on Effects
No change in spirometry,
Raw, or carbachol reactivity
No change in cell recovery
or differential counts
Possible decrease in
macrophage inactivation of
virus in vitro Possible
sensitive subgroup
Total NO2 uptake (1) 3 4 mg
(2) 5 6 mg, (3) ~3 3 mg
(4) 8 1 mg BAL fluid
analysis showed no
significant effect on total
protein or albumin There
was an apparent increase in
alpha-2-macroglobulin 3 5 h
after exposure to 0 60 ppm
(Group 1) but not after the
other protocols No changes
in percentage of lymphocytes
or neutrophils Authors
concluded that NO2 at these
concentrations neither altered
epithelial permeability nor
caused inflammatory cell
influx
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE8
Reference
Framptonetal (1991)
Framptonetal (1992)
Goings etal (1989)
(see Kulle and
Clements, 1988)
Exposure Exercise Exercise
N02 Duration Duration Vent Temp
(ppm) (nun) (nun) (L/min) (°C)
See
Groups 1,
2, and
4 above
20 360 Intermittent
10 120/day, - - 222
20 3 days
30
00
Relative Number and
Humidity Gender of Subject
(Percent) Subjects Characteristics Notes on Effects
There were no changes in
airway mechanics (FVC,
FEVj, SGaw)
Responsiveness to carbachol
was significantly increased
after 1 5 ppm NO2
(Group 4) but not after the
other exposures (Groups 1
and 2) Degree of baseline
responsiveness to carbachol
was not related to response
after 1 5 ppm
12 Healthy, NS Immediate and 18-h post-
BAL increase in PMNs
60 21 Healthy, NS, Overall trend for a slight
22 seronegative decrement in FEVj with
22 NO2 exposure ( < 1 %)
23, 21 Methacholine response
or 22 decreased with consecutive
tests, but not as a result of
NO2 exposure or infection
status Study conducted
over 3-year period NO2
did not significantly
increase viral infectivity,
although a trend was
observed This study had a
low power to detect small
differences in infection rate
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE8
Reference
Hackney et al (1978)
Exposure
NC>2 Duration
(ppm) (rmn)
10 120
(2 con-
secutive
days)
Exercise
Duration
(nun)
60
Exercise
Vent Temp
(L/min) (°C)
Light 31
Relative
Humidity
(Percent)
35
Number and
Gender of
Subjects
16
Subject
Characteristics
Healthy
Notes on Effects
Air-N02-N02 fixed
exposure sequence
A 1 5% decrease in FVC
after second day of NO2
Not clear that the decreased
FVC is an NO2 effect or an
order effect No other
effects
Hazuchaetal (1982,
1983)
01
60
21
40
15 M
23-39 years, NS No symptoms, no odor
detection, no effect on
Johnson et al (1990)
Joerres et al (1992)
Kagawa and Tsuru
(1979)
Kagawa (1982)
See
Frampton
etal
(1989b)
Groups 2
and 4
1 0 180 Intermittent - ~ - 3 M/5 F
0 15 120 60 SOW 27-29 50-60 6 M
1 0 ppm 120 60 SOW 28 55 8 M
NO
No change in a-1-protease
inhibitor after NO2
exposure
Healthy No responses
19-24 years No symptoms, no
pulmonary function effects
Suggested individual
changes in SGaw
19-24 years Suggested change in density
dependance of expired
flow
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE3
Ul
H^
ON
Reference
Kagawa (1986)
Kerretal (1979)
Kimetal (1991)
Koemgetal (1985)
Koemgetal (1987a,b)
N02
(ppm)
030
05
0 18
030
012
0 12
0 18
Exposure
Duration
(nun)
120
120
30
60
40
40
Exercise
Duration
(mm)
60
15
L10
H16
—
10
Exercise
Vent Temp
(L/nun) (°C)
SOW 28
Light/ 24
moderate
L « 25 22
H « 72
22
325 22
Relative
Humidity
(Percent)
50-60
45
45
75+
75
Number and
Gender of
Subjects
6
10
9M
4M/6F
3M/7F
4M/6F
Subject
Characteristics
19-25 years
Healthy, three
ex-smokers in
group
18-23 years,
"collegiate
athletes"
13-18 years
14-19 years
15-19 years
Notes on Effects
No effect on SGaw, other
NO2 mixtures studied but
effect of NO2 cannot be
ascertained
Decreased quasistatic
compliance Nonrandom
exposure sequence air-
NO2 No change in
spirometry or resistance
Apparent compliance
change may be due to
exposure order
No change in lung
function
No effects on lung
function
No effects of either 0 12
or 0 18 ppm NO2 on RT
Kulle(1982) 050 120 15
Linn and Hackney 40 75 L15
(1983) and Linn et al H 15
(1985b)
or spirometry
24 45 10 Normal adults Decreased static lung
compliance
L 20-29 21 50 16 M/ 9 F 18-45 years, NS No change in SR^
H 44-57 associated with NO2
Small but significant
decrease in blood
pressure, some mild
increase in symptoms
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE3
Reference
Mohsemn (1987b)
Mohsemn (1988)
Mohsemn and Gee
(1987)
Morrow and Utell
(1989)
t— i
i
-J
Muelenaer et al
(1987)
N02
(ppm)
20
20
3 0 (n =6)
4 0 (n =4)
0 0 (n =7)
03
06
Exposure
Duration
(nun)
60
120
180
225
225
240 mm
X 3 days
Exercise Exercise
Duration Vent Temp
(mm) (L/min) (°C)
21
21
60 38 21
30 «40 21
(3 X 10)
21 30-40 21
(3 XT)
60 «15 «22
(4 X 15)
Relative
Humidity
(Percent)
50
50
Dewpoint
10
40
40
«40
Number and
Gender of
Subjects
8M/3F
13 M/5 F
16 M/l F
10M/10F
10M/10F
14 M
Subject
Characteristics
18-36 years, NS
Normal, NS,
18-33 years
25 years, NS
Healthy, young,
20-48 years,
FEV^FVC
76-95%
Elderly, normal,
49-69 years,
FEV,/FVC
72-84%,
3 maleS
4 female S
NS, nonatopic,
no residential
NO2 exposure
Notes on Effects
Vitamin C blocked NO2-induced
increase in airway reactivity to
methacholine
No symptoms, no lung function
changes Increased methacholine
reactivity
45% decrease in a- 1 -protease
inhibitor in BAL fluid
No symptom, lung function or
airway reactivity responses for
the group
No symptom, lung function or
airway reactivity responses
No change in urinary hydroxy-
prohne excretion
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE3
Reference
Rehnetal (1982)
Sackner et al
(1980)
Sandstroem et al
(1989)
)•_!
i Sandstroem et al
So (1990a)
Stacy et al (1983)
Smeglm et al
(1985)
Suzuki and
Ishikawa (1965)
NO2
(ppm)
0
027
106
0 1
03
05
10
225
40
55
40
05
030
07-
20
Exposure Exercise
Duration Duration
(mm) (mm)
60
240
20 20
20 min0 20C?)
alternate
days for
12 days
240 30
240 30min
10
Exercise Relative Number and
Vent Temp Humidity Gender of
(L/min) (°C) (Percent) Subjects
22-27 25-45
_ 6
-35 - - 8
8
8
Total
n = 18
~35C?) - - 8
55 30 60 10 M
«30 - - 20
10
Subject
Characteristics
Healthy,
young M
Normal adults
Healthy, NS
Healthy, NS
26 4 years
21-48 years,
NS
Notes on Effects
Possible small increase in Raw at
0 27 ppm No change in nasal or
tracheobronchial clearance
No effects of NO2 in resting adults
Increased levels of mast cells in BAL
fluid at all concentrations Increased
numbers of lymphocytes at 4 0 and
5 5 ppm BAL 24-h postexposure
Total cell counts were reduced
Alveolar macrophages had enhanced
phagocytic activity, but fewer were
present Decreased numbers of mast
cells, T and B lymphocytes and natural
killer cells BAL 24-h postexposure
No significant effects on spirometry or
No change in lung function or airway
reactivity
Increased resistance 10 mm after
exposure
-------
TABLE 15-1 (cont'd). RESPONSES OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE EXPOSURE3
Reference
Exposure Exercise Exercise Relative Number and
NO2 Duration Duration Vent Temp Humidity Gender of Subject
(ppm) (min) (mm) (L/nun) (°C) (Percent) Subjects Characteristics
Notes on Effects
Toyamaetal 07 60
(1981)
5
3 were No effects on airway conductance or
investigators, responsiveness
22-19 years
Von Nieding et al 50 15
(1973a)
Von Nieding et al 50 120 Inter-
(1977) mittent
Von Nieding et al 50
(1979)
120 60 220
(4 X 15)
16 Healthy Decreased DLCO 18%
Light 22 55 11 M Healthy Increased resistance 60% Remained
elevated for 60 mm Possible decrease
inPaO2
22 55 11 M Healthy Resistance increased 60% Remained
elevated 60 mm after exposure
Possible decrease in earlobe PO2
Abbreviations
NO2 = Nitrogen dioxide
M = Male
F = Female
S = ACUVB hmuker
NS = Nonsmoker
FEVj = Forced expiratory volume mis
FVC = Forced vital capacity
SRaw = Specific airway resistance
BAL =
IL-1 =
TNF =
PMNs, -
n —
Kaw ~
VAR =
SGaw =
Bronchoalveolar lavage
Interleukm-1
Tumor necrosis factor
Foiymorpiionuclcar leukocytes
Airway resistance
Variable
Specific airway conductance
W
L
H
RT =
DLCO =
Pa02 =
P02 =
Watts
Light
Heavy
Total respiratory resistance
Diffusing capacity for carbon monoxide
Arterial partial pressure of oxygen
Partial pressure of oxygen
-------
first study, 14 normal patients were exposed to 5 to 8 ppm NO2 for up to 5 min on
4 separate days The airway resistance (R^) increased by an average of 0 58 cm H2O/L/s
(range 0.39 to 1 03 for the individual four-exposure mean) after the NO2 exposures It was
noted that there were no differences in response between smokers and nonsmokers
Beil and Ulmer (1976) studied the effects of 2-h exposures to 0 0, 1 0 (n = 8),
2.5 (n = 8), 5 0, and 7 5 ppm NO2 in 16 healthy resting subjects An additional group of
8 healthy resting subjects were exposed for 14 h to 5 0 ppm NO2 for 2 consecutive days
They found a small significant increase in total respiratory resistance (RT) after exposure to
2.5 ppm NO2 or greater The main response, no more than 1 cm H2O/L/s above a baseline
of 2.6 cm H2O/L/s, occurred during the first 30 min of exposure and the response was not
appreciably increased by raising the NO2 concentration to 5 0 or 7 5 ppm NO2 The
increase in RT following NO2 exposure was related to the baseline airway responsiveness to
acetylcholine Airway responsiveness to acetylchohne was increased after exposure to
7.5 ppm for 2 h or to 5 0 ppm for 14 h, but not after the 2-h exposures to 5 0 ppm or less
The pattern of response in the 14-h exposure indicated an initial increase in resistance during
the first 30 nun («30%), a slight decline in resistance over the subsequent 90 mm, and then
a modest further increase over the next 14 h (a total increase of «60%) Resistance
returned to baseline during the subsequent 10 h and this response pattern was repeated on the
second exposure day The 1982 Air Quality Criteria for Nitrogen Oxides Document cited
this study as indicating that responses were "clearly demonstrated to occur in healthy adults
with single 2-h exposures to NO2 ranging from 4,700 to 14,000 /*g/m3 (2 5 to 7 5 ppm) "
Linn et al (1985b) exposed 25 healthy, nonsmoking subjects (9 female, 16 male) for
75 min to 4 0 ppm of NO2 or purified air Subjects were exposed to each condition twice,
*i
for a total of four exposures The authors reported that approximately 11 /tg/m of
particulate nitrate was present during NO2 exposures During the exposures, subjects
performed 15 mm of light (25 L/min) and 15 mm of heavy (50 L/mm) exercise There were
no significant effects of NO2 on R^ or symptoms Although heart rate and skin
conductance were similarly unaffected, there was a slight but statistically significant
reduction in systolic blood pressure associated with NO2 exposure Although inhalation of
NO2 can result in increased blood levels of nitrite and nitrate ion, the mechanism for this
small change in systolic pressure has not been established Blood pressure readings were
15-20
-------
obtained using an automated procedure while the subjecl was seated quietly in the body
plethysmograph
Mohsemn (1987b) studied the effects of 1-h resting exposure to 2 0 ppm NO2 on
11 normal subjects to determine the effect of ascorbic acid administration prior to NO2
exposure The author hypothesized that the antioxidant properties of ascorbic acid would
modify the effect of NO2 There were a total of four exposures In the first set of clean air
and NO2 exposures, the subjects received a placebo for 3 days prior to the exposures In the
second air/NO2 exposure pair, the subjects received vitamin C In both cases, the order of
the NO2 and air exposures were randomized The blood ascorbic acid levels were increased
from 0 76 mg/dL after placebo to 1 90 mg/dL after vitamin C supplementation Neither
plethysmography nor spirometry tests indicated a significant effect of NO2 in these subjects
under placebo or vitamin C conditions There was a significant increase in airway
responsiveness to methacholine (bromide) after NO2 exposure Responsiveness to
methacholine was quantified by the dose required to reduce SGaw by 40% (PD40), this
corresponds to a 67% increase in SR^ (A 50% decrease in SGaw corresponds to a
doubling of SRaW ) After the two air exposures, PD40 averaged about 64 mg/mL, but was
reduced to 53 mg/mL after NO2 exposure and placebo treatment When the subjects were
given ascorbic acid pnor to exposure, methacholine responsiveness after NO2 exposure was
unchanged Ascorbic acid pretreatment apparently blocked the airway responsiveness
increase, which had previously been observed with NO2 exposure, although it had no effect
on baseline methacholine responsiveness However, ascorbic acid has previously been shown
to cause a decrease in methacholine responsiveness in both normals and asthmatics
(Mohsemn et al , 1983, Ogilvy et al , 1981) Thus, it is unclear whether ascorbic acid
blocks the effect of NO2 on airways responsiveness or whether there was a direct effect of
ascorbate on methacholine responsiveness subsequent to vitamin C supplementation
Mohsemn (1988) studied the response of 18 normal adults exposed to 2 ppm NO2 for
1 h at rest There were no symptoms, no changes in lung volume, no change m flow-volume
characteristics on either full or partial expiratory flow-volume (PEFV) curves, and no change
in SGaw However, airway responsiveness to methacholine was increased following exposure
in 13 of 18 subjects and decreased in only 2 of the 18 (p = 0 003) subjects The dose of
15-21
-------
methacholine needed to cause a 40% reduction in SGaw was 101 ± 44 mg/mL after air and
81 ± 45 mg/mL after NO2
Kulle and Clements (1988) studied the effects of NO2 exposure on infectivity of live
attenuated influenza A/Korea/reassortment virus in healthy nonsmoking adults (see Goings
et al., 1989) Independent control and exposure groups were exposed to clean air for 1 day
and then either clean air or NO2 (1, 2, or 3 ppm) for the next 3 consecutive days Included
in this evaluation were measurements of respiratory symptoms, lung function, and airway
reactivity to methacholine in the 2- and 3-ppm studies There were no significant changes in
respiratory or other symptoms as a result of a 3-ppm NO2 exposure The only apparently
significant changes in spirometry were observed in the control group, who showed slightly
less decrease in forced vital capacity (FVC) or forced expiratory flow at 25 to 75 % of vital
capacity (VC) (FEF25-75%) during the last of four consecutive clean air exposures Airway
responsiveness to methacholine was measured following exposure to 2 and 3 ppm The clean
air control groups showed a small significant decrease in airway responsiveness on the
second, third, and fourth days, but airway responsiveness remained unchanged in the
NO2-exposed subjects Influenza virus infection did not alter airway responsiveness in either
air or NO2 exposure groups Reactivity returned to control at 2 and 4 weeks after the
exposure series The infectivity portion of this study is discussed in Section 15 4
Frampton et al (1991) studied a group of 39 healthy nonsmokers exposed for 3 h to
either 0 60 ppm (n = 9), 1 5 ppm (n = 15), or a variable concentration protocol where three
15 min "peaks" of 2 0 ppm were added to a background level of 0 05 ppm (See Frampton
et al, 1989b, in Section 15 4 2 for details) There were no direct effects on lung function
(FVC, FEVls SGaw) after any of these exposures However, there was a small statistically
significant increase in FVC and FEVj response to carbachol challenge after the 1 5 ppm
exposure, indicating an increase in airway responsiveness There was no increase in airway
responsiveness after the 0 6 ppm or the peaks protocol However, one subject had a 20 %
greater drop in FEVj after the peaks NO2 exposure than after the air exposure This
observation suggests the possibility that some subjects may be affected by NO2 to a much
greater extent than others
15-22
-------
15.2.1.2 Concentrations Below 1.0 ppm
In NO2 exposure studies conducted at concentrations below 1 0 ppm, the findings have
been generally negative Although some authors have indicated occasional findings, there
does not appear to be a consistent pattern of response at these low NO2 concentrations that
would be indicative of short-term health effects
Kagawa and Tsuru (1979) studied six healthy men exposed to 0 15 ppm NO2 for
2 h while performing light, intermittent exercise There were no symptoms reported during
NO2 exposure Although the authors suggested that there might be some responses to NO2
exposure, the overall pattern of response does not support the conclusion that changes in lung
function were induced by NO2 These authors reported "significance" for individual
subjects, although the precise technique for making this judgment is unclear Two mean
differences were reportedly "significant" (multiple t-tesl unadjusted for multiple
comparisons), a 0 5% decrease in VC and a 16% decrease in the ratio of
FEF 75%(He)/FEF 75%(air) However, nonsignificant responses of greater magnitude were
observed under other exposure conditions (e g , air control) It appears that these
"significant" observations may only be chance occurrences out of nearly 100 t-tests, 6 of
which showed "significance " Furthermore, a temporary 05% decrease in VC is of little, if
any, physiological significance
Subsequently, Kagawa (1983a) reported the results of exposing an additional seven
subjects to 0 15 ppm NO2 (also to other pollutants, separately and in combination) for
2 h with light, intermittent exercise Using the same piotocol and exposure conditions as
those of Kagawa and Tsuru (1979) m this new data set, no statistically significant mean
changes were found to be associated with NO2 exposure in any of the plethysmographic or
spirometnc tests
Toyama and colleagues (1981) exposed five healthy subjects (two were smokers, three
were investigators) to 0 7 ppm NO2 for 60 mm while at rest They observed no responses to
this NO2 exposure that altered airway conductance or flow-volume tests
Kulle (1982) presented a reanalysis of the data previously published by Kerr et al
(1979) In this study, 10 normal, 13 asthmatic, and 7 chronic bronchitic subjects were
exposed to 0 5 ppm NO2 for 2 h, several of the subjects were smokers (3 normals,
3 asthmatics, and 5 bronchitics) There were no significant effects of NO2 exposure on
15-23
-------
pulmonary function, but it was unclear whether the change in quasistatic compliance was due
to NO2 exposure or was a statistical artifact, as the original authors (Kerr et al , 1979)
suggested Rather than compare the data across the postexposure measurements obtained
with clean air and NO2, respectively, in the reanalysis (Kulle, 1982), a difference score
(post — pre) was determined for each condition and the differences were tested for
significance All subjects perceived the odor of NO2 upon entering the exposure chamber
The author reported a significant increase in the normal subjects in the Phase IV of the single
breath nitrogen washout test However, the data suggest that the difference was probably
due to a reduced preexposure value on the NO2 exposure day, an effect that could not be
attributed to NO2. Quasistatic lung compliance was decreased after NO2 exposure in the
normal group. The absence of a change in dynamic compliance suggests that the original
authors (Kerr et al., 1979) may have been correct in concluding that "significance" was
probably due to chance alone No other spirometnc or plethysmographic measurements were
significantly altered by NO2 exposure With the exception of the apparently artifactual
change in closing volume, no new conclusions can be drawn from the reanalysis of these
data
Stacy et al (1983), as part of a large multipollutant exposure study, exposed a group of
10 men to 0 5 ppm NO2 for 4 h, including two 15-min periods of moderately heavy exercise
None of the plethysmographic or spirometnc tests showed a significant effect of NO2 The
experimental design of this study was complex, having a total of 20 treatment cells The
data were analyzed by both a multivanate analysis of variance with an adjusted p value
(a level) of 0 0026 and by individual t-tests with a less conservative p value of 0 05
Neither analysis indicated significant effects of NO2
Hazucha et al (1982, 1983) studied a group of 15 healthy adult males exposed to either
air or 0.1 ppm NO2 for 1 h Control measurements were performed on the day before and
the day after the exposure The subjects did not detect the odor of NO2, nor was there an
increase of symptoms related to the NO2 exposure There were no effects of NO2 on
spirometry, airway resistance (SRaw or RT), or methacholine responsiveness
Rehn et al (1982) reported a small (17%) increase in SR^ after exposure of eight
•a
healthy men to 0 27 ppm (500 j«g/m ) for 1 h However, no response was seen at 1 06 ppm
15-24
-------
(2,000 jug/m3) This was reported in a technical paper (in Swedish) and has not yet been
published in a peer-reviewed journal
Bylin et al (1985) exposed eight normal subjects to 230, 460, and 910 ^g/m
(0 12, 0 24, and 0 48 ppm) for 20 mm An analysis of variance did not reveal any
significant effects of NO2 on changes in SR^, but specific comparisons indicated a
significant 11 % increase in SR^ at 0 24 ppm and a 9% decrease in SR^ at 0 48 ppm
Even though statistically significant, such small changes (±15%) in airway resistance are
well within the normal variation of 10 to 20% (Pelzer and Thomson, 1966, Skoogh, 1973)
Histamine bronchial responsiveness was tested after the 0 48-ppm exposure, but there were
no changes
Koemg et al (1987a) exposed normal subjects to (1) 0 12 ppm NO2 for two 30-min
periods at rest, (2) 0 12 ppm for 30 mm at rest plus 10 mm with exercise, and (3) 0 18 ppm
for 30 mm at rest and 10 nun during exercise For the at-rest 0 12-ppm NO2 exposures,
there were no significant changes in lung function, symptoms, or arterial oxygen saturation
Nor were there significant NO2 effects on lung function with the mild exercise exposures to
0 12 and 0 18 ppm NO2
Morrow and Utell (1989) studied both young (20 to 48) healthy subjects and elderly
(49 to 69) healthy subjects exposed to 0 3 ppm NO2 for 3 75 h The young subjects
performed a total of 30 mm of moderate exercise during exposure and the older subjects
exercised for 21 nun There were no differences between air exposure and NO2 exposure
for symptom responses, changes in lung function, or in airway responsiveness to carbachol in
either young or older subjects
The effects of 0 60-ppm NO2 exposures on young men and women during 1 h of
continuous heavy exercise were studied by Adams et al (1987) There were no significant
effects of NO2 exposure on airway resistance, symptoms, spirometry, or exercise responses
Kim et al (1991) studied nine athletes exposed to filtered air, 0 18, and 0 30 ppm NO2
for 30 mm while exercising (running and walking) Sixteen minutes were spent running at a
ventilation of about 72 L/min, 10 of the remaining 14 nun were spent walking Overall
ventilation is estimated to have averaged about 50 L/min There were no significant changes
in respiratory symptoms, FEV1} RT, peak expiratory flow rate, or ventilation (V50%vc) as a
result of NO2 exposure in this group of athletic male subjects
15-25
-------
In another study, young (18 to 26) and older (51 to 76) men and women were exposed
to 0.60 ppm NO2 by Drechsler-Parks et al (1987) and Drechsler-Parks (1987) Subjects
performed light, intermittent exercise during the 2-h exposures There were no effects on
spirometry or symptoms None of the individual pre—post exposure differences in NO2
(as compared to air) for FVC or FEV^ were outside of the normal range (i e , there were no
individual subjects who appeared reactive to NO2)
15.2.1.3 Respiratory Symptom and Sensory Effects of Nitrogen Dioxide Exposure
Several studies reported in the previous section also examined symptomatic responses of
subjects exposed to NO2 None of the studies of NO2 exposure in normal subjects, including
exposure for as long as 75 min to 4 0 ppm NC>2 resulted in a significant increase in
respiratory symptoms Sensory effects were examined in at least two studies (Bykn et al,
1985, Hazucha et al, 1983) The subjects in the study of Hazucha et al (1983) were unable
to detect the odor of 0 1 ppm NO2 Bylin et al (1985) reported an odor threshold of
0.04 ppm for normals and 0 08 ppm for asthmatics
15.2.1.4 Mucociliary Clearance After Nitrogen Dioxide Exposure
Rehn et al (1982) examined the effects of NO2 exposure on mucocihary clearance in
both the nose and lung Nasal clearance was determined using the rate of saccharin
transport from nares to oropharynx Tracheobronchial clearance was determined by
monitoring the rate of disappearance of radiolabeled Teflon aerosol After a 1-h exposure to
either 0 27 or 1 06 ppm (500 or 2,000 /ig/m3) NO2, there were no changes in either nasal or
tracheobronchial clearance rates
15.2.2 Effects of Nitric Oxide
In addition to NO2, Kagawa (1982) examined the effects of 1 ppm NO exposure for
2 h in eight normal subjects The data were analyzed by multiple t-tests using individual
data. All changes were referenced to the mean baseline (i e , mean of the preexposure
measurement for the air and the NO exposure) value rather than the corresponding
measurement tune from the clean air exposure Three "significant" individual changes in
SGaw were reported at 1 h of exposure one increase and two decreases After 2 h of
15-26
-------
exposure, there were three decreases and one increase, and in the postexposure period, there
were two increases and two decreases, all reported to be "significant". These statistical
analyses may not be the most appropriate Analysis of the mean data using similar
procedures (i e , multiple t-test referenced to the mean baseline level) produced only one
significant change an 11% decrease in the ratio HeV50/air V50 Given that this effect
(1) occurred only at one of the three measurement points for the NO exposure, (2) was one
of 98 paired t-tests, and (3) was significant at only the p < 0 05 level, it is reasonable to
suggest that the effect may have occurred by chance
A study of the effects of a mixture of NO2 (0 3 ppm) and NO (0 6 ppm) was recently
reported by Kagawa (1990) Exposures lasted 120 min and included mild (50 W)
intermittent exercise There were no significant changes in pulmonary function (airway
conductance [Gaw], V50%vc» slope of alveolar nitrogen concentration), symptoms, or airway
responsiveness to acetylcholine
Von Nieding et al (1973b) exposed healthy subjects and smokers to 15 to 39 ppm NO
for 15 min Total respiratory resistance increased significantly (»10 to 12%) after exposure
to ^20 ppm NO Diffusing capacity was not changed, but a small decrease (7 to 8 torr) in
PaO2 was noted
15.2.3 Effects of Nitrogen Dioxide Gas or Gas/Aerosol Mixtures on Lung
Function in Normal Subjects
Several studies of NO2-containing pollutant mixtures were previously discussed in the
Air Quality Criteria for Oxides of Nitrogen Document (U S Environmental Protection
Agency, 1982a) The general finding of these studies was that NO2 did not enhance the
effects caused by other oxidants, notably O3 (Hackney et al, 1975a,b,c, Von Nieding et al,
1977, Horvath and Folinsbee, 1979 [preliminary report of Folinsbee et al, 1981], Suzuki
and Ishikawa, 1965) On the other hand, Abe (1967) studied NO2-SO2 mixtures (4 to
5 ppm) and reported that their effects were additive, with both gases causing
bronchoconstaction Independently, the effect of SO2 was immediate and short-lasting,
whereas the effect of NO2 was delayed and more persistent The effect of the NO2-SO2
mixture was intermediate between the two responses Other reports suggesting possible
interactions of NO2, principally with some type of particle, included Schlipkoeter and
15-27
-------
Brockhaus (1963) and Nakamura (1964, cited in U S Environmental Protection Agency,
1982a.) These studies are reviewed extensively in Air Quality Criteria for Oxides of
Nitrogen Document (U S Environmental Protection Agency, 1982a)
Table 15-2 summarizes studies of healthy subjects exposed to NO2-contaming pollutant
mixtures The pollutant mixture of greatest interest, in terms of research effort, has been the
combination of NO2 and O3 (Adams et al, 1987, Drechsler-Parks, 1987, Drechsler-Parks
et al, 1989, Folinsbee et al, 1981, Kagawa and Tsuru, 1979, Kagawa, 1983a,b, Toyama
et al., 1981) Also reported were several studies of NO2-SO2 combination exposures
(Kagawa, 1983a,b, Kleinman et al, 1985, Linn et al, 1980a) Other pollutant mixtures
containing NO2 were also studied (Kagawa, 1986, Islam and Ulmer, 1979a,b, Stacy et al,
1983; Klemman et al, 1985)
Several recent studies evaluated the effects of O3 and NO2 in combination Three
studies investigated the effects of 0 5 to 0 6 ppm NO2 in combination with 0 3 to 0 5 ppm
O3 (Adams et al, 1987, Folinsbee et al , 1981, Drechsler-Parks, 1987) In these studies,
NO2 alone had no effect on measured health end points and the significant effects of O3 on
lung function were not altered by the presence of NO2 Kagawa and Tsuru (1979) and
Kagawa (1983a) reported conflicting results In their first study, it was suggested that the
effects of NO2 and O3 were "more than additive" However, in the second study, they
reported no significant enhancement of the effect of O3 by the mixture of other pollutants
(All pollutants—NO2, O3, and SO2—were at a concentration of 0 15 ppm) All of the above
studies included exercise during the 1- to 2-h exposures Toyama et al (1981) also studied
subjects exposed to NO2-O3 mixtures (0 5 ppm of each gas) Because the concentration of
each gas was 0 7 ppm when the exposures were to a single pollutant, it is impossible to
determine if there was an additive effect The above studies taken as a whole suggest
strongly that there is no interaction between NO2 and O3 that would result in enhancement of
acute O3-induced changes in lung function in normal subjects following short duration
exposures at NO2 concentrations less than 1 ppm However, this conclusion is not
necessarily applicable to other measured end points
Hazucha et al (1992) studied the effects of exposure to NO2 followed by exposure to
O3 in a group of healthy nonsmokers Subjects were exposed to 0 60 ppm NO2 and then,
3 h later, were exposed to 0 3 ppm O3 Mild intermittent exercise was performed during the
15-28
-------
TABLE 15-2. EXPOSURE OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE MIXTURES8
to
Reference
Adams et al (1987)
Avoletal (1983)
Avoletal (1985a),
Avoletal (1987)
Avoletal (1985b)
Drechsler-Parks
(1987)
Folinsbee et al
(1981)
Hackney et al
(1975b)
Exposure
Concentration
(ppm)
0 60 NO2
+ 0 30 03
0 05 NO2
(Amb)
0 04 N02
(Amb)
0 055 NO2
(Amb)
0 60 NO2
+ 0 45 O3
0 60 N02
(+0 45 O3)
0 50 NO2
(+0 5 O3)
(a) 0 50 03 +
0 29 NO2
(b) 0 50 O3
+ 0 29 NO2
+ 30 00 CO
Exposure
Duration
(nun)
60
60
60
60
120
120
120
240
(2 con-
secutive
days of
exposure
to each
mixture)
Exercise Exercise
Duration Vent Temp
(mm) (L/min) (°C)
60 70 21-25
50
60 56 32
60 22 4 32 7
60 32 32
60 25 24
60 25 24
30 40 25
30
35
40
120 -20 31
Relative
Humidity
(Percent)
45-60
43
43
45
55
55
45
85
40
50
35
Number and
Gender of
Subjects
20 M
20 F
42 M/8 F
33 M/33 F
46 M/13 F
8M/8F
8M/8F
8M/8F
10 M
10 M
10 M
10 M
4
Subject
Characteristics
Healthy young
adults
Healthy young
adult cyclists
Children,
8-11 years
Adolescents,
12-15 years
18-26 years, NS
51-76 years
51-76 years
Young adults, NS
Young adults, NS
Young adults, NS
Young adults, NS
Healthy
Notes on Effects
No additional effect of
NO2 over and above
effect of O3
No apparent effect over
and above that of O3
alone
No effects of ambient
air exposures
Ambient air exposure
effect attributed to O3
No significant changes
attributable to NO2
Tendency (p > 0 05)
for NO2 + O3 to be
greater than O3 alone
FEVj, decreased by
8-14% No differences
between O3 + NO2 ana
O3 alone
With each group,
minimal alterations in
pulmonary function
caused by O3 exposure
Effects were not
increased by addition of
NO2 or NO2 and CO to
test atmospheres
\
-------
TABLE 15-2 (cont'd). EXPOSURE OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE MIXTURES'
Reference
Hackney et al
(1975c)
Hazucha et al
(1992)
h— *
Vj* Islam and Ulmer
g (19798)
Islam and Ulmer
(1979b)
Kagawa (1983b)
Exposure
Concentration
(ppm)
(a) 0 25 O3
+ 0 29 NO2
(b) 0 25 03
+ 0 29 NO2
+ 30 00 CO
0 60 N02
followed by
03003
50NO2
+ 0 103
+ 5 0 SO2
0 16 NO2
0 34 SO2
008O3
0 15 NO2
0 15 O3
0 15 SO2
Exposure Exercise Exercise
Duration Duration Vent Temp
(nun) (mm) (L/min) (°C)
120 60 -20 31
(2 consecutive
days of
exposure)
120 60 40 22
3-h rest
120 60 40 22
120 60 ' 22
480 0 - 22
120 60 -25
Relative Number and
Humidity Gender of
(Percent) Subjects
35 7
40 15 M
40
60 8M
8M
8M
60 15
7M
Subject
Characteristics
Healthy
Healthy, NS
<30 years
30-40 years
>49 years
16-26 years
19-23 years
Notes on Effects
Little or no change in
pulmonary function found
with O3 alone Addition of
NO2 or of NO2 and CO
did not noticeably increase
the effect Seven subjects
included, some believed to
be unusually reactive to
respiratory irritants
NO2-O3 sequence increased
effect of O3 on airway
responsiveness
FVC (-5%), FEV!
(-11 7%), decreased with
exercise exposure to this
mixture in <30 years
group
No change in FVC, acetyl-
choline airway reactivity
No significant enhancement
of the effects of O3 and
SO2 by presence of NO2
-------
TABLE 15-2 (cont'd). EXPOSURE OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE MIXTURES8
Reference
Kagawa (1986)
Kleinman et al
(1985)
Lmnetal (1980b)
Exposure Exposure Exercise Exercise
Concentration Duration Duration Vent Temp
(ppm) (mm) (mm) (L/rnin) (°C)
030NO2 120 20 -25 28-29
+ 0 30 O3
+ 200 jug/m3
H2S04
0 15 NO2 120 60 -25 28-29
+ 0 15 O3
+ 200 /ig/m3
H2S04
0 15 N02 120 60 -25 28
+ 0 15 O3
+ 0 15 SOo
+ 200 mg/m3
H2S04
050NO2 135 60 -20 20
+ 05 SO^
+ 20 /ig/m"
ZnSO4
(NH4)2 S04
+ 330 jitg/m
NaCl
007 Ambient 120 60 -20 33
and other
pollutants
Relative Number and
Humidity Gender of Subject
(Percent) Subjects Characteristics
50-60 6 Japanese men
(some smokers)
6 Japanese men
(some smokers)
59-60 3 Japanese men
(some smokers)
85 11M/9F 20-53 years
32 14 M/20 F 29 years
Notes on Effects
Possible small decrease in
SGaw
Possible small decrease in
SGaw
Possible small decrease in
FEVj
No effects on function,
possible symptom
responses NO2 effects
not discernible from
mixture
Small decreases in FVC,
FEVj, in ambient air
mostly attributable to O3
No association of NO2
levels with lung function
change
-------
TABLE 15-2 (cont'd). EXPOSURE OF HEALTHY SUBJECTS TO NITROGEN DIOXIDE MIXTURES"
Reference
Exposure Exposure Exercise Exercise Relative Number and
Concentration Duration Duration Vent Temp Humidity Gender of Subject
(ppm) (mm) (nun) (L/nun) (°C) (Percent) Subjects Characteristics
Notes on Effects
to
Linnetal (1980a) 0 50 N02 120
+ 0 50 SO2
60 -20 31
40
10M/14F
26 ±4 years,
21 NS,
3 S
No significant effect on lung
function in normals Trend
for a slight decrease m FVC
after combined exposure
Von Nieduig et al 50
(1979) + 5 0 S02
+ 01 O3
0 1N02
+ 0 3 SO2
Abbreviations
NO2 = Nitrogen dioxide
03 = Ozone
M = Male
F = Female
Amb = Ambient
NS = Nonsmoker
FEVj = Forced expiratory volume in 1 s
CO = Carbon monoxide
SO2 = Sul&r dioxide
120
120
FVC
H2SO4
ZnSC^CNH^
NaCl
S
W
RT
PaO2
60 -20 22 55
(70W)
60 -20 22 55
= Forced vital capacity
= Sulfuncacid
= Specific airway conductance
3O4 = Zinc ammonium sulfate
= Sodium chloride
= Active smoker
= Watts
= Total respiratory resistance
= Arterial partial pressure of oxygen
23-38 years, RT increased from 1 5 to
two atopic 2 4 (p < 0 01),
questionable decrease in
PaO2 (8 ton)
23-38 years, No effects at all
two atopic
-------
exposures Nitrogen dioxide alone caused no significant lung function responses, but there
was a slightly greater decrease in FEVj after the NO2-O3 sequence than after the air-O3
sequence Methacholine airway responsiveness was significantly increased with the NO2-O3
sequence compared to an air-O3 sequence, indicating that prior NO2 exposure enhanced the
airway responsiveness increase typically found with O3 exposure
Linn et al (I980a) exposed a group of normal subjects to a mixture of 0 5 ppm NO2
and 0 5 ppm SO2 for a 2-h period, during which light, intermittent exercise was performed
There were no lung function (spirometry, closing volume, RT) responses There was an
overall increase in symptoms due to the NO2-SO2 exposure, but no significant increase in
any specific symptom category Using the same 2-h intermittent, light exercise exposure
protocol, Kleinman et al (1985) examined the effects of similar NO2-SO2 levels in
combination with sodium chloride (NaCl) aerosol (330 jug/m ) and zinc ammonium sulfate
(20 jwg/m ) They found a slight increase in symptoms in the aerosol plus gas exposure
compared to the aerosol alone, suggesting that the mixture was slightly more irritating
There were no pulmonary function effects (spirometry, closing volume, RT) of this exposure
regimen Kagawa (1983a,b) reported results of SO2-NO2 (0 15 ppm each) exposures in
normal subjects for 2 h with light, intermittent exercise Unfortunately, the analysis of
differences (using repeated t-tests) is confusing If a reasonable alpha level (<0 01) is used
to determine significance (based on the large number of comparisons), then there were no
statistically significant changes in Gaw in response to the NO2-SO2 mixture
Islam and Ulmer (1979a) examined the effects of a mixture of 5 ppm NO2, 5 ppm SO2,
and 0 1 ppm O3 on a group of 24 healthy subjects divided into three groups according to
age There were two series of 2-h exposures one at rest, the other including exercise
In subjects <30 years, R^ increased 48%, FVC decreased 5%, and FEVj decreased
11 7%, but PaO2 (determined from ear lobe blood samples) was unchanged Similar effects
occurred in the older subjects, but the changes were of A smaller magnitude Arterial oxygen
partial pressure fell (6 8 and 8 3 torr) during the exposures in the older subjects, but it also
decreased (4 5 and 4 3 torr) during the control exposures The methods of data analysis
were not presented in the paper so that the statistical significance of the observed changes
cannot be evaluated Furthermore, the additivity of effects due to the different pollutants
cannot be determined
15-33
-------
Islam and Ulmer (1979b) also studied 15 healthy subjects exposed to 0 34 ppm SO2,
0 16 ppm NO2, and 0 08 ppm O3 for 8 h at rest on four successive days This mixture did
not cause any changes in lung function, blood gases, or blood chemistry
Studies using several gas and/or aerosol mixtures were conducted by Stacy et al (1983)
and Kagawa (1986) Stacy et al (1983) exposed healthy, young males to mixtures of NO2
(0 5 ppm) and aerosols of sulfunc acid (H2SO4, 100 ^g/m3), ammonium sulfate
33 3
(133 /tg/m ), ammonium bisulfate (116 /*g/m ), or ammonium nitrate (NH4NO3, 80 jug/m )
There were no effects of any of the pollutant mixtures on spirometry, plethysmography, or
symptoms. Kagawa (1986) studied the effects of several NO2 mixtures (A) NO2
(0 30 ppm), O3 (0 30 ppm), and H2SO4 (200 /xg/m3), (B) NO2 (0 15 ppm), O3 (0 15 ppm),
and H2SO4 (200 /tg/m3), or (C) NO2 (0 15 ppm), O3 (0 15 ppm), SO2 (0 15 ppm), and
*3
H2SO4 (200 jug/m ) Exposure A included 20 mm of exercise (total) and exposures B and C
included 60 min of exercise over 2 h Symptoms were attributed to O3 exposure Small,
possibly significant decreases (<10%) in Gaw were observed after exposure to mixtures A
and B. A possible decrease in FEV^ (unknown magnitude) was observed after exposure C
The differences observed with these mixtures were no different than responses observed with
O3 alone, indicating no enhanced response due to the presence of NO2 in the mixture
Several reports of exposure to NO2-contaming ambient air mixtures have been
published by the Rancho Los Amigos group (Linn et al, 1980b, Linn and Hackney, 1983,
Avol et al , 1983, Avol et al, 1985a, Avol et al, 1987) The mean NO2 level in the
ambient air (from the Los Angeles Air Basin) ranged from 0 04 to 0 07 ppm during the
approximately 2-h exposure periods These studies were conducted during the summer smog
seasons of 1978-84, but were not specifically designed to test for the effects of NO2 on lung
function In the Linn et al (1980b) study, there was no association between NO2 levels and
symptom or lung function effects either in normal or asthmatic subjects In the Linn et al
(1982) study, the O3 levels were only 0 03 ppm, and there were no significant effects
associated with ambient exposure in normal or asthmatic subjects There was a relationship
between O3 concentration and change in FEVj in the Avol et al (1983) report There were
few differences between the responses of asthmatics and normal subjects in these studies and
no apparent influence of NO2 level Ambient air exposure of adolescents (Avol et al,
1985a) was associated with decreased FEVj in male and female adolescents, with a
15-34
-------
somewhat larger response in female subjects (—7 5%) than for males (—3 4%), but the
responses tended to be associated with the O3 levels A similar study (Avol et al, 1987) was
conducted with exercising children, who showed a trend to larger pulmonary function
decrements with increasing O3 levels Although these ambient air exposure studies were not
designed to test for the interaction of NO2 with other pollutants, even though Los Angeles
has the highest NO2 levels in the United States, they do illustrate that the lung function
effects of ambient air exposure (in Los Angeles) appear to be primarily accounted for by the
presence of O3
There has been one study of pulmonary effects of mtnc acid (HNO3) vapor followed by
exposure to O3 (Aris et al, 1991b) Ten healthy men, previously determined to be sensitive
to O3 (> 10% AFEVj after exposure to 0 20 ppm O3 for 3 h), were exposed on different
days for 2 h to either air, distilled water fog, or fog containing 500 jwg/m3 (»200 ppb) of
HNO3 One hour after each of these three randomly ordered exposures, the subjects were
exposed to 0 20 ppm O3 for 3 h During exposure, subjects exercised for 50 min/h at a
ventilation of »40 L/min The decrease in FEV^ after the O3 exposure tended to be slightly
larger after the air preexposure (—26%) than with either of the fog exposures (—17% and
— 18%, respectively) Neither SR^, methacholine responsiveness, nor symptoms were
different among exposure conditions Nitac acid vapoi or water fog alone did not induce
any symptoms or changes in pulmonary function The results of this study do not support
the hypothesis that HNO3 exposure will increase responses to O3
There appears to be no obvious synergistic or moire than additive interactions between
NO2 and other pollutant gases or particles that have been evaluated to date Pulmonary
function responses to O3, for example, do not appear to be increased by the addition of low
levels (< 0 6 ppm) of NO2 The variety of physiologic end points used to evaluate
combination exposures have, however, been limited primarily to spirometry and
plethysmography
15.2.4 Summary
In the studies summarized in this section, several observations indicated that, at
concentrations in excess of 2 0 ppm, there were functional changes in the lungs of healthy
normal volunteers that could be attributed to NO2 exposure Increases in Raw were reported
15-35
-------
at concentrations of 5 to 8 ppm from both short (5-min) and longer (1- to 2-h) exposures
(Von Nieding et al, 1979, Von Nieding and Wagner, 1977, Von Nieding et al, 1980, Islam
and Ulmer, 1979a,b) At slightly lower concentrations (2 to 4 ppm), no changes were seen
in resistance or spirometry (Linn et al, 1985b, Mohsenin, 1987b, 1988) However,
Mohsemn reported an increase in airway responsiveness to methachohne (bromide) after
exposure to 2 ppm, Frampton et al (1991) reported increased carbachol response after
exposure to 1.5 ppm, and Hazucha et al (1992) reported augmentation of ozone-induced
airways hyperresponsiveness by prior NO2 (0 6 ppm) exposure None of the seven studies of
exposure to less than 1 0 ppm in normal subjects demonstrated clear responses to NO2
There were several instances of isolated observations indicating statistical significance, but
there was no consistent pattern of response For example, Kagawa and Tsuru (1979)
reported small changes in VC at 0 15 ppm, but did not verify this observation in two
subsequent studies (Kagawa, 1983a,b, 1986), even at 0 30 ppm Furthermore, other studies
(Fohnsbee et al., 1978, Toyama et al, 1981, Stacy et al, 1983, Adams et al, 1987, and
Drechsler-Parks et al , 1987), conducted at higher concentrations (0 5 to 0 7 ppm), found no
evidence of lung function effects
15.3 THE EFFECTS OF NITROGEN OXIDE EXPOSURE IN
SENSITIVE SUBJECTS
Certain groups within the population may be more responsive to the effects of NO2
exposure, including persons with respiratory disease, children, the elderly, and other
individuals not readily identified as members of a specific group The reasons for paying
special attention to these groups is that potential for NO2-induced responses or exacerbation
of disease may be much higher than in healthy young adults Studies on other NOX gases
and NOX mixtures are also discussed
The airways of asthmatic subjects may be hyperresponsive to a variety of inhaled
materials, including pollens, cold-dry air, allergens, and air pollutants Asthmatics have the
potential to be among the most susceptible members of the population with regard to
respiratory responses to NO2 (Section 15 3 1) On the average, asthmatics are much more
sensitive to inhaled bronchoconstnctors such as histamine, methachohne, or carbachol The
15-36
-------
potential addition of an NO2-induced increase in airway response to the already heightened
responsiveness to other substances raises the possibility of exacerbation of this pulmonary
disease by NO2 One of the potential mechanisms by which NO2 could affect asthmatics is
via a change in airways responsiveness This is discussed in Section 15 4
Other potentially susceptible groups include patients with COPD, these subjects are
discussed in Section 15 3 2 A major concern with COPD patients is the absence of an
adequate pulmonary reserve Any alteration in lung function in these patients can potentially
cause serious problems
15.3.1 The Effects of Nitrogen Dioxide on Asthmatics
An important issue in the evaluation of human clinical exposure studies involving
asthmatics is the variability in response between, and even within, laboratories In the
absence of significant differences in the exposure protocol or exposure dose, an explanation
frequently invoked to explain the differences in response is that the characteristics or seventy
of the disease may differ from one subject group to another
The Expert Panel Report from the National Asthma Education Program (National Heart
Lung and Blood Institute, National Institutes of Health, 1991) has recently defined asthma in
the following manner
Asthma is a lung disease with the following characteristics (1) airway
obstruction that is reversible (but not completely so in some patients) either
spontaneously or with treatment, (2) airway inflammation, and (3) increased
airway responsiveness to a variety of stimuli
According to the National Institutes of Health (1991), about 10 million people, or 4% of the
population of the United States, have asthma The prevalence is higher among African
Americans, older (8 to 11 years) children, and urban residents (Schwartz et al, 1990)
There is a broad range of seventy of asthma ranging from mild to severe (see Table 15-3)
Common symptoms include cough, wheezing, shortness of breath, chest tightness, and
sputum production A positive response (skin test) to common inhalant allergens is a typical
feature of asthma Asthma is characterized by an exaggerated bronchoconstnctor response to
15-37
-------
TABLE 15-3. CLASSIFICATION OF ASTHMA BY SEVERITY OF DISEASE3
Characteristics
Mild
Moderate
Severe
A Pretreatment
Frequency of
exacerbations
Frequency of
symptoms
Degree of exercise
tolerance
Frequency of
nocturnal asthma
School or work
attendance
Pulmonary function
• Peak Expiratory
Flow Rate (PEFR)
• Spirometry
Exacerbations of cough and
wheezing no more often than
1-2 times/week
Exacerbation of cough and
wheezing on a more frequent basis
than 1-2 times/week Could have
history of severe exacerbations, but
infrequent Urgent care treatment
in hospital emergency department
or doctor's office <3 times/year
Few clinical signs or
symptoms of asthma between
exacerbations
Good exercise tolerance but
may not tolerate vigorous
exercise, especially prolonged
running
Symptoms of nocturnal
asthma occur no more often
than 1-2 times/month
Good school or work
attendance
' Methachohne
sensitivity
PEFR > 80% predicted
Variability <20%
Minimal or no evidence of
airway obstruction on
spirometry Normal
expiratory flow volume
curve, lung volumes not
increased Usually a > 15%
response to acute aerosol
bronchodilator administration,
even though baseline near
normal
Methachohne PC^g
> 20 mg/mL °
Cough and low grade wheezing
between acute exacerbations often
present
Exercise tolerance diminished
Symptoms of nocturnal asthma
present 2-3 times/week
Virtually daily wheezing Exacerbations
frequent, often severe Tendency to have
sudden severe exacerbations Urgent visits to
hospital emergency departments or doctor's
office >3 times/year Hospitahzation
>2 times/year, perhaps with respiratory
insufficiency or, rarely, respiratory failure and
history of intubation May have had cough
syncope or hypoxic seizures
Continuous albeit low-grade cough and
wheezing almost always present
Very poor exercise tolerance with marked
limitation of activity
Considerable, almost nightly sleep interruption
due to asthma Chest tight in early morning
School or work attendance may be Poor school or work attendance
affected
PEFR 60-80% predicted
Variability 20-30%
Signs of airway obstruction on
spirometry are evident Flow
volume curve shows reduced
expiratory flow at low lung
volumes Lung volumes often
increased Usually a >15%
response to acute aerosol
bronchodilator administration
Methachohne PC2Q between 2 and
20 mg/mL
PEFR < 60% predicted
Variability > 30%
Substantial degree of airway obstruction on
spirometry Flow volume curve shows marked
concavity Spirometry may not be normalized
even with high dose steroids May have
substantial increase in lung volumes and marked
unevenness of ventilation Incomplete
reversibility to acute aerosol bronchodilator
administration
Methachohne PC2Q < 2 mg/mL
12-24 h Regular drug
therapy not usually required
except for short periods of
time
B After optimal treatment is established
Response to and Exacerbations respond to Periodic use of bronchodilators Requires continuous, multiple around-the-clock
duration of therapy broncodilators without the use required during exacerbations for drug therapy including daily corticosteroids,
of systemic corticosteroids in a week or more Systemic steroids either aerosol or systemic, often in high doses
usually required for exacerbations
as well Continuous around-the-
clock drug therapy required
Regular use of anti-inflammatory
agents may be required for
prolonged periods of time
"Characteristics are general, because asthma is highly variable, these characteristics may overlap Furthermore, an individual may switch
into different categories over time
Variability means the difference either between a morning and evening measure or among morning peak flow measurements each day for a
week
Although the degree of methacholine/histamme sensitivity generally correlates with seventy of symptoms and medication requirements,
there are exceptions
Source National Institutes of Health (1991)
15-38
-------
many physical changes (e g , cold or dry air, exercise) and chemical and pharmacologic
agents (e g , histamine or methacholine) Asthma is typically associated with airway
inflammation and epithelial injury (National Institutes of Health, 1991, Beasley et al, 1989,
Laitinen et al , 1985, Wardlaw et al, 1988)
In addition to basic anthropometnc information such as age, height, weight, gender,
and race, other information may be useful in characterizing asthmatics In order to evaluate
differences between subject populations from one studj to another, useful information might
include baseline lung function, frequency of asthma episodes, nonspecific bronchial
responsiveness, reversibility of bronchoconstnction, types of medication and frequency of
use, specific serum immunoglobulin E (IgE) levels, skin test responses, response to exercise
challenge, duration of disease, and factors that precipitate or aggravate the disease
In many cases of chamber exposures of asthmatics, the exposures are accompanied by
moderate exercise The potential for an increase in airway resistance or decline in lung
volumes or forced expiratory flow caused by exercise alone is an important covanate in these
studies Exercise, even of moderate intensity, can induce some increase in airway resistance,
even in clean air at normal room temperature and relative humidity (RH) (i e , at 20 °C,
50% RH) In order to determine the true effect of an air pollutant in exercising asthmatics,
the response to exercise must be considered Accordingly, in all studies summarized in this
section, a control exposure to clean air was performed, including exercise when appropriate
Asthmatics who participate in controlled human exposure studies typically have mild
allergic asthma In many cases, these individuals can go without medication altogether or
can discontinue medication for brief periods of time if exposures are conducted outside their
normal allergy season
Controlled human exposure studies that evaluated respiratory effects of NO2 exposure
of asthmatics are summarized below in two tables one that describes characteristics of the
asthmatic subjects tested (Table 15-4), and another one that presents the exposure conditions
used and observed responses to NO2 (Table 15-5)
Symptomatic effects were observed in asthmatics exposed to 0 5 ppm for 2 h in a study
reported by Kerr et al (1979) However, only four of the subjects reported symptoms of
respiratory discomfort, and the authors concluded that "The symptoms reported were
minimal, did not correlate with functional changes, and are of doubtful significance "
15-39
-------
TABLE 15-4. CHARACTERISTICS OF ASTHMATIC SUBJECTS EXPOSED TO NITROGEN DIOXIDE3
Age
Reference Number (years)
Ahmed et al 20 M/34 F 18-39
(1983b)
Ahmed et al 9 20-51
(1983a)
Avoletal (1988) 27M/32F 18-50
Avoletal (1989) 24M/10F 10-16
5| Bauer etal (1986a) 15 20-45
Bylmetal (1985) 6M/2F 17-45
Bylmetal (1988) 8M/12F 17-56
Hazucha et al 15 M 21-46
(1983, 1982)
Joerres and 10 M/4 F 20-55
Magnussen (1990)
Kerretal (1979) 9M/4F 19-50
also Kulle (1982)
FEV1/FVCb SR^ IgEc
% (L»cmH2O/L/s) Elevated Reversibility Allergy0 Medications
58-97 34-201 - - 10/20
53-96 43-149 - - Ragweed -
43-98 3 8-21 2 Y Y Y 14 OB
30 IB/OB
15 IB
61-98 3-15 History 33/34 10 IB/OB
6 OB
10 None
49-83 439 - - 13 IB
7 OB
699 Y Y 4/8 5 Occasional
IB/OB
3 OB-daily
3 76 ± 0 33 Some Y 12/20 2 IB, OB
(occasional)
— 6 73 — History — None daily
38-83 28-182 - - 12/14 11/14 IB
SOB
1ST
6ICS
2CROM
4IPB
«71 =74 - -
Medication
Witholding5
—
24-48 h
48hAH
8 h IB, OB
(Some
noncomphance)
48hAH
24hOB
12hffi
12hIB
24hOB
6h
12hOB
8hIB
48 h
lOhffi
lOhlPB
10 h CROM
16hOB
10 h ICS
-
Airway
Reactivity
MET
HIST
Effi
COLD
Ragweed
antigen
MET
COLD
MET
Effi
COLD
HIST
HIST
MET
Histamme
S02
-
Notes
_
"History of bronchial
asthma"
Some moderate
asthmatics
No cromolyn,
No steroid
Mild asymptomatic
Very mild asymptomatic
Very mild asymptomatic
Mild or inactive disease
"Mild asthma", all
subjects asymptomatic
except one (FEVj/FVC
ratio 38%)
3 Smokers
-------
TABLE 15-4 (cont'd). CHARACTERISTICS OF ASTHMATIC SUBJECTS EXPOSED TO NITROGEN DIOXIDE3
Reference
Klemman et al
(1983)
Joerres and
Magnussen (1991)
Koemg etal (1985)
Koemg et al
(1987a,b)
Koemg et al
(1989a)
Koemg et al
(1989b)
Linnetal (1980a)
(mixed NO2 +
SOj)
Linn and Hackney
(1984)
Linnetal (1985b)
Linnetal (1986)
Number
12 M/19 F
9M/2F
1 4 M/6 F
H 4 M/6 F
m 7 M/3 F
6M/3 F
5M/4F
7 M/12 F
6 Ex-
smokers
12M/11 F
12M/11F
15 M/6 F
Age
(years)
18-55
18-55
12-18
11-19
12-18
12-18
12-17
33
18-34
18-34
20-34
FEVj/FVC15 SRaw IgE°
% (L»cmH2O/L/s) Elevated Reversibility Allergy*
»74 Rt = 262 - - 16/31
67-96 39-107 7/11
FEVj Rt = 4 57 Y Y Y
1 95-4 10
23-42 Rt = 507 Y Y Y
17-46 Rt = 470 Y Y Y
24-54 - - - 5/9
(FEVj)
224-40 - - - 9/9
2 32 3 45 - - Y
(FEVj) Rt = 3 45
67-100 5 48 + 2 33
67-100 5 48 ± 2 33
3 16 4 21 13/21
(FEVj)
>180
Medication
Medications Witholding^
8 None 4 h
21 IB, OB
8 ST, 1 CROM
6 None 8 h
SOB
8ffi 4h
6 OB
7 IB «4h
SOB
6 IB «4h
7 OB
-
5ffi
1 None
3 CROM
3 OB
3 AH
«4h
—
~4h
10 IB »4h
5 None
SOB
1ST
Airway
Reactivity
MET
MET
EIB
MET
EIB
MET
EIB
MET
MET
EIB
MET
Effi
—
SO2
SO2
COLD
19/21
EIB 10/21
S02 17/21
Notes
Wide range of clinical
seventy
Mild asymptomatic
asthmatics
Asymptomatic extrinsic
allergic asthmatics
Physician diagnosed
asthma
Physician diagnosed
asthma
Mild asthmatics
-------
TABLE 15-4 (cont'd). CHARACTERISTICS OF ASTHMATIC SUBJECTS EXPOSED TO NITROGEN DIOXIDE3
N>
Age
Reference Number (years)
Mohsenm (1987b) 10 22-44
Morrow and Utell 10 M/10 F 19-54
(1989)
Oreheketal (1976) 13 M/7 F 15-44
Oreheketal (1981) 6M/1F 31 1
Roger etal (1990) A 13 M 19-35
B 21 19-30
Rubinstein et al 5 M/4 F 23-34
(1990)
Abbreviations
FEVj = Forced expiratory volume in 1 s
FVC = Forced vital capacity
SRaw = Specific airway resistance
IgE = ImmunoglobulinE
M = Male
F = Female
MET = Methacholme
HIST = Histamme
EIB = Exercise-induced bronchospasm
COLD = Cold air
F^/FVC0 SRaw IgE6
% (L»cmH2O/L/s) Elevated Reversibility Allergy6
60-93
42-86
55-81
59-85
51-85
IB
AH
ST
ICS
CROM
IPB
Rt
N02
CARB
38-132 - Y -
(50) - Y 14/20
3 95-14 5
x = (11 3)
929
68 + 06 - - 16/20
54+11
2 6-13 5 80-2,040 Y 12/13
3 2-10 6 38-2,040 Y 18/21
48-120 - -
= Inhaled /3 agonist
= Antihistamme
= Oral corticosteroid
= Inhaled corticosteroid
= Cromolyn sodium
= Ipratropnum bromide
= Sulfur dioxide
= Forced oscillation
= Nitrogen dioxide
= Carbachol
Medications
No steroids
No caffeine
No vitamin C
4IB
SOB
10 IB, OB
No steroids
-
IB, OB
IB, OB
9IB
4 OB
4AH
Medication Airway
Witholding6 Reactivity Notes
24 h MET Mild asthmatics
CARB
24 h CARB Duration asthma
(1-24 years), 6 smokers
13 mild/7 moderate
- Grass 1 smoker,
Pollen 3 asthmatics,
4 allergic
48 h, OB MET Mild asthmatics,
12 h, IB no cromolyn,
no steroids
8 h, IB MET No respiratory illness
12 h, OB, AH within 4 weeks
4h, CAP
OB = Oral theophylme
Ratio of FEVypVC m percent
CElevation of IgE levels above those reported for the normal population
TJeversibility of bronchoconstnction tested with bronchodilator
eAllergy—number of subjects with one or more known allergies—by history or skin testing—Y indicates that subjects were classified as "allergic asthmatics"
Standard medications and number of subjects using them (see list), none indicates no regular use of medication during the study period
Period for which specific types of medication were withheld prior to the study (in hours)
Airway reactivity was tested using one or more standard methods (see list)
-------
TABLE 15-5. EXPOSURE CONDITIONS AND RESPONSES IN ASTHMATICS EXPOSED
TO NITROGEN DIOXIDE3
Reference
Exposure Exercise Exercise Relative
NO2 Duration Duration Ventilation Temp Humidity
(ppm) (mm) (nun) (L/min) (°C) (Percent)
Effects
Ahmed et al (1983b) 0 1 60
Ahmed et al (1983a) 0 1 60
Avoletal (1988)
03 120
ut Avoletal (1989)
06
03
Bauer et al (1986a)
03
Bylinetal (1985)
0 12
025
120
180
30
20
20
60
60
90
40
41
30
10
30
No significant effect on SGaw and FEVj,
variable effect on carbachol reactivity No
information on controlled exposure
No effect of NO2 on FEV1; SGaw, or bronchial
reactivity to ragweed antigen, either immediately or
24-h postexposure
22 46 Exercise-related increases in symptoms Possible
NO2 related decrease in FEVj and PEFR Increased
cold air response after 0 3 ppm
More consistent increases in SRaw at 0 6 ppm, but
not significantly different from air and 0 3 ppm
24 45 After 60 mm of exposure, FEV, FVC, and PEFR
were significantly reduced (-3 4, -4 0, and -56%,
respectively) No change in airways responsiveness
to cold air challenge SRaw increased 17 % after
NO2 exposure After 180 mm of exposure, the
responses had returned to baselme levels
20 9-14 Resting 20-min exposures produced no effects
Slight excess decrease in FEVj and PEFR in NO2
plus exercise above that caused by exercise alone
PEFR -16% (air), -28% (NO2), FEV{ -55%
(air), -9 3 % (NO2) Significantly increased response
to cold air after NO2 exposure
22 35 No significant change in SRaw at any NO2 levels
Histamine reactivity tended to increase
05
20
-------
TABLE 15-5 (cont'd). EXPOSURE CONDITIONS AND RESPONSES IN ASTHMATICS EXPOSED
TO NITROGEN DIOXIDE3
Reference
Byhnetal (1988)
Hazuchaetal (1982)
Hazuchaetal (1983)
Joerres and Magnussen
(1990)
Klemmanetal (1983)
Koemg etal (1985)
Koemg et al
(1987a,b)
Koemg etal (1989b)
Koemg etal (1989a)
Kulle (1982)
(same as Kerr et al ,
1979)
Joerres and Magnussen
(1991)
Exposure
NO2 Duration
(ppm) (mm)
014
027
054
0 10
025
020
0 12
I 012
n o 12
m o is
50 ppb
HN03
57 ppb
HN03
050
025
30
60
30
120
60
60
40
40
40
45
120
30
Exercise Exercise
Duration Ventilation Temp
(nun) (L/min) (°C)
25 9
21
94
•™ ^*T
60 «20 22
22
22
10 33 22
10 39 22
10 « 25-30 25
30 25-30 22
15 -- 24
10 30 26
Relative
Humidity
(Percent) Effects
43
40
50
50
75
75
75
75
65
65
45
20
Overall trend for SR^ to decline during exposure
period, not related to NO2 concentration Histamine
bronchial reactivity tended to mcrease after 0 14 and
0 27 ppm NO2 exposure
No significant changes associated with NO2
exposure
After resting breathing of 0 25 ppm NO2,
responsiveness to inhaled SO2 was increased No
effect of NO2 alone on SR^
No effects on spirometry or airway resistance
Airway reactivity to methacholine results variable—
tended to mcrease with exposure
No significant responses in pulmonary function due
to NO2 Increased symptoms after NO2 exposures
(Same as Koemg et al , 1985 ) No change in FEVj,
RT increased 10 4% (NS), 3% decrease in FEVt
(p < 0 06)
FEVj decreased 44% after HNO3 and 1 7% after
HNO3 air exposure RT increased 22 5% after
HNO3 and 74% after air exposure
FEVj decreased 33% after NO2 exposure and 17%
after air exposure (difference NS) Reduction of oral
ammonia did not mcrease response (-1 7%)
Increased respiratory symptoms in 4/13 subjects
Increased static lung compliance Impossible to
determine amount of effect due to NO2
Mouthpiece exposure system No changes in
methachohne responsiveness were observed after
NO2 exposure in these mild asthmatics
-------
TABLE 15-5 (cont'd). EXPOSURE CONDITIONS AND RESPONSES IN ASTHMATICS EXPOSED
TO NITROGEN DIOXIDE3
Reference
Lmnetal (1980a)
Lmn and Hackney
(1984)
Linnetal (1985b)
Lmnetal (1986)
N02
(ppm)
05 +
03 ppm
SO2
40
03
Exposure
Duration
(mm)
120
75
60
Exercise
Duration
(mm)
60
a 15
b 15
30
Exercise
Ventilation
(L/min)
«20
a 25
b 49
41
Temp
31
21
22
Relative
Humidity
(Percent)
40
50
50
Effects
No significant effect on spirometry or RT
No NO2 effects on
skin conductance
pressure
No effect of NO2
SRaw, symptoms,
Small decrease in
heart rate, or
systolic blood
Exercise-related increase in SRaw
under all conditions
Mohsenin (1987b)
1 0
30
05
60
60
60
30
30
—
41
41
—
22
22
21
50
50
50
No change in symp
toms Significant
group mean
;
Morrow and Utell
(1989)
Oreheketal (1976)
030
0 11
(n = 20)
(0 09-0 13)
026
(n = 4)
increase in responsiveness to methacholrne after N02
exposure No other function changes
225 30 30-40 21 0 40 Group findings indicated no significant responses
(3 X 10) No change in lung function, symptoms, or carbachol
reactivity Subjects previously studied (Bauer et al ,
1986a) showed possible responses to NO2 New
subject subgroup showed significantly greater
response in air exposures
60 — — — — 13/20 subjects had enhanced responses to carbachol
after 0 llppmNO2
1/4 subjects had enhanced responses to carbachol
after 0 26 ppm NO2
-------
ON
TABLE 15-5 (cont'd). EXPOSURE CONDITIONS AND RESPONSES IN ASTHMATICS EXPOSED
TO NITROGEN DIOXIDE8
Reference
Oreheketal (1981)
Roger etal (1990)
Rubinstein et al
(1990)
N02
(ppm)
Oil
(007-
0 16)
A 03
B 0 15
030
060
030
Exposure
Duration
(nun)
60
110
75
30
Exercise
Duration
(mm)
~
60
30
20
Exercise
Ventilation Temp
(L/min) (°C)
_ „
42 20
42 20
3X 22
(-30)
Relative
Humidify
(Percent)
-
40
40
55
Effects
No change in SRaw or in responsiveness to grass
pollen in 3 allergic asthmatics and 4 allergic subjects
FEVj decreased 11% in NO2 but only 7% in air,
after first 10 min of exercise Smaller changes later
in exposure
No increase in airway reactivity to methacholine
2 h after exposure No change in FEVj or SRaw as
a result at NO2 exposure
No changes in SRaw, FVC, FEVj, SBN2 or
symptoms after NO2 exposure NO2 exposure did
not increase airways responsiveness to SO2
Abbreviations
NC>2 = Nitrogen dioxide
SGaw = Specific airway conductance
FEVj = Forced expiiatory volume mis
= Volume of isoflow
PEFR = Peak expiratory flow
SRaw = Specific airway resistance
FVC = Forced vital capacity
RT = Total respiratory resistance
NS = Not significant
HNO3 = Nitnc acid
SO2 = Sulfur dioxide
SBN2 = Single breath nitrogen washout
-------
Avol et al (1988) studied a group of 59 moderate-to-severe asthmatics exposed to clean
air, 0 3 ppm, and 0 6 ppm NO2 for 2 h while performing moderate (minute ventilation
[VE] = 41 L/min), intermittent (6 X 10 mm) exercise Each subject was exposed once each
to clean air, 0 30 ppm, and 0 6 ppm There were significant changes in SRaw and FEV1 as
a function of exposure duration for all exposure conditions, but there was no significant
effect of NO2 exposure on these measures of pulmonary function Cold air bronchial
reactivity (assessed by the decrease in FEVl after breathing cold-dry air) was measured
1 h postexposure and then again the following day There was a significant interaction
between response and tune of testing (i e , 1 h postexposure and 24 h postexposure),
suggesting a slightly increased response after exposure to 0 30 ppm, but not after 0 6 ppm
There were no respiratory symptom responses attributable to NO2 exposure A post hoc
analysis of a subgroup of subjects with the most abnormal lung function (i e , FEVyFVC
ratios < 0 65) revealed no statistically significant effects of NO2 In addition to the
controlled exposures, 36 subjects also were exposed to ambient air containing 0 09 ppm NO2
and low levels of other pollutants Neither lung function, cold-air reactivity, nor symptom
responses were significantly different in ambient air than in clean air
Bauer et al (1986a) reported a statistically significant spirometnc response to NO2 in a
group of 15 asthmatics exposed to 0 3 ppm NO2 by mouthpiece for 20 min at rest followed
by 10 mm of exercise (30 L/min) These subjects were characterized as having "mild
obstructive lung disease (asthma) " All subjects had elevated response to cold air
bronchoprovocation Nitrogen dioxide deposition studies indicated that 72 % (at rest) and
87 % (during exercise) of the inhaled NO2 was deposited within the respiratory tract
According to the authors, the measurements of NO2 deposition were in general agreement
with the model predictions of Miller et al (1982) (see also Section 13 2 1) After NO2
exposure, 9 of 15 asthmatics had a decrease in FEVj relative to their postexercise FEVj in
clean air The postexercise 1PEV1 was 4 1 % lower aftei NO2 (mean = 2,788 mL) than after
air (mean = 2,906 mL) exposure, the pre- to postexposure difference on the NO2 day
(10 1 %) and the pre- to post-NO2 minus the pre- to posl-air (i e , delta-delta) differences
(6%) were significant using a paired t-test These differences were no longer present by
60 min after the exposure Maximum expiratory flow at 60 % total lung capacity (TLC)
(PEFV curve) was also decreased more after NO2 exposure than after air exposure Changes
15-47
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in FVC and SGaw were not different between air and NO2 exposures Airway
responsiveness to cold air in this study was determined as follows At each ventilation rate
of cold air breathing, the respiratory heat exchange (RHE) was calculated From the
relationship of the log RHE versus the percentage decrease in FEVj, the RHE, which caused
a 10% decrease in FEVls was linearly interpolated and is referred to as PD10RHE
(provocative dose in RHE units needed to decrease FEVj by 10%) Of the 12 subjects for
whom the PDioRHE could be determined, 9 showed an increased response to cold air after
the NO2 exposure The average PD10RHE decreased from 0 83 kcal/min after air exposure
to 0.54 kcal/min after NO2 exposure
One of the factors that may have led to the demonstration of increased response after
exposure to a low concentration of NO2 in this group of asthmatics could be the fact that a
mouthpiece exposure system containing relatively dry air (RH of 9 to 14% at 20 °C) was
used, and that there was possibly some interaction between the NO2 effect and airway
drying It is well known that breathing dry (cold) air will induce bronchoconstnction in
asthmatics, and that the effect of SO2 on asthmatics is exacerbated by cold-dry air breathed
via the mouth (U S Environmental Protection Agency, 1986, SO2 document addendum)
Concern over this possible confounding effect is tempered by the fact that Bauer et al
(1986a) controlled for the airway drying effect by exposmg subjects to clean air at the same
temperature and RH However, if the formation of HNO3 or nitrous acid is potentially
involved in the observed responses, the air chemistry could be strongly influenced by RH
(Sequestration of HNO3 on surfaces is increased with increased ambient water vapor
content)
Eight asthmatics exposed to 0 0, 0 1, 0 25, and 0 5 ppm NO2 for 20 mm were studied
by Bylin et al (1985) Exposures were conducted in a body plethysmograph and the range
of concentrations was +18% to -26% of the target concentration Changes in SRaw during
the four exposures averaged +3%, +9%, -2%, and -14%, respectively, the CV for the
SRaW measurements was 19 % for these subjects A three-way analysis of variance revealed
no significant differences in SR^ due to NO2 exposure There was a tendency for the pre-
to postexposure difference for thoracic gas volume (TGV) to be larger for the NO2 exposures
(9 to 10%) However, the absolute volume of TGV was at most 3 to 4% lower than at
comparable tunes in other NO2 exposures and only 2% less than the air exposure The
15-48
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significance of this difference was in the higher preexposure values for the 0 1- and 0 5-ppm
NO2 exposures, such an effect, if real, should not be attributed to NO2 There were no
significant changes in tidal volume or respiratory rate, which would have been suggestive of
an irritant response At the highest concentration tested (0 5 ppm), histamine bronchial
responsiveness was also evaluated after exposure The authors reported a significant increase
in histamine responsiveness due to NO2 exposure Significance was evaluated by a sign test
(p < 0 04, responsiveness increased in five subjects and was unchanged in three)
However, this finding should be interpreted cautiously because the sham (air) exposure
histamine challenge had to be discontinued in two subjects, one of whom was later classified
as having increased responsiveness Five of the eight asthmatics had several months
previously been hyperreactive to histamine but were not at the tune of the NO2 exposures
This paper suggested possible increased histamine reactivity after 0 50 ppm NO2 exposure of
asthmatics but no direct effect of NO2 on Raw at concenl rations up to 0 5 ppm for 20 mm
Bylin et al (1988) also reported the effects of 260, 510, and 1,000 /*g/m3 (0 14, 0 27,
0 53 ppm, respectively) on a group of 20 mild asthmatics There were no significant
changes in SR^, although there was a general trend for SR^ to fall throughout the period
of exposure regardless of the pollutant level There was, however, a significant increase
(p = 0 03) in airway responsiveness to histamine after 30-min exposure to the middle
o
concentration (i e., 510 jug/m ), but not at the lowest and highest concentration (see note
below) The absence of a concentration-related increase in responsiveness, and the fact that
the significance of these findings is based on repeated application of a nonparametnc pair
comparison test using an alpha level (p value) not adjusted for multiple comparisons,
suggests that these results should be interpreted with caution This observation contrasts with
the earlier observation (Bylin et al , 1985) that suggested a possible increased responsiveness
after exposure to 910 /*g/m (Note, however, the discussion above regarding the statistical
approach used in the 1985 study) The raw data presented in the paper were subjected to
reanalysis (data available on request to EPA) using a Friedman nonparametnc analogue of an
F test, which is probably more appropriate for these data than a senes of Wilcoxon matched
pairs signed rank tests The Friedman test showed no difference across treatment groups
(i e , there was no statistically significant increase in histamine responsiveness as a result of
NO2 exposure)
15-49
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In a study that was an important precedent to a number of studies, Orehek et al (1976)
studied the effects of low levels of NO2 exposure on the bronchial sensitivity of mild
asthmatic patients to carbachol, a bronchoconstricting agent Exposures took place in an
airtight room Nitrogen dioxide concentration started at 246 ^g/m (0 13 ppm) and declined
-------
in that NO2 was added to an exposure room with a stalling concentration of 0 16 ppm, which
was allowed to decay during the exposure to a concentration of 0 07 ppm There was no
change in R^ or symptoms as a result of the NO2 exposure There was also no difference
in the Raw response to allergen challenge in these subjects (i e , NO2 did not act
synergistically with allergen challenge) Furthermore, there was no difference between the
responses of the three asthmatics and the other four subjects
Hazucha et al (1982, 1983) published two reports that contain complementary data
from a study in which the Orehek protocol was repeated In contrast to the report of Orehek
et al (1976), Hazucha et al (1982, 1983) found no statistically significant change in airway
reactivity to methacholine (another bronchoconstncting agent) in a group of 20 methachokne-
reactive mild asthmatics following a 1-h resting exposure to 0 1 ppm NO2 A small (8%)
increase in SRaw (p = 0 23) was observed after NO2 exposure Three of the 15 subjects had
a greater than 20% decrease in the dose of methacholine required to double SRaw (PD100)
However, at least three of the subjects had a change of similar magnitude in the opposite
direction, judging from the graphical presentation of the methacholine dose-response curves
Respiratory system resistance measured by the forced oscillation method was not changed by
NO2 exposure Hazucha et al (1983) suggested that the difference in the conclusions
regarding statistical significance reached by Orehek et al (1976), despite similar findings,
was because "the statistical approach used by Orehek was not appropriate " Hazucha et al
(1983) discussed the factors that led to their conclusions that, had they analyzed their data in
a similar manner to Orehek et al (1976), the findings would have been comparable
The hypothesis that NO2 exposure may cause airway hyperresponsiveness was also
examined by Kleinman et al (1983), who employed a different experimental design than
Orehek et al (1976) and Hazucha et al (1983) They studied 31 mild-to-moderate
asthmatics who were exposed to 0 2 ppm NO2 for 2 h while performing light, intermittent
exercise There were no significant effects of NO2 exposure on forced expired spirometry
Total RT (forced oscillation) tended to increase (9%) alter NO2 exposure, but the difference
was not significant (p = 0 11) Symptom responses tended to be slightly higher after air
exposures A number of different methods were used to evaluate the methacholine challenge
data The general tendency was for greater responsiveness to methachohne after NO2
exposure The determination of the dose that would cause a 10% decrease in FEVl (D10)
15-51
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was the most "conventional" approach (see O'Connor et al, 1987, for a discussion of
techniques) to assessing methacholine responsiveness In the 21 subjects in which this dose
could be ascertained, D10 was 8 6 ± 16 2 /*g on the air day and 3 0 ± 6 2 /*g on the NO2
days (p < 0 05 by t-test and Wilcoxon test) Thus, it appears that the results of this study
suggest a possible increase in airway responsiveness after a 2-h exposure to 0 20 ppm NO2
Koenig et al (1985) have studied the effects of a 1-h resting exposure of asthmatic
adolescents to 0 12 ppm NO2 There were no "consistent significant changes in pulmonary
functional parameters" after NO2 exposure Although symptom data were not presented, the
authors indicated that subjects had more symptoms after NO2 exposure but that the trend was
not significant
Subsequent studies by Koenig et al (1987a,b) of mouthpiece exposures to 0 12 ppm
NO2, which incorporated exercise (30-min rest followed by 10-min exercise), indicated
increases in RT and decreases in FEVj after both air and NO2 exposure These changes
were apparently due to exercise alone (RT increased 8 1 % with air and 10 4% with NO2,
postexercise FEVi was decreased 74% with air and 4 1 % with NO2) In the final phase of
the study, subjects were exposed to 0 18 ppm NO2 using the same exercise protocol In this
case, no differences in RT were seen and FEVj decreases were 1 3 and 3 3 % for air and
NO2, respectively This difference (p = 0 06) may indicate a possible response trend
There were no differences in symptoms between exposure conditions in either the 0 12- or
0 18-ppm NO2 exercise exposure studies
Morrow and Utell (1989) studied a group of 20 asthmatics exposed to 0 30 ppm NO2
for 3.75 h The exposure included three 10-mm periods of moderate exercise There were
no statistically significant group changes in symptoms, spirometry, plethysmography, or
airway reactivity to carbachol as a result of the NO2 exposure Some of the subjects (n = 7)
had participated in the Bauer et al (1986a) study The 13 remaining (new) subjects were
judged to have more severe asthma than the "repeaters " Although the repeaters tended to
have responses that were similar to those m the previous study (larger FEVj decrements in
NO2 than in air), the new subjects had significantly greater FEVj decrements during the air
exposures.
Linn et al (1985b) and Linn and Hackney (1984) exposed a group of 23 mild
asthmatics to 4 ppm NO2 Subjects completed a total of four exposures (two each to NO2
15-52
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and clean air) separated by 1 week Exposures lasted for 75 min and included two 15-min
exercise periods separated by a 25-min rest period The first exercise was light (25 L/min)
and the second was heavy (49 L/min) All subjects were responsive to inhaling 0 75 ppm
SO2 during exercise Mean baseline preexposure SRaw measurements varied from 5 48 and
5 59 on the air exposure days to 6 14 and 6 44 on the NO2 exposure days, although it is
unlikely that the slightly higher baseline values on the NO2 exposure days affected the
subjects' responses Airway resistance increased after exercise and more so after the heavy
(57 2%) than after the light (17 6%) exercise (percentages represent mean values collapsed
across all exposure conditions) There was no significant difference in lung function that
could be attributed to NO2, if anything, SR^ tended to be slightly lower with the NO2
exposures Other physiological tests, such as skin conductance and heart rate, were not
different between exposure conditions As with the group of normal subjects studied under
similar conditions, these asthmatics had a slightly, but significantly, lower systolic blood
pressure towards the end of the NO2 exposure The authors suggested the possibility that
NO2 deposited in the respiratory tract may form a vasoactive substance such as an organic or
inorganic nitrate Nitrate formation after NO2 inhalation has been observed in animal studies
(Postlethwait and Mustafa, 1981) However, measurements of blood levels of nitrate were
not performed by Linn et al (1985b) Both symptoms and state-trait anxiety scores were
evaluated during and after exposure, there were no significant variations that could be
attributed to NO2 exposure
It is difficult to explain the differences between this group of asthmatics exposed to
4 ppm for 75 mm (with exercise) compared to the group exposed to 0 30 ppm for 30 min
with exercise studied by Bauer et al (1986a) The subjects of Bauer et al were exposed to
NO2 in dry air through a mouthpiece, which could have caused some "drying" of the upper
airways This would not be a factor in the Linn et al (1985b) study, where a chamber
exposure was used Another possible explanation is that the asthmatics studied by Linn et al
were accustomed to NO2 exposure because of their place of residence (although the ambient
levels in Los Angeles are, of course, much lower than 4 ppm) However, the indoor
environment can be an important avenue of NO2 exposure, but is not known for either
group Secondly, the asthmatics in the Linn et al study, although reactive to SO2, tended to
have milder disease, none used regular asthma medications and all but three subjects had an
15-53
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FEVj/FVC ratio in excess of 75 % All of the subjects in the Bauer et al study used some
form of bronchodilator (oral or inhaled) and 9 of 15 subjects had a baseline FEVyFVC ratio
less than 75% It is not clear whether the effects of NO2 could have been confounded by
exposure to an ambient aeroallergen Although subjects in the Linn et al study were
exposed in March, a time when outdoor pollen aeroallergens would have tended to be
minimal for the several preceding months, winter is the peak season for fungal aeroallergens
(Street and Hamburger, 1976, McLean et al, 1991) Also, increased bronchial reactivity to
cold air was an important finding in the Bauer et al study, but it was not measured in the
Linn et al. study
Further studies were conducted by Linn et al (1986) on 21 (minimal to mild)
asthmatics exposed to 0, 0 30, 1 0, and 3 0 ppm NO2 for 1 h The exposures included
intermittent, moderate exercise (VE= 41 L/min) This group was characterized as "clinically
mild extrinsic (allergic)" asthmatics who required infrequent, if any, medication As in the
previous study with 4 0 ppm NO2 exposures, there were no significant effects of NO2 on
spirometry, SRaw, or symptoms Furthermore, there was no significant effect on airway
reactivity as measured by cold-air challenge (see Section 15 4) In order to examine the
suggestion that the seventy of response to NO2 may be related to the clinical seventy of
asthma, the authors selected three subjects whom they characterized as having more severe
illness. There was no indication that the responses of these subjects were related to NO2
exposure, although they expenenced markedly larger changes in resistance than other milder
asthmatics under all exposure conditions Heart rate or minute ventilation did not vary
significantly with NO2 exposure The previously observed decrease in systolic pressure,
associated with 4 0 ppm NO2 exposure, was not examined in these subjects
Mohsemn (1987a) studied 10 mild asthmatics exposed to 0 5 ppm NO2 for 1 h at rest in
an environmental chamber There were no changes in symptoms, spirometry, or
plethysmography that could be attributed to NO2 exposure The response to methacholine
(bromide) was evaluated with partial expiratory flow at 40% VC (PEF40%VC), rather than
changes in SRaw or FEVl3 to test for "small airway abnormality" without the influence of
prior deep breaths There was a significant increase in airway responsiveness to
methachohne after the NO2 exposure The dose of methachohne required to decrease
PEF40%VC by 40% was 9 2 ± 15 after air and 4 6 + 8 2 after NO2 (p = 0 042)
15-54
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Roger et al (1990) reported the results of NO2 exposure in mild asthmatics The first
was a pilot study of 12 mild asthmatics exposed to 0 30 ppm for 110 min, including three
10-min periods of exercise After the first 10 min of exercise in NOz, they found an 11 %
decrement in FEVl5 which was significantly larger than the 7% decrease seen after the clean
air exposure These differences between air and NO2 exposure persisted for the remainder
of the exposure period, although the overall responses were progressively less with
successive periods of exercise, as is common with exercise-induced asthma when the exercise
stimulus is intermittent
A concentration-response study was subsequently conducted (Roger et al, 1990) with
21 mild asthmatics, including 6 subjects from the pilot study, who were exposed to 0 0,
0 15, 0 30, and 0 60 ppm NO2 The 75-min exposures included three 10-min exercise
periods In contrast to the pilot study, there were no differences in response between the air
and NO2 exposure at any exposure concentration or time during the exposure Bronchial
reactivity to methacholine, tested 2 h after the exposures, was similar for air and NO2
exposures There were no significant differences in symptom scores across the four exposure
conditions The authors were unable to specifically identify factors that could have caused
the difference in response between the pilot study and the larger, more comprehensive
concentration-response study They suggested that the pilot study asthmatics may have had
more reactive airways, based on their poorer baseline lung function and greater airway
responsiveness to methacholine compared to the subjects in the concentration-response study
Furthermore, the studies were conducted during different seasons, which may account for
some of the variability in response
Rasmussen et al (1990) presented a preliminary report of a concentration-response
study of healthy asthmatic subjects exposed to 0 1, 0 2 and 0 8 ppm NO2 Exposures lasted
120 min and included 10 mm of exercise There were no significant changes in lung
function (SRaw, FEV^) or airway responsiveness to histamine resulting from NO2 exposure at
any concentration in either normal or asthmatic subjects Also assessed were acoustic
rhinometry, nasal mucocihary clearance, and alveolar epithelial permeability, these results
were not reported
A series of abstracts have been presented by investigators from Mt Sinai Medical
Center in Miami (Sackner et al , 1980, Ahmed et al , 1983a,b), these reports have not
15-55
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appeared in the peer-reviewed literature but are available as technical reports (Ahmed et al,
1983a,b). The latter report presents data that are qualitatively similar to Orehek et al (1981)
and Hazucha et al (1983) in that some subjects (13 out of 20) showed increased airways
responsiveness to carbachol after NO2 exposure and some (7 out of 20) did not Even with
the post hoc separation of subjects into "reactive" and "nonreactive" groups, the increase in
airway responsiveness in the reactive group (n = 13) was not statistically significant There
were no significant changes in lung function Adequate characterization of the exposure
conditions was not presented The former report (Ahmed et al, 1983a) dealt with effects of
NO2 on nine ragweed-sensitive asthmatics There were no group mean changes in Gaw or
FEVj after NO2 exposure There was also no change in bronchial responsiveness to a
ragweed antigen inhalation challenge either immediately or 24 h after exposure to 0 1 ppm
NO2
The effects of prior NO2 exposure on SO2-induced bronchoconstriction has been
examined in two studies (Torres and Magnussen, 1990, Rubinstein et al, 1990) Torres and
Magnussen (1990) exposed 14 mild-to-moderate asthmatic subjects to 0 25 ppm NO2 for
30 mm while breathing through a mouthpiece at rest There were no changes in SRaw as a
result of the exposure After the exposure, airways responsiveness to SO2 was assessed by
isocapnic hyperventilation of 0 75 ppm SO2 using stepwise increases in ventilation, the initial
level was 15 L/min with subsequent mcreases to 30, 45, 60 L/min, and so forth After each
3-min period of hyperventilation, SR^ was determined The ventilation of SO2 required to
produce a 100% increase in SRaw (PV100SRaw[SO2]) was estimated using interpolation of
ventilation versus SRaw (dose-response) curves The PV100SRaw(SO2) was significantly
reduced after NO2 exposure compared to after filtered air exposure, suggesting that the
airways were more responsive to SO2 as a result of the prior NO2 exposure
Rubinstein et al (1990) exposed nine asthmatics to 0 30 ppm NO2 for 30 mm
(including 20-min light exercise) There were no significant effects of NO2 exposure on lung
function (single breath nitrogen washout, SR^, FVC, FEV{) or respiratory symptoms,
although a slight increase m SRaw was observed as a result of exercise An SO2-
bronchoprovocation test was administered after exercise, but using a different technique than
Torres and Magnussen (1990) Increasing amounts of SO2 were administered by successive
doubling of the SO2 concentration (0 25, 0 5, 1 0, 2 0, 4 0 ppm) at a constant, isocapnic
15-56
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ventilation of 20 L/min, maintained for 4 mm Specific airway resistance was measured
after each step increase in SO2 concentration The concentration of SO2 required to increase
SRaW by 8 units (PD8uSO2) was interpolated from a dose-response curve of SO2
concentration versus SRaw The PD8uSO2 was 1 25 + 0 70 ppm after air exposure and
1 31 ± 0 75 after NO2 exposure, indicating no mean change in responsiveness to SO2 Only
one subject showed a tendency toward increased responsiveness to SO2 after NO2 exposure
(see also Section 15 4)
The contrasting findings in these two studies is somewhat puzzling because the subjects
of Rubinstein et al (1990) were exposed to a higher NO2 concentration and exercised during
exposure However, Jorres and Magnussen's subjects appeared to have had slightly more
severe asthma and were somewhat older The modest increase in SRaw induced by exercise
in the Rubinstein et al study may have interfered with the response to SO2 (i e , the subjects
may have been in a refractory state) Finally, the different method of administering the SO2
bronchoprovocation test (i e , increased VE at constant SO2 vs increasing SO2 at constant
VE) may produce a different response because hyperventilation alone could contribute to the
increase in SR^ (Deal et al , 1979, Eschenbacher and Sheppard, 1985) Thus, although
similar, the two SO2 challenges are not necessarily comparable
15.3.1.1 Effects of Nitric Acid Vapor on Asthmatics
Koemg and associates (1988, 1989a,b) have recently reported preliminary results of
a study of adolescent asthmatics exposed to HNO3 vapor In the first report (Koemg et al,
1988), subjects were exposed to 50 and 100 ppb HNO3 and to 50 ppb HNO3 plus 68 /xg/m3
H2SO4 The average FEVj decreased following exposure (30-min rest followed by 10-min
mild exercise) under all three conditions, although there were no significant differences
among the responses to these exposures
Koemg et al (1989a) reported the responses of adolescent asthmatics to a 40-min
exposure to 50 ppb (2 /*M/m3) HNO3 vapor exposure via a mouthpiece exposure system
In this study, after 30 min of rest and 10 mm of exercise while breathing HNO^ vapor, there
was a 4 4% decrease in FEVj compared to 1 8% decrease after air breathing A 22 5%
increase in total respiratory resistance was also observed after HNO3, compared to a 7 5%
increase after air
15-57
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In further studies by Koemg et al (1989b), subjects were exposed to air and
57 ± 16 ppb HNO3 twice once without and once with a preliminary gargle of lemonade,
intended to reduce oral ammonia (NH3) levels During the 45-min exposure, subjects
exercised twice for 15 min at a ventilation of about 25 L/min Baseline oral NH3 of
318 ± 84 ppb was reduced to 113 ± 98 ppb after lemonade gargle There were small, but
not statistically significant, decrements in FEVj after all exposures, —33% after HNO3
alone and —1.7% after both air and HNO3 plus lemonade Similar trends (—94%, HNO3,
—55%, HNO3 plus lemonade, —51%, air) were observed for V50%Vc The data did not
support the hypothesis that reduction of oral ammonia (by usmg a lemonade gargle) would
increase the response to HNO3 because HNO3, in the absence of ammonia, would not be
converted to NH4NO3 in the upper airway Nevertheless, the authors made the interesting
suggestion that, in mixtures of HNO3 vapor and H2SO4 aerosol, gaseous NH3 may react
more rapidly with the gaseous HNO3 than with the aerosol, thus reducing the potential
neutralization of H2SO4 It should be emphasized that this is speculation based on the
physicochemical properties of HNO3 vapor and H2SO4 aerosol and is not supported by
experimental observations A complete report of these studies is not currently available (i e ,
as of October 1992)
15.3.2 Effects of Nitrogen Dioxide on Patients with Chronic Obstructive
Lung Disease
Patients with COPD represent an important potentially sensitive population group
Some of these patients have airways hyperresponsiveness to physical and chemical stimuli
In addition, because of their already compromised lung function, they have much less reserve
than people with normal lung function The poor distribution of ventilation in patients with
COPD may lead to a greater delivery of NO2 to the segment of the lung that is well
ventilated, thus resulting in a greater regional tissue dose Tables 15-6 and 15-7 summarize
these studies
In a review of their studies, Von Nieding and Wagner (1979) summarized previously
reported findings The main observations were that R^ increased in chronic bronchitics
exposed to 2 0 ppm NO2 or greater and that, after exposure to 4 to 5 ppm NO2, PaO2 was
decreased and the alveolar-arterial oxygen gradient was widened
15-58
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VO
TABLE 15-6. SUBJECT CHARACTERISTICS FOR PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY
DISEASE EXPOSED TO NITROGEN DIOXIDE3
Number
and
Gender of
Reference Subjects
Kerretal (1979) 7
Linn et al (1985a) 13 M/9 F
Morrow and Utell 13 M/7 F
(1989)
Von Nieding and 1 16
Wagner (1979)
14
Von Nieding et al 84 M
(1973a)
Von Nieding et al 88
(1971, 1970)
Abbreviations
FEVj = Forced expiratory volume in Is
FVC = Forced vital capacity
SRaw = Specific airway resistance
SGaw = Specific airway conductance
CBR = Chronic bronchitis
M = Male
FEV^FVC SR^ or SGaw
Age (Percent) (L'cml^O/L/s) Dyspnea
24-53 75 5 10
48-69 29-67 166 19/22
47-70 43-75 0 30-2 87b Y
3 48-33 3
25-74
RT = 5 93
30-72 - 3 5-10
—
EM = Emphysema
AS = Asthma
IB = Inhaled /3 agonist
OB = Oral bronchodilator (theophylhne)
ST = Oral corticosteroid
COPD = Chronic obstructive pulmonary disease
Diagnosis Medications
CBR
6 CBR 11 IB
21 EM 12 OB
4 AS 2 ST
COPD
Chronic
nonspecific
lung
disease
CBR
COPD
CBR
Medication Airway
Witholding Reactivity Notes
Daily cough
for three
consecutive
months
4 h IB
8 mild
12 moderate
Chronic
nonspecific
lung disease
_.
; Female
SGaw = Specific airway conductance
-------
TABLE 15-7. EXPOSURE CONDITIONS AND RESPONSES IN COPD PATIENTS EXPOSED
TO NITROGEN DIOXIDE3
Reference
Kerretal (1979)
Linnetal (1985a)
Morrow and Utell (1989)
Von Nieding and Wagner
(1979)
Von Nieding et al (1973a)
Von Nieding et al (1971,
1970)
Cone
(ppm)
05
05
10
20
030
1-8
(a) 1-5
(b)5
05-5
Exposure Exercise Exercise
Duration Duration Ventilation Temp
(nun) (nun) (L/min) (°C)
120 15 25 24
60 30 16 225
225 21 25 21
(3 X 7)
5-60
30 breaths - -
(15 mm)
60
15
Relative
Humidity
(Percent) Responses
45 No effects in bronchitics alone Possible
decrease in quasistatic compliance
49 No change in FVC, FEVj, etc , at any NO2
level SR^ tended to increase after first
exercise period Possible decrease in peak
flow at 2 ppm No symptom changes No
change in SaO2
40 Total NO2 inhaled dose 1 215 mg Decrease
of 9 6% m FVC after exposure 52%
decline m FEVj significant after «4-h
exposure
At 4-5 ppm for 15 nun, PaO2 decreased
(artenalized capillary blood) Raw increased
with exposure to 1 6 ppm or greater
Increase m Raw related to NO2 concentration
No effect on Raw below 1 5 ppm
Changes in PO2 of earlobe capillary blood
Change occurred m first 15 mm, effect did
not increase with further exposure
Decrease m earlobe blood PO2 at 4 0 ppm
and above Increased Raw at concentrations
of 1 6 ppm and above
"Abbreviations
FVC = Forced vital capacity
FEVj = Forced expiratory volume mis
NC>2 = Nitrogen dioxide
SRaw = Specific airway resistance
SaC>2 = Arterial oxygen saturation
Pa(>2 = Arterial partial pressure of oxygen
Raw = Airway resistance
PC>2 = Partial pressure of oxygen
-------
The results of two NO2 exposure studies were discussed in Von Nieding et al (1980)
In the first study, 14 healthy and 14 bronchitic patients were exposed to 5 to 8 ppm NO2 for
up to 5 min on 4 separate days The mean increase in R^ was 1 07 cm H2O/L/s Except
for three subjects with an increase in Raw of greater thai 2 0 cm H2O/L/s, the responses of
the bronchitics were similar to the healthy subjects
In the second study (Von Nieding et al, 1980), 30 healthy subjects and 40 bronchitic
subjects were exposed to 5 ppm NO2 for 5 mm The subjects were divided into clusters
according to their preexposure RaWs, which ranged from less than 1 cm H2O/L/s to greater
than 4 0 cm H2O/L/s There was a tendency for the response to NO2 to be greater in the
subjects with the highest baseline R^ In subjects with baseline R^ > 4 0 cm H2O/L/s, the
increase m R^ averaged just less than 1 5 cm H2O/L/s, the increase was less than 0 5 cm
H2O/L/s in subjects with baseline R^ < 1cm H2O/L/s Percentage changes ranged from
approximately 25 to 50% Unfortunately, this synopsis does not provide a more
comprehensive review of the data
More recently, Linn and co-workers (1985a) studied a diverse group of 22 COPD
patients, including men and women with emphysema and chronic bronchitis, exposed, while
exercising intermittently, for 1 h to 0 5, 1 0, and 2 0 ppm NO2 In agreement with the
previous Von Nieding and Wagner (1979) study, no changes in arterial oxygenation (ear
oximetry measurements of hemoglobin saturation) were observed Also, no changes in lung
function (spirometry, plethysmography) were observed that could be attributed to NO2 The
only exception was the tendency (not statistically significant) for peak flow to be slightly
lower (about 5%) during the 2 0-ppm exposures No increase in symptoms was reported
Morrow and Utell (1989) examined the responses of 20 patients with COPD who were
exposed to 0 3 ppm NO2 for 3 75 h, during which tune they performed mild exercise for
three 7-min penods Forced vital capacity (FVC) showed progressive and significant
decreases dunng and following NO2 exposure, with the largest change (—9 6%) occurring
after 3 75 h of exposure Smaller decrements were seen in FEVX (-52%) at the end of
exposure There was no effect on SGaw or diffusing capacity as a result of NO2 exposure
The differences between subjects with more severe disease (FEVj < 60 % predicted) and
those with milder disease (FEVj > 60 % predicted) in terms of their relative responses to
NO2 were generally not significant, except for a possible slightly greater decrease in FEVX in
15-61
-------
the milder COPD group When the COPD patients were compared with healthy, elderly
nonsmokers, there was an apparently significant difference between the two groups in their
response to NO2, that is, the COPD patients showed a decrement, but the healthy
nonsmokers showed an improvement in FEY^ There were also apparent differences in NO2
response between healthy, elderly smokers and healthy, elderly nonsmokers These
exploratory post hoc analyses generate interesting hypotheses, but they do not explain
whether the COPD patients responded to NO2 because of their current or previous smoking
habit or because of some predisposition to NO2 effects caused by their lung disease The
reasons for the marked difference in response between these subjects and those of Linn et al
(1985a) are unclear Possible explanations include differences in ambient concentrations due
to the place of residence (Los Angeles vs Rochester, but see discussion in Section 15 3 1
regarding this factor) and, more importantly, duration of exposure (4 h vs 1 h) However,
the higher concentrations used in the Linn et al (1985a) study could be expected to produce
greater effects in equally reactive subjects Differences in the seventy of COPD could be
related to the differences in response, although the subjects of Linn et al had similar or
worse lung function than the subjects of Morrow and Utell Note also that the mild COPD
subjects of Morrow and Utell had greater responses
15.3.3 Summary
Although findings in asthmatics have been mixed, the pulmonary function responses to
NO2, within the ambient range, are relatively small when compared to SO2 exposure (see
Table 15-8 and Figures 15-1 and 15-2) Several studies (Bauer et al , 1986b, Roger et al,
1985; Koenig et al, 1987a,b, Avol et al, 1986) suggest possible small changes in
spirometry or plethysmography at concentrations in the range of 0 1 to 0 5 ppm However,
the absence of changes at higher NO2 concentrations, failing to suggest a concentration-
response relationship (Avol et al, 1986, Byhn et al , 1985, Linn et al, 1985b, Linn et al,
1986), is problematic Patients with COPD experience pulmonary function changes with
brief exposure to high concentrations (5 to 8 ppm for 5 mm) or with more prolonged
exposure to lower concentrations (0 3 ppm for 3 75 h) In both asthmatic and COPD
populations, there remain several unanswered questions regarding the interaction of disease
state and exposure variables
15-62
-------
TABLE 15-8. AIRWAY RESISTANCE AND FORCED EXPIRATORY VOLUME IN ONE SECOND CHANGES IN
ASTHMATICS EXPOSED TO NITROGEN DIOXIDE3
ON
Reference
Avoletal (1988)
Bauer et al (1986a)
Bylinetal (1985)
Byhnetal (1988)
Hazuchaetal (1982,
1983)
Kleinman et al (1983)
Koemgetal (1985)
Koemgetal (1987a,b)
Lmnetal (1985b)
Percent A FEVj
NO2 Air N02
03 -10 -11
06 -11 -12
03 -41 -101
K-7 0)]
0 12
025
050
0 14
027
053
0 1
02 -33 -2 61
0 12 0 -30
0 12 -63 -61
018 -13 -33
4 ppm
(twice)
Percent A SR^
Air
+34
+34
NA
+30
+30
+30
-12
-12
-12
-19
RT = +1
RT = -53
RT = -43
RT = +5 1
+37
+ 17
lw or SGaw or ARTb
NO2
405
78
NA
+4 1
-39
-230
-75
-88
-135
+66
+9
-44
+ 103
+ 116
+ 15
+22
Notes
ASRaw is mean for last
two measurements
Shows the directional
change in response
Air (10 FEVj s decreased,
5 increased) NO2 (all
FEVj s decreased)
Air (10-5+), NO2 (15-)
[Excludes two anomalous
subjects ]
Data given for 20 mm of
exposure
NC
NC
Greater %AFEV! due to
higher baseline on NO2
day
NC
Average of two exposures
-------
TABLE 15-8 (cont'd). AIRWAY RESISTANCE AND FORCED EXPIRATORY VOLUME IN ONE SECOND
CHANGES IN ASTHMATICS EXPOSED TO NITROGEN DIOXIDE3
Percent A FEVj
Reference N02 Air NO2
Linnetal (1986) 03 -25 -03
i— >
£
Roger
10
30
etal (1990) 03 -6 -12
0 15 -35 -39
030 -17
0 60 -37
Percent A SRaw or SGaw or ARTb
Air N02
+63 +64
-1 9 +35
+0 6 +29
-
+52 +69
+54
+55
Notes
NC
Change after 3rd
exercise period
Abbreviations
FEVj = Forced expiratory volume mis
SRaW = Specific airway resistance
SGaw = Specific airway conductance
RT = Total respiratory resistance
NA = Not available
NC
= No comment
SGaw changes were converted to SRaw for comparison (SRaw = l/SGaw) positive changes indicate increased resistance
-------
§
§
2
o.
E
b
a
4
2
0
ULJ „
U- -4
-6
-8
-10
M L
LR A A
K w
R A
A'
B*
DAT;
Study
Avol etal, 1988
Avol etal, 1989
Bauer et al, 1986
Kc«nigetal,1987a,b
Linn et al , 1986
Mohsenln 1987a
Roger etal, 1990
\
Symbol
A
A'
B
K
L
M
R
ppmxL
972
1,944
1,080
162
135
477
1,590
4,770
360
265
530
1,060
L
%A
-12
+09
-33
-41*
-03
+03
+13
+23
+15
-1 1
+04
+01
1,000
'Statistically significant
2,000 3,000
ppm x L
4,000
5,000
Figure 15-1. Percent change (post-air vs. post-NO2) in FEVj vs. NO2 dose in ppm x L
in asthmatics.
^k *•" .
i 15 -
y
W
c
O
15
1
I
E
§
&
j=
Function
A
10 -
•
DATA
A
RM
-
-JQI
8 5-l L A'
f:
n
I :
^ -5-
-
-10-
-15-
» *m.\J
B'K R
B L
A'R A
BB'
Study
Avol etal, 1988
Avol etal, 1989
By lln etal, 1985
By lin etal, 1988
Koanig et al , 1987a,b
Unn etal, 1986
Mohsenln 1987a
Roger etal, 1990
Symbol
A
A
B
B
K
L
M
R
ppm xL
972
1,944
1,080
360
29
59
116
50
98
191
121
477
1,590
4,770
360
265
530
1,060
-1 6
+176
+ 44
-15
+ 43
-37
+ 06
+ 52
+ 21
-48
+ 21
+ 45
+ 07
-96
+102
+ 94
-21
+ 21
L
I ' l ' l ' l
1,000
2,000 3,000 4,000
ppm x L
Figure 15-2. Percent change ([post-NO2 - post-air]/post-air) hi resistance (RaW, SRaw,
5,000
or
vs. NO2 dose in ppm x L in asthmatics.
15-65
-------
15.4 EFFECTS OF NITROGEN DIOXIDE EXPOSURE ON AIRWAY
RESPONSIVENESS
Physiological changes in the airways induced by a variety of inhaled substances have
been used to assess the "responsiveness" or "reactivity" of the airways Comparing results
across studies is difficult because of the variety of types of airway challenges, the variety of
methods used to administer the tests, the different physiological end points used to quantify
the responses, differences in waiting period after the exposure to determine reactivity, and
whether or not the exposure involved exercise A variety of stimuli have been used to
challenge the airways, including (1) chemical mediators such as histamine, methacholine,
carbachol, or hypertonic saline, (2) physical methods such as exercise or isocapnic hyperpnea
with cold air; (3) other pollutants such as SO2, or (4) specific antigenic substances such as
ragweed or grass pollen In this section, the effects of NO2 on measures of airway
responsiveness are discussed A more detailed discussion of overall aspects of most of the
studies, including exposure conditions and measurement of other variables, are presented in
Sections 15 2 and 15 3 and the tables associated with those sections
Despite the absence of bronchial or airway hyperresponsiveness in some asthmatics and
its presence in some nonasthmatics (Pattemore et al, 1990), there is a correlation between
increased asthma symptoms or increased medication usage and increased airway
responsiveness (Bntton et al, 1988) Alterations in airway responsiveness may also occur as
a result of repeated challenges with methacholine (Beckett et al , 1992), histamine (Hamielec
et al , 1988), hypertonic saline (Belcher et al, 1987), exercise (Stearns et al, 1981), or, in
some cases, by interaction between two different challenges either histamine (Hamielec
et al , 1988) or hypertonic saline challenge (Belcher et al , 1987) administered before an
exercise challenge can reduce the airway response to exercise Prior exercise-induced
bronchoconstnction can reduce responsiveness to hypertonic saline (Belcher et al, 1987), but
not, apparently, to histamine (Belcher et al, 1987, Hamielec et al , 1988) Thus, although
the responses to a number of airway challenges may be correlated (Chatham et al, 1982),
they cannot be considered equivalent
15-66
-------
15.4.1 Healthy Subjects
In a small number of recent studies, the effects of NO2 on airway responsiveness in
healthy, normal subjects have been reported Airway responsiveness has been shown to
increase in normal subjects after exposure to NO2 concentrations in excess of 1 0 ppm Bed
and Ulmer (1976) found increased responsiveness to acetylcholine in subjects exposed to
either 7 5 ppm for 2 h or to 5 0 ppm for 14 h Mohsemn (1987b, 1988) found increased
responsiveness to methachohne after 1-h exposures to 2 0 ppm and Frampton et al (1991)
reported increased carbachol responsiveness after 3-h exposures to 1 5 ppm. Mohsemn
(1987b) also reported that the increased airway responsiveness post-NO2 exposure could be
blocked by elevation of serum ascorbate levels through vitamin C pretreatment (see
Section 15 2 1 1) In contrast, using subjects exposed to 0 1 ppm at rest, Hazucha et al
(1982) and Ahmed et al (1983b) found no significant change in airway responsiveness to
cholinergic agonists (methacholine and carbachol, respectively) A 20-min exposure to
0 48 ppm similarly had no significant effect on airway responsiveness to histamine (Bylin
et al, 1985) Kulle and Clements (1988) examined airway responsiveness after 2-h
exposures to 2 0 and 3.0 ppm NO2 on 3 consecutive days Nasal inoculation with influenza
virus occurred on the second day of NO2 exposure Although there was a significant trend
for airway responsiveness to decline in one group of subjects exposed to clean air, there was
no trend for airway responsiveness to increase after exposure to either NO2 concentration
Responses were not altered after virus inoculation Thus, in three studies, NO2
concentrations of at least 1 5 ppm NO2 inhaled over at least 60 min were associated with
increased responsiveness to either cholinergic or histanunergic agonists The mechanism for
this increase in responsiveness needs to be established before the clinical implications of this
finding can be ascertained
15.4.2 Asthmatic Subjects
A change in airway responsiveness appears to be one of the more sensitive indicators of
response to NO2 exposure in asthmatics The findings from the various studies reported here
are summarized in Table 15-9 See Section 15 3 1 for a more detailed description of the
exposure and measurement methodology
15-67
-------
TABLE 15-9. CHANGES IN AIRWAY RESPONSIVENESS ASSOCIA1
WITH NITROGEN DIOXIDE EXPOSURE3
oo
Number
ASTHMATICS
Ahmed et al (1983b)
Ahmed et al (1983a)
Hazuchaetal (1983)
Oreheketal (1976)
Rasmussen et al (1990)
Oreheketal (1981)
(4 allergic + 3 asthmatic)
Bylinetal (1988)
Roger etal (1990)
Kleinman et al (1983)
Rasmussen et al (1990)
Joerres and Magnussen
(1990)
Joerres and Magnussen
(1991)
Bylinetal (1988)
Avoletal (1988)
Avoletal (1989)
20
19
15
20
20
7
20
19
31
20
14
11
20
37
34
N02
(ppm)
01
01
0 1
0 1
01
0 11
014
0 15
020
020
025
025
027
030
030
Duration
of Exp Challenge
(mm) Type
60
60
60
60
120
60
30
80
120
120
30
30
30
120
30
GARB
RAG
METH
CARB
METH
GRASS
HIST
METH
METH
METH
SO2
METH
HIST
COLD
COLD
End
Point
SGaw
SGaw
SRaw
SRaw
FEVj
SRaw
SRaw
SRaw
FEVj
FEVj
SRaw
SRaw
SRaw
FEVjL
FEVjL
Time
Postexp
(mm)
-
-
20
IM
EM
IM
25
60
IM
IM
27
60
25
60
60
Exercise
N
N
N
N
Y
N
N
Y
Y
Y
N
Y
N
Y
Y
Change in AR
+
13
10
6
14
7
8
7
3
Average PD ± SDC
Air NO2 Notes
604
8 97 ± 24 7
19 ±04
056
267
3 36 ± 4 63
20 ± 10
036
(AIR-NO2 = 0 00)
-
14
10
20
-
6
7
7
12 ±03
-
33 ± 07
86 ± 16
13 ±03
-
3 1 ±07
30 ± 62
(AIR-NO2 = 0 02)
11
7
14
11
12
2
4
6
16
21
46 5 ± 5 1
0 41 ± 1 6
-
-8 4 ± 11
-5 3 ± 12
37 7 ± 3 5
0 41 ± 1 6
-
-10 7 ± 12 Delta FEV!
-4 7 ± 13 Delta FEVi
-------
TABLE 15-9 (cont'd). CHANGES IN AIRWAY RESPONSIVENESS ASSOCIATED
WITH NITROGEN DIOXIDE EXPOSURE3
ON
Bauer et al (1986a)
Linnetal (1986)
Morrow and Utell (1989)
Roger etal (1990)
Rubinstein et al (1990)
Bylinetal (1985)
Mohsemn (1987a)
Bylinetal (1988)
Avoletal (1988)
Roger etal (1990)
Rasmussen et al (1990)
Linnetal (1986)
HEALTHY SUBJECTS
Ahmed etal (1983b)
Hazucha et al (1983)
Bylinetal (1985)
Framptonetal (1991)
Number
12
21
20
19
9
8
10
20
37
19
20
21
20
15
8
15
N02
(ppm)
030
030
030
030
030
048
050
053
060
060
080
100
0 1
0 1
048
15
Duration
of Exp Challenge End
(mm) Type Point
30
120
225
80
30
20
60
30
120
80
120
120
60
60
20
180
COLD FEV!
COLD FEVi
CARB SGaw
METH SR^
S02 SR^
HIST SRaw
METH SGaw
HIST SRaW
COLD FEV!
METH SRaW
METH FEVj
COLD FEVi
CARB SGaw
METH SRaW
HIST SRaw
CARB FEVi
Time
Postexp
(nun)
60
IM
-
60
60
20
IM
25
60
60
IM
IM
20
20
30
Exercise
Y
Y
Y
Y
Y
N
N
N
Y
Y
Y
Y
N
N
N
Y
Change in AR
9
-
-
8
4
5
7
12
13
11
-
10
6
1
11
3
-
-
9
5
-
2
7
16
8
-
10
7
2
4
Average PD + SD°
Air NO2
0 83 ± 0 42
-114
33 ±07
1 3 ± 07
-
92 ± 1 5
-
-84 ± 11
33+07
(AIR-NO2
-11 4
207
16 2 ± 2 7
-
-48 ± 1
0 54 ± 0 33
-121
33 ± 08
13+08
-
46 ± 80
-
-10 4+14
37 ± 1 1
= -006)
-112
196
18 3 + 3 0
-
-75 + 1
Notes
PDioRHEd
Delta FEVi
Delta FEVi
Delta FEVi
Delta FEVi
-------
TABLE 15-9 (cont'd). CHANGES IN AIRWAY RESPONSIVENESS ASSOCIA1
WITH NITROGEN DIOXIDE EXPOSURE3
Kulle and Clements (1988)
Kulle and Clements (1988)
Mohsemn (1988)
Beil and Ulmer (1976)
Bed and Ulmer (1976)
Beil and Ulmer (1976)
Number
21
21
18
16
16
16
N02
(ppm)
20
30
20
25
50
75
Duration
of Exp
(mm)
120
120
60
120
120
120
Challenge
Type
METH
METH
METH
ACH
ACH
ACH
End
Point
FEVl
FEV!
SGaw
RT
Jvr
RT
Time
Postexp
(mm)
IM
IM
IM
IM
IM
IM
Change in AR
Exercise +
N
N
N 12 2
N
N
N
Average
Air
-10 7e
-70
101 ± 44
08
09
07
PD ± SDC
NO2 Notes
-12 7 Delta FEVj
-9 9 Delta FEVj
81 ±45
06
09
10
Abbreviations
NC>2 = Nitrogen dioxide
CARB = Carbachol
SGaw = Specific airway conductance
N = Rest
RAG = Ragweed
METH = Methacholme
SRaw = Specific airway resistance
IM = Immediately after exposure
FEVi = F°rced expiratory volume in 1 s
Y = Included exercise
GRASS = Grass pollen
HIST = Histamme
SC>2 = Sulfur dioxide
COLD = Cold-dry air
ACH = Acetylcholme
RT = Total respiratory resistance
Change in AR + = increased airway responsiveness (AR) after NC>2 compared to air, - = decreased AR after NC>2 compared to air
°PD + SD = Mean + Standard deviation (SD) of provocative dose (PD) (±SEM = standard error of mean) See individual papers for calculation of PD
CTDioRHE = Respiratory heat exchange (loss) for 10% drop in FEVj
Separate control and exposure groups First day of three consecutive daily exposures
-------
There have been several studies of NO2-exposed asthmatics in which the airway
responsiveness was evaluated using cholinergic agonists (carbachol, acetylchohne,
methacholine) Subjects were exposed to 0 1 to 0 2 ppm NO2 in five such studies
Of these, both Hazucha et al (1983) and Roger et al (1990) found no significant change in
group mean response to methacholine challenge Ahmed et al (1983b) reported a trend for
airway responsiveness to carbachol to increase after a 1 -h exposure to 0 1 ppm NO2, but the
trend was not significant (p = 0 07) Nevertheless, some subjects appeared to be more
responsive than others Orehek et al (1976) reported that 13 of 20 subjects exposed to
0 1 ppm NO2 experienced an increased airway responsiveness to carbachol In these
13 subjects, the mean PD10o decreased from 0 66 to 0 36 mg However, in the seven
"nonresponders", the PD10o of 0 36 mg remained unchanged A number of questions have
been raised about the analytical approach used in this study, and these are discussed in more
detail in Section 15 3 1 Klemman et al (1983) also evaluated airway responsiveness to
methacholine after a 2-h exposure to 0 2 ppm NO2 The dose of methacholine required to
cause a 10 % drop in FEV^ decreased from 8 6 to 3 0 /.eg As a group, these studies appear
to suggest that some individuals, if not a subgroup of asthmatics, may experience increased
airway responsiveness after NO2 exposure
It might be anticipated, when there is a trend for a response at a low concentration, that
exposure to increased concentrations would tend to confirm the trend by producing a less
equivocal response Mohsemn (1987a) found a significant decrease in the dose of
methacholine required to produce a 40% decrease in flow at 40% of VC on a partial flow
volume curve, the PD40 decreased from 9 2 after air to 4 6 after exposure to 0 5 NO2 for
1 h On the other hand, at both 0 3 and 0 6 ppm for 110 mm, Roger et al (1990) found no
difference in airway responsiveness to methacholine Morrow and Utell (1989) also found
no change in airway responsiveness to carbachol after a 3 75-h exposure to 0 3 ppm These
differences cannot be explained either on the basis of NO2 concentration or total NO2 dose
because the total dose in the Mohsemn (1987a) study was lower than either of the other two
studies
Histamine airway challenges have been used in three studies following NO2 exposure
Two studies by Bylin et al (1985, 1988), at NO2 concentrations ranging from 0 14 to
0 53 ppm, suggest possible increased responsiveness to histamine after a 20- to 30-rmn
15-71
-------
resting NO2 exposure In the first study, 5 of 8 subjects showed an increase in response
after a 0.48-ppm exposure, and in the second study, 14 of 20 subjects showed an increase in
response after a 0 27-ppm exposure However, the second, larger study (n = 20) did not
confirm the observations (at 0 53 ppm) of the first study and a somewhat more conservative
statistical approach (Friedman nonparametnc test) failed to confirm the significance of these
findings In a preliminary report, Rasmussen et al (1990) examined the effects of 3-h
exposures to 0 1, 02, and 0 8 ppm NO2 on airway responsiveness to histamine They found
no significant group mean change in airway responsiveness Again, these results are
suggestive that some asthmatics may experience increased airway responsiveness after NO2
exposure, but the inconsistent nature of the findings from study to study and the absence of a
dose-response relationship is problematic
Bauer et al (1986a), Linn et al (1986), and Avol et al (1988, 1989) have examined
the effects of NO2 exposure on airway responsiveness to cold air inhalation Bauer et al
(1986a) found an increase in cold air airway responsiveness after a 30-min exposure to
0.30 ppm NO2 The airway responsiveness was expressed as the quantity of respiratory heat
loss required to produce a 10% drop in FEVl5 this averaged 0 83 kcal/min after air exposure
and 0 54 kcal/min (p < 0 05) after NO2 exposure, indicating an increase in airway
responsiveness Linn et al (1986) found no change in airway responsiveness to cold air after
1-h exposures to 0.3, 1 0, or 3 0 ppm NO2 in a group of 21 asthmatics Avol et al (1988)
found a trend for a group mean increase m airway responsiveness to cold air after 0 3 ppm,
but not after 0 60 ppm, NO2 exposure, this increased response was observed in only 11 of
the 29 subjects at 0 30 ppm In a study of young asthmatics, also exposed to 0 30 ppm NO2
for 1 h, Avol et al (1989) found no mean change in cold air airway responsiveness Indeed,
only 12 of 33 subjects demonstrated a change m cold air airway responsiveness in the
direction indicative of increased responsiveness Again, these cannot be explained on the
basis of NO2 concentration or total NO2 exposure dose because both were lower in the Bauer
et al (1986a) study, where a significant change in the airway responsiveness was observed
Comparison of the studies in Table 15-9 and 15-4 indicates that the Bauer et al (1986a)
study was shorter, included less exercise, and utilized a mouthpiece exposure system
Additional discussion is presented m Section 15 3 1
15-72
-------
Airway responsiveness to SO2 has been evaluated after 30 nun of exposure to 0 25 to
0 30 ppm NO2 in two studies Torres and Magnussen (1990) found an increased airway
responsiveness to SO2 after a resting exposure to 0 25 ppm NO2, but Rubinstein et al (1990)
found no change in airway responsiveness to SO2 after a 0 30-ppm NO2 exposure of sunilar
duration that included 20 mm of exercise The SO2 challenges were administered by
different techniques, Torres and Magnussen used a series of increasing levels of ventilation at
a constant SO2 concentration, whereas Rubinstein et al (1990) used increasing concentrations
of SO2 at a constant ventilation
The effects of NO2 on airway responsiveness to a specific antigen have been examined
in only two studies Ahmed et al (1983a) reported no increase in airway responsiveness to
ragweed antigen in a group of allergic asthmatics following 60 min of exposure to 0 1 ppm
NO2 Orehek et al (1981) found no change in airway responsiveness to grass pollen in a
group of allergic subjects (including three asthmatics) after a 60-min exposure to 0 11 ppm
NO2.
From the studies for which individual data were readily available, the number of
subjects whose airway responsiveness increased and whose airway responsiveness decreased
is listed in Table 15-9 Tabulation of data from this table provided information regarding the
direction of the change (i e , increase or decrease) in airway responsiveness following NO2
exposure One of the problems in this kind of analysis is that it is often difficult to
distinguish between a negative and a no-change situation (i e , it is less likely that airway
responsiveness would decrease from its baseline level than increase)
Of the 105 subjects exposed to < 0 20 ppm, the overall data indicated 67 subjects with
increased airway responsiveness and 38 with decreased airway responsiveness Sunilar ratios
were observed for exercise and rest exposures For the studies of exposure to 0 20 to
0 30 ppm, using all types of challenges, airway responsiveness increased in 96 subjects and
decreased in 73 For studies involving exercise during the exposure, airway responsiveness
increased in 71 and decreased in 65 However, both studies involving resting exposure
showed significant increases in airway responsiveness, whereas only two of nine studies
using exercise exposures were significant In the resting studies, airway responsiveness
increased in 25 subjects and decreased in 8 subjects These studies also were of shorter
duration («30 min) than many of the exercise studies Airway responsiveness increased in
15-73
-------
29 and decreased in 13 subjects in studies of 30-min duration, whereas there were 41
increases and 53 decreases in exposures lasting 60 min or longer At concentrations greater
than 0.30 ppm, the overall total indicated 48 increases and 33 decreases in airway
responsiveness For resting studies, 24 subjects had increased airway responsiveness and
only 9 showed decreased airway responsiveness (5 did not change), whereas 23 increased and
24 decreased in the exercise studies These data are summarized in Table 15-10
The studies in which the change in airway responsiveness was assessed after NO2
exposure are presented in three concentration ranges in Table 15-10 and are divided
according to whether or not exercise was involved in the exposure The data are presented
as the fraction of the total number of subjects with increased airway responsiveness The
increase in airway responsiveness does not appear to be associated with any particular type of
airway challenge The overall percentage of increased airway responsiveness in
NO2-exposed subjects was 59% This is accounted for almost entirely by the resting studies,
with an overall percentage of 69% (p < 0 01) (106 increased and 48 decreased), because, in
the exercising studies, responses were about equally balanced between increased and
decreased responsiveness (104 increased and 96 decreased) There was a trend (p < 0 05)
for a slightly larger percentage («75%) of subjects to have increased airway responsiveness
after NO2 exposure when the exposure is performed both under resting conditions and at
concentrations above 0 20 ppm In fact, of the six studies reporting a significant response
(Klemman et al , 1983, Bauer et al , 1986a, Bylin et al , 1988, Torres and Magnussen, 1990,
Mohsemn, 1987a, Bylin et al, 1985), four were resting exposures and, in four, the exposure
duration was 30 mm or less
The implication of this trend is unclear because the brief duration and low ventilation
during exposure indicate that the NO2 exposure dose in these studies is relatively low If this
trend is real, some interesting hypotheses could be generated Is it possible that exercise
during exposure somehow interferes with the mechanism causing increased airway
responsiveness9 It is known, for example, that repeated exercise induces a refractory state
such that the subject is less sensitive to exercise-induced bronchoconstnction (Edmunds
et al, 1978, Ben-Dov et al, 1982) In many cases of NO2 exposures involving exercise,
repeated bouts of exercise were performed during exposure, which could possibly have made
the subjects refractory to the effects of NO2 During exercise, the responsiveness to
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TABLE 15-10. FRACTION OF NITROGEN DIOXIDE-EXPOSED SUBJECTS WITH
INCREASED AIRWAY RESPONSIVENESS3
Nitrogen Dioxide
Concentration ([NO2]) (ppm)
ASTHMATICS
0 05 < [N02] < 0 20
0 20 < [NO2] <, 0 30
0 30 < [NO2]
All [N02]
HEALTHY
[NO2] < 1 0
£ [NO2] > 1 0
All Exposures
0 64 (105)b
0 57 (169)
0 59 (81)
0 59 (355)b
0 47 (36)
0 79 (29)b
Exposures
with Exercise
0 59 (17)
0 52 (136)
0 49 (48)
0 52 (201)
0 73 (15)
Exposure
at Rest
0 65 (88)b
0 76 (33)b
0 73 (33)c
0 69 (154)b
0 47 (36)
0 86 (14)c
aData are fraction of subjects indicating an increase in airways responsiveness above the value for clean air Numbers in parenthesis indicate actual number
of subjects in each category Total number = 354
"p < 0 01 two-tailed sign test
°p < 0 05 two-tailed sign test
-------
methacholuie is reduced substantially (Inman et al, 1990) and exercise causes a more rapid
reversal of methacholine-induced bronchoconstnction than occurs at rest (Freedman et al,
1988) On the other hand, is there a biphasic response of NO2 causing increased airway
responsiveness at low exposure doses (for example, causing mast cell degranulation, vis a vis
Sandstroem et al [1990a]), with a reversal of this response occurring at higher exposure
doses, possibly through a direct relaxing effect on airway smooth muscle9 For example,
nitrites formed in the lungs of NO2-exposed animals (Postlethwait and Mustafa, 1981) may
have a direct relaxing effect on smooth muscle, including bronchial smooth muscle
In healthy subjects, an increase in airway responsiveness clearly occurs at higher NO2
exposure concentrations (Beil and Ulmer, 1976, Frampton et al , 1991, Mohsemn, 1988)
In the normal subjects at all concentrations, there were 37 airway responsiveness increases
and 23 airway responsiveness decreases At greater than 1 0 ppm, there were 23 increases
and 6 decreases, that is, a ratio of 0 79 (p < 0 01)
15.5 EFFECTS OF NITROGEN DIOXIDE OR NITRIC ACID
EXPOSURE ON BLOOD, URINE, AND BRONCHOALVEOLAR
LAVAGE FLUID BIOCHEMISTRY
The effects of NO2 on the constituents of bronchoalveolar lavage (BAL) fluid, blood,
and urine have been examined, both in vivo and in vitro The general purpose of these
studies has been to examine mechanisms of pulmonary effects or to determine NO2-induced
alterations in body fluids that could potentially result in systemic effects Investigations have
been aimed at determining the effects of NO2 on levels of serum enzymes and antioxidants,
as well as direct effects on red blood cells and hemoglobin Studies of the effects of NO^ on
airway lining fluids have focused on changes in alphaj-antitrypsin levels Potential effects of
NO2 on collagen metabolism have been investigated by examining urinary excretion of
collagen metabolites
15.5.1 Biochemical Effects in Blood
Chaney et al (1981) examined the effects of 0 20 ppm NO2 on various blood
parameters in 19 healthy subjects exposed for 2 h while exercising intermittently A control
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group of 15 subjects was exposed to clean air They observed a significant increase in
glutathione (GSH) levels after exposure None of the other blood parameters (red blood cell
GSH reductase, 2,3-diphosphoglycerate, methemoglobm, vitamin E, immunoglobulin, and
complement C3) were changed significantly The significance of the response reported in
this study appears to be the result of a difference between the control group and the exposure
group in general (different subjects were used in each group) The changes in GSH were
small and were within the normal range, with the average baseline level of GSH being
approximately 38 5 mg/dL The postexposure average of the air group was
36 4 mg/dL ± 1 35 (standard error of the mean [SEM]) and of the NO2 groups was
40 3 ± 1 19 (SEM) mg/dL The authors suggested that the increased level of GSH may be
in response to oxidation of hemoglobin to methemoglobm by NO2 However, Gohil et al
(1988) have recently demonstrated substantial decreases in GSH levels during prolonged
submaximal exercise, which was followed by elevated GSH levels in the postexercise penod,
GSH levels varied from 0 15 mM during exercise to 0 6 mM 3 days postexercise, varying
about a baseline level of approximately 0 4 mM It is not clear to what extent the
observations of Chaney et al (1981) may have been confounded by this exercise effect
It should be noted that Posin et al (1978) found no association between NO2 exposure
(1 ppm for 2 5 h) and GSH levels, although there were apparent changes in blood
biochemistry including increased levels of GSH reductase However, it is not clear from the
Posin et al (1978) study that any of the observed "effects" can be attributed to NO2
exposure, there was no concentration-response relationship, effects were not reproducible
from concentration to concentration, and similar effects were seen with clean air exposures
In vitro exposure of human blood to high levels of NC>2 (6 and 45 ppm) resulted in
methemoglobm formation (Chiodi et al, 1983) However, Borland et al (1985) were unable
to demonstrate increased methemoglobm levels in smokers exposed to high NO levels from
cigarette smoke Methemoglobm is also formed during m vitro exposure to NO (1,000 ppm)
(Chiodi and Mohler, 1985) These observations appear to have no relevance to the potential
effects of ambient NO2
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15.5.2 Bronchoalveolar Lavage Fluid Biochemistry
Mohsemn and Gee (1987) have reported that subjects exposed to 3 to 4 ppm NO2 for
3 h had a 45 % decrease in the activity of alpha-1-protease inhibitor (c^PI) the major lung
protease inhibitor of the enzyme elastase These levels were measured in BAL fluid obtained
3 5 to 4 h after exposure Alpha-1-protease inhibitor is "important in protecting the lung
from proteolytic damage, particularly from the elastase of neutrophils " The mean elastase
inhibitory capacity decreased from 95 ± 12% 111 the air group to 55% in the NO2-exposed
group. (Due to analytical impurities in the standard, the 95% inhibition measured in the
air-exposed group was presumed equivalent to 100%, thus the 45% difference) The authors
noted that even a 50 % reduction in o^PI activity is not associated with an increased risk of
emphysema (Kabiraj et al, 1982) However, reduction in protease inhibition could result in
connective tissue damage and could conceivably be important in individuals with an
cx-1-antitrypsin deficiency
Johnson et al (1990) also examined the response of c^PI to in vivo NO2 exposure in a
group of 24 healthy nonsmokers The subjects were exposed to either 1 5 ppm NO2 for
3 h or to a variable concentration consisting of a baseline level of 0 05 ppm NO2 with three
15-min "peaks" of 2 0 ppm Details of the exposure protocol and subject characteristics are
provided in Frampton et al (1989b) (discussed in Table 15-1, Section 15 2)
Bronchoalveolar lavage was performed 3 5 h after exposure and the fluid was frozen for
subsequent analysis. The functional activity of o^PI was taken to be the elastase inhibitory
activity corrected for the concentration of otjPI determined by immunoassay Neither the
levels of o^PI, as determined by immunoreactivity, nor its functional activity were
significantly changed by NO2 exposure
The different findings by Johnson et al (1990) and Mohsemn and Gee (1987) with
regard to a{PI activity may be accounted for by the considerably larger (about two- to
threefold) exposure levels in the latter study Furthermore, different methods were used to
handle the BAL fluid and to quantify cqPI concentrations in the two studies As discussed
fay Mohsemn and Gee (1987), there appears to be a large range of c^PI activity that is
compatible with lung health, and there is broad range of activity of o^PI in relation to its
concentration The importance of small changes in c^PI is not clearly established, and
15-78
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therefore, the usefulness of changes in ajPI activity as a marker of NO2 exposure will
require additional research
15.5.3 Urine Biochemistry
Muelenaer et al (1987) studied normal males exposed to 0 6 ppm NO2 for 4 h/day on
three consecutive days to examine the possibility that NO2 exposure caused diffuse
pulmonary injury They used hydroxyproline excretion as a marker of increased collagen
catabolism or connective tissue injury Subjects had no residential NO2 exposure, no
allergies or infections that might have produced inflammatory responses, and were minimally
exposed to environmental tobacco smoke Despite controlling for these potentially
confounding variables, the authors observed no significant changes in hydroxyprohne
excretion as a result of NO2 exposure, either immediately or for up to 9 days after exposure
15.6 EFFECTS OF NITROGEN DIOXIDE OR NITRIC ACID VAPOR
EXPOSURE ON HUMAN PULMONARY HOST DEFENSE
RESPONSES
From the epidemiological (Chapter 14) and animal toxicology (Section 1322 I)
literature, it is clear that there is considerable concern regarding the role NO2 exposure may
play in potentiating susceptibility to both bacterial and viral infections Important host
defenses that may be affected by NO2 exposure include the mucociliary clearance system,
alveolar macrophages (e g , altered viral inactivation), and humoral and cell-mediated
immune responses (e g , changes in antibodies and changes in cell populations and their
activities in the lung) The effects of NO2 exposure on viral infectivity have been studied in
human volunteers The effects of NO2 on macrophage functions have been examined using
macrophages from NO2-exposed subjects or macrophages exposed to NO2 in vitro The
effects of NO2 on mucociliary clearance in humans are discussed in Section 15 2 1 4
Kulle and Clements (1988) and Goings et al (1989) (two reports of the same study)
examined the effect of NO2 exposure on the infectivity rate of live attenuated influenza
A/Korea/reassortment virus in healthy, nonsmoking adults exposed to NO2 Seven separate
groups were exposed to either clean air (n = 23, 21, 21) or to NO2 at 1 0 (n = 22),
15-79
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2.0 (n = 21, 22), or 3 0 ppm (n = 22) The exposures consisted of 1 preliminary day of
clean air exposure and then 3 consecutive days of the treatment (i e , either NO2 or clean
air). The virus was administered mtranasally after the second exposure day (i e , the third of
the four days) Infectivity was defined as evidence of virus recovery or a rise in either nasal
wash or serum antibody liters after virus inoculation Infectivity rates in the three clean air
groups were 65, 71, and 71%, respectively, and were 77, 57, 91, and 91% in the 3 0, 2 0,
2 0, and 1 0 ppm NO2 exposure groups, respectively Although the rates of infection were
elevated after NO2 exposure in three of four NO2-exposed groups, these changes were not
significant The investigators and an expert review committee (Kulle and Clements, 1988)
concluded that the results of the study were inconclusive rather than negative, this implies
that the hypothesis that NO2 exposure may alter the frequency or seventy of viral infections
was neither confirmed nor denied by the results of this study
Goings et al (1989) have further elaborated on the results of the above study They
made the point that the experimental design had a low power to detect a 20 % difference in
infection rate of influenza A/Korea (ie,71%vs 91%) and, thus, the lack of statistical
significance is not unexpected At least 70 subjects would be required to detect such a
difference using this virus, which has a relatively high rate of Infectivity Finally, the
influenza A/Korea/reassortment virus is not likely to infect the lower respiratory tract where
most of the NO2 deposition occurs There is also the possibility that the results may have
been confounded by an influenza epidemic, which occurred concurrently with this study,
although caused by a different but related virus The epidemic occurred between Year 1 and
Year 2
Frampton et al (1989a) studied two groups of normal subjects exposed to NO2 under
two different protocols that had the same concentration x time (C x T) product One group
was exposed continuously for 3 h to 0 60 ppm and the other was exposed to a background
level of 0 05 ppm with three "spikes" of 2 0 ppm for 15 mm each The C x T product for
each of these two protocols was the same The major amis of this study were to test the
hypothesis that the ability of alveolar macrophages to inactivate influenza virus was reduced
by NO2 exposure, and to examine the possibility that a series of peak exposures would cause
more impairment than a constant concentration (see also Section 13 2 2 1) Healthy, normal
nonsmokers with no history of airway hyperresponsiveness or of recent upper respiratory
15-80
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infection were exposed to both air and NO2 in random .sequence Exposures included six
10-mm exercise periods, coinciding with the "spikes" in the second protocol There were no
significant effects of these exposures on spirometry or plethysmography under either
protocol Alveolar macrophages obtained by BAL were tested in vitro for their ability to
inactivate influenza (A/AA/Marton/43 H1N1) virus and for the in vitro production of
Interleukin-1 (IL-1) by virus-exposed macrophages Interleukin-1 is an important
proinflammatory protein produced by macrophages that performs a number of functions,
including induction of fibroblast proliferation and activation of lymphocytes, and is
chemotactic for monocytes during the immune response to infection There were no
differences in total cell recovery, viability, or differential cell counts between air- and
NO2-exposed samples for either protocol There was a trend (p < 0 07) for less effective
inactivation of virus by macrophages obtained from subjects exposed continuously to
0 60 ppm NO2 This trend was due to the responses of only four of the nine subjects The
macrophages harvested from these four subjects also showed an increase in IL-1 production
not seen in macrophages from the other subjects No effects of virus inactivation were seen
in the subjects exposed to the 2 0-ppm spikes Although the results of this study were not
statistically significant, the study had relatively low power to detect an effect The findings
are provocative and suggest that further work is necessary to test the hypothesis that NO2
may influence host defense mechanisms in humans
Frampton et al (1989b) also analyzed the protein content of BAL fluid obtained from
NO2-exposed subjects at either 3 5 or 18 h postexposure Three different exposure protocols
were used 3-h exposure to 0 60 ppm or 1 5 ppm NO2 or a 3-h variable concentration
exposure where three 15-min "peaks" of 2 0 ppm were superimposed on a background of
0 05 ppm NO2 Exposures included 10 min of exercise during each half-hour of exposure
There were no significant changes in pulmonary function or respiratory symptoms observed
after NO2 exposure Airway reactivity, assessed by caibachol inhalation, was increased after
the 1 5 ppm NO2 exposure (Frampton et al, 1991) Two groups of subjects were exposed
to 0 60 ppm so that BAL could be obtained either at 3 5 or 18 h postexposure Analysis of
BAL fluid obtained 3 5 h after a 0 60-ppm exposure indicated an increase in alpha-2-
macroglobulin (a2-M), a regulatory protein that has antiprotease activity and
immunoregulatory effects The observed increase in a2-M appears to be transient (no change
15-81
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was seen at 18 h postexposure) and was not observed at a higher NO2 concentration
(15 ppm) Further information appears to be necessary to establish the implications of this
finding
The effects of NO2 on macrophage function in NO2-exposed animals is discussed in
Section 13 22 I Nitrogen dioxide-induced changes have been noted in macrophages
harvested from animals exposed to NO2 concentrations less than 1 0 ppm These studies are
discussed in detail in the animal toxicology chapter (Chapter 13)
The effect of in vitro exposure to NO2 on alveolar macrophages harvested by BAL was
examined by Pinkston et al (1988) Fifteen healthy adults underwent BAL to provide
macrophages for culture After an 18-h incubation, the cells were exposed to 5, 10, or
15 ppm NO2 or 5 % carbon dioxide as a control for an additional 3 h Following the
exposure, some of the cells were incubated for an additional 24 h, and cell-free supernatants
were then obtained for analysis of neutrophil chemotactic factor (NCF) Other macrophage
cultures were incubated for 24 h with influenza virus and the supernatant was then obtained
for analysis of IL-1 There were no changes in macrophage viability, determined by trypan
blue exclusion, in cells exposed to any of the three NO2 concentrations There were no
changes in release of NCF in any of the NO2-exposed cell cultures Furthermore, NO2
exposure did not impair the ability of cells to release NCF after stimulation with activated
zymosan Nitrogen dioxide exposure did not stimulate release of IL-1 from exposed
macrophages Influenza virus stimulated the release of IL-1, but there were no significant
differences between NO2-exposed and air-exposed macrophage cultures Therefore, NO2
exposure triggered neither the release of NCF, which would attract neutrophils to the
airways, nor the release of IL-1, which activates lymphocytes (among other functions)
Equally important, NO2 exposure did not impair the ability of macrophages to produce either
EL-1 or NCF in response to conventional stimuli
Sandstroem et al (1989) exposed a group of 18 healthy nonsmokers to 2 25, 4 0,
and/or 5 5 ppm (n = 8 in each concentration group) for 20 mm of moderate exercise
(VE « 35 L/min) in an exposure chamber Bronchoalveolar lavage was performed at least
3 weeks before and 24 h after each exposure Increased levels of mast cells in BAL fluid
were observed after all NO2 exposures Increased levels of lymphocytes were observed only
at the two higher concentrations
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In order to determine the time course of this response, Sandstroem et al (1990a)
exposed 32 subjects to 4 ppm NO2 for 20 mm, including 15 mm of mild exercise, and then
performed BAL at 4, 8, 24, or 72 h postexposure (in four different groups of eight subjects)
Increased levels of mast cells and lymphocytes were observed at 4, 8, and 24 h, but not at
72 h postexposure There was no change in macrophage numbers nor in albumin
concentration in BAL fluid Eosinophils, neutrophils, and epithelial cell counts were not
altered as a result of NO2 exposure Unpleasant odor and mild nasopharyngeal irritation
were typical symptoms There were no changes in spirometry The observation of increased
numbers of mast cells appears to be unique to this study, although other investigators
(Frampton et al , 1989a,b) may not have looked for changes in mast cell numbers The
authors considered the increased numbers of mast cells and lymphocytes to represent a
nonspecific inflammatory response
Rasmussen et al (1992) studied 14 healthy nonsmoking adult subjects exposed to
2 3 ppm NO2 and to clean air for 5 h with a 1-week interval between exposure Indications
of a decrease in alveolar permeability were observed alter the NO2 exposure The results
support the assumption that a delayed response is a feature of the human response to NO2
and stresses the importance of an extended period of observation in future NO2 exposure
studies
Three recent studies examined the effects of multihour exposures to 1 to 2 ppm NO2 on
lavaged cells and mediators Devlin et al (1992) studied healthy subjects exposed to
2 0 ppm NO2 for 4 h with alternating 15 min periods of rest and moderate exercise One of
the main findings after NO2 exposure was a threefold increase in polymorphonuclear
leukocytes (PMNs) in the first lavage sample representing predominantly bronchial cells and
fluid In addition, macrophages recovered from the predominantly alveolar fraction showed
a 42% decrease in ability to phagocytose Candida albicans and a 72% decrease in release of
superoxide anion Frampton et al (1992) exposed exercising subjects to 2 0 ppm NO2 for
6 h Bronchoalveolar lavage was performed either immediately or 18 h postexposure There
was a modest increase in lavage fluid PMN levels (< twofold increase) but no change in
lymphocytes Alveolar macrophage production of superoxide anion was not altered in these
subjects These two studies suggest that NO2 exposure may induce a mild bronchial
inflammation and may also lead to impaired macrophage function Torres et al (1992)
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examined both healthy and asthmatic subjects exposed to 1 ppm NO2 for 3 h, but observed
no changes in cells or mediators in BAL fluid or in the appearance of bronchial mucosal
biopsies after this exposure Neither macrophage function nor a specific bronchial washing
were examined in this study
Boushey et al (1988) studied five healthy volunteers exposed to 0 60 ppm NO2 on
4 days over a 6-day period Exposures lasted 2 h each and included alternating 15-min
periods of rest and exercise (V^ ** 30 to 40 L/min) On the final (fourth) day of NO2
exposure, venous blood samples were obtained and a BAL was performed Baseline BAL
and pulmonary function data were obtained on a separate occasion There were no effects of
repeated NO2 exposure on pulmonary function (SRaw, FVC, FEVj) or respiratory symptoms
Following the fourth day of NO2 exposure, a slight mcrease in circulating (venous blood)
O *>
lymphocytes was observed (1792 ± 544/mm post-NO2 vs 1598 + 549/mm baseline)
The only change observed in BAL cells was an apparent increase (p < 0 04) in natural killer
(NK) cells from 42 ±24% (baseline) to 7 2 ± 3 1 % (post-NO2) The authors expressed
reservations that the apparent increase in NK cells may have been an artifact of the cell
separation process Interleukin-1 and tumor necrosis factor levels in BAL fluid were not
detectable Tumor necrosis factor is another proinflammatory protein that, among other
activities, promotes adherence of PMNs to endothekal cells and enhances their phagocytic
activity
Sandstroem et al (1990b) studied a group of eight healthy nonsmokers exposed to
4.0 ppm NO2 for 20 nun/day (moderate exercise, VE « 35 L/min) on alternate days over a
12-day period (seven exposures total) Bronchoalveolar lavage was performed 2 weeks
before the first exposure and 24 h after the last exposure The first 20 mL of BAL fluid was
treated separately and presumed to represent primarily bronchial cells and secretions After
NO2 exposure, there was a reduction in numbers of macrophages in the bronchoalveolar
portion, although on a per cell basis, alveolar macrophage phagocytic activity was increased
There were decreased numbers of mast cells in the bronchial portion of the lavage fluid
In addition, there were reduced numbers of T-suppressor, B lymphocyte, and NK cells in the
alveolar portion of the BAL fluid compared to the baseline lavage These observations
contrast with those seen by Sandstrom et al (1989) after single NO2 exposures, suggesting
some alteration in bronchial and alveolar cell populations after repeated NO2 exposure The
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most obvious difference between Sandstrom et al (1990b) and Boushey et al (1988) is the
higher NO2 concentration and the longer duration of the former study Further work is
necessary to confirm these observations, to determine the time course of response to repeated
exposure, and to determine the NO2 exposure dose necessary to invoke modification of
bronchoalveolar cell populations
The effects of HNO3 vapor exposure (in vivo) have been examined in two recent
studies Becker et al (1992) exposed nine healthy subjects to 200 /-ig/m3 (80 ppb) of HNO3
vapor for 120 mm, including 100 min of moderate exercise (VE « 42 L/mm)
Bronchoalveolar lavage performed 18 h postexposure indicated an increased phagocytic
activity of macrophages harvested from the HNO3-exposed lung Alveolar macrophages also
showed increased resistance to infection with respiratory syncytial virus Compared to air
exposure, there were no increases in inflammatory mediators (such as prostaglandm E^,
leukotnene B4, C3a, or neutrophils) or in cell damage indicators such as lactate
dehydrogenase (LDH) or lavage fluid protein The absence of markers of tissue damage
(LDH) or permeability (lavage fluid protein), suggest that, under these exposure conditions,
HNO3 did not cause frank tissue damage
3
Ans et al (199 la) exposed 10 healthy subjects to 500 jttg/m HNO3 vapor for 4 h,
including moderate exercise Lavage fluid was obtained from both bronchial as well as
bronchoalveolar washings, and bronchial biopsy specimens were obtained No change m
LDH levels or lavage protein were observed as a result of HNO3 exposure These
investigators found no differences in differential cell counts in the lavage fluid of both
bronchial and bronchoalveolar washings They also exposed a different group of subjects to
3
500 jttg/m HNO3 plus 0 20 ppm O3 and found no potentiation of the O3-induced
inflammatory response by the addition of HNO3 vapor 1o the exposure Their data suggest
that, at these concentrations, HNO3 does not cause tissue injury, nor does HNO3 alter the
inflammatory response typical of O3 exposure
15.7 EFFECTS OF NITRATES ON HUMAN LUNG FUNCTION
Five studies have been conducted on human exposure to nitrate aerosols since 1979
(see Table 15-11) These studies have been discussed in the Acid Aerosol Issues Paper (U S
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TABLE 15-11. EXPOSURE CONDITIONS AND RESPONSES IN SUBJECTS EXPOSED TO NITRATES3
Ul
I
00
o\
Reference
Kleinman et al
(1980)
Sackner et al
(1979)
Stacy etal (1983)
Utelletal (1979)
Utelletal (1980)
Nitrate Species
and Cone
Otg/m3)
NH4N03
200
NaNO3
10,100,100
1,000
1,000
80 (NEySTOg)
80 (NUjNOg)
+0 5 ppm
NO2
NaNO3
7,000
NaN03
7,000
Exposure Exercise Exercise
Duration Duration Ventilation
(mm) (nun) (L/nun)
120 60 «20
10
240 30 55
16 (X2)
(32 Total)
16 (X2)
(32 Total)
Relative
Temp Humidity Number of
(°C) (Percent) Subjects
31 40 20
19
5
5
6
6
6
6
30 60 12
12
25 10
11
25 11
Subject
Char
Normal
Asthma
Normal
Asthma
Normal
Asthma
Normal
Asthma
Normal
Normal
Normal
Mild
Asthmatics
Influenza
Patients
Aerosol
MMAD Effects
11 No significant changes in
normals or asthmatics
except possible decrease in
Rp No symptom effects
02 No changes
0 55 No effects
0 46 No effects
0 49 No symptoms SGaw
decreased 17% and max
40% TLC decreased 12%
after nitrate, within 2 days
of onset of illness
Similar effects 1 week
later, but not 3 weeks
later
Abbreviations
MMAD = Mass median aerodynamic diameter NaNC>3 = Sodium nitrate
NH4NC>3 = Ammonium nitrate SGaw = Specific airway conductance
RT = Total respiratory resistance
max40%TLC = Maximum expiratory flow at 40% of TLC on
a PEFV curve
-------
Environmental Protection Agency, 1989) The only obvious effect was a decrease in G,
aw
and in PEFV curves in normal subjects with influenza exposed to 7,000 jug/m of sodium
nitrate (NaNO3) aerosol This is probably three orders of magnitude (i e , approximately
1,000 times) above the nitrate concentration that could exist in the ambient air These
studies indicate that, at least as far as lung function is concerned, there is no present concern
for adverse effects from current ambient levels of nitrate aerosols
Sackner et al (1979) studied a diverse group of normal and asthmatic subjects exposed
o
to concentrations reaching 1 ,000 /tg/m of NaNO3 for 10 mm at rest There were no
significant effects on an extensive battery of pulmonary function tests
Utell et al (1979) studied both normal and asthmatic volunteers exposed to
7,000 jwg/m3 of 0 46 ^m NaNO3 aerosol for 16 mm via mouthpiece The major health effect
end points measured m their study included R^w, both full and PEFV curves, airway
reactivity to carbachol, and aerosol deposition Aerosol deposition as a percentage of inhaled
aerosol averaged about 50% for normals and about 56% for asthmatics, the group differences
were not significant The exposure to NaNO3 aerosol was indistinguishable from the control
NaCl exposure in normals Similarly, there were no effects of NaNO3 exposure on
asthmatics
Utell et al (1980) subsequently studied 11 subjects with influenza exposed to the same
NaNO3 regimen as above The subjects were initially exposed at the time of illness and then
were reexposed 1,3, and 6 weeks later Aerosol deposition ranged from 45 to 50% over the
four exposure sessions All subjects had cough and fever, and 10 of 11 subjects had viral or
immunologic evidence of acute influenza Baseline measurements of FVC and FEVj were
within normal limits and did not change throughout the 6-week period There were small but
significant decreases in Gaw following NaNO3 inhalation, but not after NaCl exposure This
difference was present during acute illness and 1 week later, but was not seen at 3 and
6 weeks after illness The decrease in SGaw seen on the initial exposure was accompanied
by a decrease in partial expiratory flow at 40%TLC, this was also observed at the 1-week
follow-up exposure This study suggests that the presence of an acute viral respiratory tract
infection may render humans more susceptible to the acute effects of nitrate aerosols
Nevertheless, the concentration of nitrates used m this exposure study exceed maximum
ambient levels by more than 100-fold
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In addition to NaNO3 aerosols, ammonium nitrate (NH4NO3) exposure has been studied
by Kleinman and associates (1980) Twenty normal and 19 asthmatic subjects were exposed
to a nominal 200 /^g/m of 1 1 /im NH4NO3 aerosol The 2-h exposures included mild,
intermittent exercise and were conducted under warm conditions (31 °C, 40% RH) There
were no significant physiologically meaningful effects of the NH4NO3 exposure in either
subject group
Stacy et al (1983) also studied the effects of 80 jwg/m3 of NH4NO3 in a group of
healthy male adults As in the Kleinman et al (1980) study, there were no changes in lung
function or symptoms
15.8 CONCLUSIONS AND DISCUSSION
At the beginning of this chapter, a series of questions were posed concerning the
potential biological responses to NO2 exposure in humans Some of these questions can be
answered in part using the data presented in this section, others will clearly require additional
research.
Symptoms associated with NO2 exposure in healthy subjects have been limited to
detection of the odor of NO2, in some cases at surprisingly low concentrations, less than
0.1 ppm (Bykn et al, 1985) Few of the studies examined in this review noted a significant
increase in respiratory symptoms Sandstrom et al (1990a) noted mild nasopharyngeal
irritation after exposure to 4 ppm for 20 mm
Nitrogen dioxide exposure at sufficiently high concentrations produces changes in lung
function in healthy subjects A number of investigators have reported increased airway
resistance after exposure to NO2 concentrations exceeding 2 5 ppm (Beil and Ulmer, 1976,
Von Nieding et al., 1979, Von Niedmg and Wagner, 1977, Von Nieding et al, 1980)
However, at concentrations of NO2 between 2 and 4 ppm, some investigators have not
observed any NO2-induced changes in airway resistance or spirometry (Linn et al , 1985b,
Mohsenin, 1987b, Mohsemn, 1988, Sandstroem et al, 1990a) At NO2 exposure
concentrations below 1 0 ppm, there is little if any convincing evidence of change in lung
volumes, flow-volume characteristics of the lung, or airways resistance in healthy subjects
Nitrogen dioxide is believed to have its primary effect on small airways However, routine
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spirometry and airway resistance measurements are not sensitive indicators of small airways
function Thus, the absence of change in these physiological indicators of large airways
function at low NO2 concentrations should not be viewed as evidence that NO2 has no effects
on lung function Further developments will be necessary to permit sensitive, reproducible,
nomnvasive evaluation of small airways, the primary site of NO2 deposition in the lung
Nitrogen dioxide exposure does result in increased airway responsiveness in normal
subjects exposed to concentrations in excess of 1 0 ppm Mohsenin (1987b) and Frampton
et al (1991) reported an increase in airway responsiveness after exposure to 2 0 and
1 5 ppm, respectively Repeated bouts of airway inflammation could promote deleterious
long-term changes in the lung, such as loss of elasticity and acceleration of age-related
changes in lung function However, the development of such responses is only speculative,
given the present level of scientific evidence
Potentially sensitive subjects in the population include children, older adults, patients
with asthma or COPD, or individuals who may be unusually sensitive to NO2 for other
reasons There are insufficient data on children, adolescents, or older adults, either healthy
or with asthma, to determine their NO2 responsiveness relative to healthy young adults
At the concentrations that may fall within the ambient range (eg, < 1 0 ppm), the
effects of NO2 on lung function (i e , spirometry, airway resistance) in asthmatics have
tended to be small For example, Bauer et al (1986a) observed a 4 to 6% decline in FEVj
in asthmatics exposed to 0 3 ppm NO2 for 30 min Koenig et al (1988) reported a 4%
decrease in FVC, but no significant change in other spirometry variables, after exposure of
adolescent asthmatics to 0 30 ppm NO2 On the other hand, several other investigators
(Avol et al, 1988, Bylin et al, 1985, Hazucha et al , 1982, 1983, Kleinman et al, 1983,
Koemg et al, 1985, Linn et al, 1985b, 1986, Mohsenin, 1987a, Roger et al, 1990) have
not found any significant changes in spirometry or airway resistance of asthmatics exposed to
concentrations < 1 0 ppm Again, spirometry and airway resistance are not sensitive
measures of small airways function, where NO2 is known to be primarily deposited
A second important category of sensitive subjects includes patients with COPD, who
have shown mcreased airway resistance after brief exposures to greater than 1 6 ppm NO2
(Von Nieding et al , 1970, 1971, 1973a) (see Table 15-6) In addition, during a longer (4-h)
exposure, Morrow and Utell (1989) reported decreased (approx 5 %) FVC in COPD patients
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exposed to 0 30 ppm Other investigators (Linn et al, 1985a, Kerr et al, 1979) did not find
responses in COPD patients even with exposures to levels as high as 2 0 ppm It appears
that brief acute exposure to relatively high concentrations of NO2 (> 2 ppm) will cause
bronchoconstnction in some COPD patients and that these responses may also be observed
with longer exposures to lower concentrations
An unresolved issue with the current data base is the existence of NO2-induced
pulmonary responses in asthmatics that have been reported at low but not at high
NO2 exposures Although small functional responses have been observed in studies from
various laboratories, effects are not consistently present and demonstrating reproducibility of
responses has been difficult, even within the same laboratory Furthermore, all responses to
NO2 that have been observed in asthmatics have occurred at concentrations between 0 2 and
0.5 ppm. Changes hi lung function or airway reactivity have not been seen even at much
higher concentrations (i e , up to 4 ppm) There is, at present, no plausible explanation for
this apparent lack of a concentration-response relationship There is a possibility that a
portion of the variability in response to NO2 may be attributed to differences in the seventy
of asthma This is a complex issue and has not been studied adequately at this time
In patients with chronic obstructive lung disease, Bauer et al (1987) and Morrow and Utell
(1989) have observed decreased lung function (FVC, FEV^) after exposure to 0 30 ppm for
4 h, but Linn et al (1985a) and Von Nieding and Wagner (1979) found no effects in COPD
patients from short duration exposures below 2 0 ppm It appears that further work will be
necessary to provide enough information to estimate the concentration-response relationships
for NO2 exposure of asthmatics and COPD patients, who appear to be the sensitive
subpopulations
In several studies of asthmatics exposed to NO2, airway responsiveness to a variety of
agents has been demonstrated However, in many other studies using similar experimental
exposures, there was no significant change in airway responsiveness In order to evaluate
this apparent dilemma, a meta-analysis was utilized as descnbed in Section 15 4 Without
regard to the type of airway challenge, NO2 concentration, exposure duration, or other
variables, the overall trend was for airway responsiveness to increase (59% of 354 subjects
increased). This trend was somewhat more convincing for exposures conducted under
nonexercising conditions (69% of 154 subjects increased), indeed, the excess positive
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responses were almost entirely accounted for by exposures conducted under resting
conditions The implications of this overall trend are unclear and will require further
investigations to verify if there is an interaction with exercise-induced changes in lung
function that may possibly obscure changes in airway responsiveness due to NO2 exposure
Increased airway responsiveness could potentially lead to temporary exacerbation of asthma,
possibly leading to increased medication usage or even increased hospital admissions The
lowest observed effect level for this response appears to be in the 0 2- to 0 3-ppm range
Several recent studies have examined the possibility that NO2 could induce a pulmonary
inflammatory response and/or alter immune system host defenses These studies typically
include collection of cells and airways fluids from the lung using BAL In contrast to
O3 exposure, NO2 does not, at the concentrations studied, induce an increase in BAL levels
of neutrophils, or eosinophils, the typical markers of inflammation following O3 exposure
However, Devlin et al (1992) have reported increased PMNs in bronchial washings
Sandstroem et al (1990a) have observed an increase in mast cells and lymphocytes in BAL
fluid, which they attribute to a nonspecific inflammatory response Boushey et al (1988)
have reported an increase in natural killer lymphocytes in BAL fluid Macrophage numbers
have not been increased by NO2 exposure, nor did then ability to kill virus appear to have
been altered by exposure, although Frampton et al (1989a) suggested that, in some subjects,
macrophage responses may have been unpaired Rasmussen et al (1992) observed
indications of a decrease in alveolar permeability after exposure to 2 3 ppm NO2 for 5 h
Mucociliary clearance was not altered after NO2 exposure in the one study in which it was
measured (Rehn et al , 1982) Nitrogen dioxide was found to cause a reduction in alpha-1-
antiprotease activity in one study (Mohsenin and Gee, 1987), but not in another (Johnson
et al, 1990) Following NO2 exposure, Frampton et al (1989b) found an increase in alpha-
2-macroglobulin, a molecule that has immunoregulatory as well as antiprotease activity
Immunological responses to NO2 exposure are just beginning to be elucidated and additional
research will be required to determine whether these responses have any implications for
epidemiologically determined associations between NO2 exposure and increased respiratory
tract infections
The effects of repeated NO2 exposure have been examined in two studies (Sandstroem
et al , 1990b, Boushey et al, 1988) Boushey et al (1988) reported only a slight increase
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(12%) in circulating lymphocytes and a possible increase in natural killer lymphocytes after
four 2-h exposures to 0 60 ppm There were no detectable changes in inflammatory
mediators Sandstroem et al (1990b), on the other hand, found decreased numbers of mast
cells, macrophages, and lymphocytes in the BAL fluid Despite the decreased numbers, the
phagocytic activity of alveolar macrophages was enhanced These observations suggest that
host defense responses are different after repeated exposure than after a single acute
exposure More research appears to be necessary to confirm and expand these observations
because of the important potential connection between altered host defense responses and
increased respiratory infectivity
In healthy adults, a variety of mixtures of other pollutants with NO2 have been
examined, primarily using spirometry and airway resistance measurements as end points
In general, NO2 does not cause significant exacerbation of responses to other pollutants, such
as O3, SO2, or particulate matter In other words, there is no more than an additive
response when NO2 is included in the pollutant mixture However, further investigation of
NO2 mixtures appears warranted using other biological markers, including measures of
epithelial permeability, clearance, airway responsiveness, airway inflammation, and measures
that are sensitive to changes in small airways function In asthmatics, there is a tendency for
increased responsiveness to cold air, methacholme, carbachol, and histamine after NO2
exposure (see previous discussion) In one study, asthmatics were also more responsive to
SO2 after a previous exposure to NO2 (Torres and Magnussen, 1990) In addition to
interactions with other pollutants, NO2 exposure could potentially enhance (or inhibit)
responses to other substances, particularly airborne antigens In two studies (Ahmed et al,
1983a, Orehek et al, 1981), the response to grass pollen inhalation was examined in
sensitive subjects after exposure to 0 1 ppm NO2, but no significant difference in the
response after air and NO2 exposures was observed Given the increase in responsiveness to
nonantigemc substances such as methacholine, histamine, SO2, or cold air discussed
previously, it may be worthwhile to reexamine this hypothesis using higher NO2
concentrations or more prolonged exposures
Responses to other NOX species have also been studied Nitric oxide does not appear
to cause any lung function effects at low concentrations (< 1 0 ppm) either alone (Kagawa,
1982) or combined with NO2 (Kagawa, 1990) Von Nieding et al (1973b) reported
15-92
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increased airways resistance in subjects exposed to excessively high concentrations of NO
(>20 ppm) Responses to HNO3 vapor have been studied in adolescent asthmatics (Koemg
et al , 1989a,b) and m healthy adults (Aris et al , 199Ib, Becker et al, 1992) Further
investigation is needed to examine the responses to HNO3 vapor Nitrates (e g , sodium
nitrate) have not been found to cause any deleterious effects (Utell et al , 1979, 1980,
Kleinman et al , 1980, Stacy et al , 1983) at levels that might be expected in the atmosphere
The following conclusions may be drawn from the studies discussed here
1 Nitrogen dioxide causes decrements in lung function, particularly increased airway
resistance in healthy subjects at concentrations exceeding 2 0 ppm for 2 h
2 Nitrogen dioxide exposure results m increased airway responsiveness in healthy,
nonsmoking subjects exposed to concentrations exceeding 1 0 ppm for exposure
durations of 1 h or longer
3 Nitrogen dioxide exposure at levels above 1 5 ppm may alter numbers and types of
inflammatory cells m the distal airways or alveoli, but these responses depend upon
exposure concentration, duration, and frequency Nitrogen dioxide may alter
function of cells within the lung and production of mediators that may be important
in lung host defenses
4 Nitrogen dioxide exposure of asthmatics causes, in some subjects, increased airway
responsiveness to a variety of provocative mediators, including cholinergic and
histaminergic chemicals, SO2, and cold air Howevei, the presence of these
responses appears to be influenced by the exposure protocol, particularly whether or
not the exposure includes exercise
5 Modest decrements in spirometnc measures of lung function (3 to 8 %) may occur in
some asthmatics and COPD patients under certain NO2 exposure conditions
6 Nitric acid levels in the range of 50 to 200 ppb may cause some pulmonary function
responses in adolescent asthmatics, but not m healthy adults Other commonly
occurring NOX species do not appear to cause any pulmonary function responses at
concentrations expected in the ambient environment, even at higher levels than m
worst-case scenarios However, not all nitrogen oxides acid species have been
studied sufficiently
7 No association between lung function responses and respiratory symptom responses
were observed Furthermore, there is little evidence of a concentration-response
relationship for changes in lung function, airway responsiveness, or symptoms at the
NO2 levels that are reviewed here
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16. HEALTH EFFECTS ASSOCIATED
WITH EXPOSURE TO NITROGEN DIOXIDE
16.1 INTRODUCTION
This chapter concisely summarizes and integrates key information and conclusions from
preceding chapters into a coherent framework or perspective upon which to base
interpretations concerning human health risks posed by ambient or near-ambient levels of
nitrogen dioxide (NO2) in the United States Toward this end, the chapter is organized into
several sections, each of which discusses one or more major components of an overall health
risk evaluation (1) qualitative and quantitative characterization of key health effects of NO2
and their biological bases, (2) identification of population groups potentially at enhanced risk
for health effects associated with NO2 exposure, (3) ambient and indoor NO2 levels and
related exposure aspects, and (4) a summary of NO2 concentration-health effect relationships
16.2 KEY HEALTH EFFECTS OF NITROGEN DIOXIDE
This section concisely discusses two key types of health effects that are of most concern
at ambient or near-ambient concentrations of NO2 (1) increases in airway responsiveness of
asthmatic individuals after short-term exposures, and (2) increased occurrence of respiratory
illness among children associated with longer term exposures to NO2 A third category of
NO2 effects, emphysema, is also discussed but appears to be only of major concern with.
exposures to much higher than ambient levels of NO2
16.2.1 Airway Responsiveness in Asthmatics and Short-Term
(One- to Three-Hour) Exposure to Nitrogen Dioxide
Asthmatics have airway hyperresponsiveness to a variety of chemical and physical
stimuli and are considered to be one of the most NO2-responsive groups in the population
The physiological end point that, to date, appears to be the most sensitive indicator of
response to NO2 in asthmatics is a change in airway responsiveness Airway inhalation
challenge tests are used to evaluate the "responsiveness" of a subject's airways to inhaled
16-1
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materials To test for the degree of airway responsiveness, a pharmacologically active
chemical (such as histamine, methacholine, or carbachol) that causes constriction of the
airways is used Responses are usually measured by evaluating changes in airway resistance
or spirometry after each dose of the challenge is administered Airway hyperresponsiveness
is an abnormal degree of airway narrowing, caused primarily by airway smooth muscle
shortening in response to nonspecific stimuli An extensive discussion of such responses is
presented in Chapter 15
The Expert Panel Report from the U S National Asthma Education Program
(National Institutes of Health, 1991) has recently defined asthma
Asthma is a lung disease with the following characteristics (1) airway
obstruction that is reversible (but not completely so in some patients) either
spontaneously or with treatment, (2) airway inflammation, and (3) increased
airway responsiveness to a variety of stimuli
About 10 million people in the United States, or 4% of the population, have asthma
(National Institutes of Health, 1991) The prevalence is higher among African Americans,
older (8 to 11 years) children, and urban residents (Schwartz et al, 1990) There is a broad
range of seventy of asthma, ranging from mild to severe Common symptoms include
cough, wheezing, shortness of breath, chest tightness, and sputum production A positive
response (skin test) to common inhalant allergens is a typical feature of asthma Asthma is
also associated with airway inflammation and epithelial injury (National Institutes of Health,
1991, Beasley et al, 1989, Laitmen et al, 1985, Wardlaw et al, 1988) Asthma is further
characterized by an exaggerated bronchoconstnctor response to many physical changes
(e.g., cold or dry air, exercise) and to chemical/pharmacologic agents (e g , histamine or
methacholine). The differences in airway responsiveness may span several orders of
magnitude (at least 100-fold) between normal and asthmatic individuals (O'Connor et al ,
1987). Despite the absence of airway hyperresponsiveness in some asthmatics and the
presence of airway hyperresponsiveness in some nonasthmatics (Pattemore et al, 1990),
there is a correlation between increased asthma symptoms or increased medication usage and
increased airway responsiveness (Britton et al, 1988)
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At concentrations below 1 0 ppm NO2, there is little (if any) convincing evidence of
lung function decrements or changes in airway responsiveness in healthy individuals There
is, however, some evidence that acute exposure to NO2 may cause an increase in airway
responsiveness in asthmatics This response has been observed only at relatively low NO2
concentrations, mostly within the range of 0 20 to 0 30 ppm NO2, which is of concern within
the ambient environment Analysis of data on asthmatics experimentally exposed to NO2 in
studies using various challenges produced airway responsiveness increases in 96 subjects and
decreases in 73 subjects (Folinsbee, 1992) In the concentration range between 0 20 and
0 30 ppm, the excess increase in airway responsiveness was attributable to subjects exposed
to NO2 at rest Because NO2 does not appear to cause airway inflammation at these levels
(although modest responses occur at higher [> 1 5-ppni] concentrations) and the increase in
airway responsiveness appears to be fully reversible, the implications of the observed
increase in responsiveness are unclear Although it is conceivable that increased nonspecific
airway responsiveness caused by NO2 could lead to increased responses to a specific antigen,
there is presently no plausible evidence to support this hypothesis On the other hand, it is
possible that persistence of airway hyperresponsiveness may be associated with an accelerated
rate of decline in pulmonary function with age (O'Connor et al, 1987)
An unresolved issue with the current data base is the existence of NO2-induced
pulmonary function changes in asthmatics that have been reported at low, but not at high,
NO2 concentrations Although small changes in spirometry or airway resistance have been
observed in studies from various laboratories, effects are not consistently present and
demonstrating reproducibility of responses has been difficult, even within the same
laboratory Furthermore, most responses to NO2 that have been observed in asthmatics have
occurred at concentrations between 0 2 and 0 5 ppm Changes in lung function or airway
responsiveness have not been observed even at much higher concentrations (i e , up to
4 ppm) There is, at present, no plausible explanation for this apparent lack of a
concentration-response relationship for both airway responsiveness and pulmonary function
changes
In summary, controlled human exposure studies ai e limited to acute, fully reversible
functional and/or symptomatic responses Although it is clear that some asthmatics are more
susceptible than nonasthmatics to NO2, the observed effects do not follow a
16-3
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concentration-response relationship Therefore, the findings do not provide clear quantitative
conclusions about the health effects of short-term exposure to NO2
16.2.2 Respiratory Morbidity in Children Associated with Exposure to
Nitrogen Dioxide
The effects of NO2 on respiratory illness and the factors determining occurrence and
severity are important public health concerns because of the potential for exposure to NO2
and because childhood respiratory illness is very common (Samet et al , 1983, Samet and
Utell, 1990) This takes on added importance because recurrent childhood respiratory illness
may be a risk factor for later susceptibility to lung damage (Glezen, 1989, Samet et al ,
1983, Gold et al, 1989)
The discussion of epidemiological findings in Chapter 14 indicates that the combined
evidence is supportive of an effect of estimated exposure to NO2 on respiratory symptoms
and disease in children aged 5 to 12 years as indicated by the EPA meta-analysis of nine
selected indoor studies presented in Chapter 14 However, in the individual studies of
infants 2 years of age and younger, no consistent relationship was found between estimates of
NO2 exposure and the prevalence of respiratory symptoms and disease Based on a meta-
analyses of these infant studies, the overall combined estimate is positive, however, it clearly
contains the no-effect value of 1 0, (i e , is not statistically significant), and so we cannot
conclude that the evidence suggests an effect in infants
Several uncertainties need to be considered in interpreting the subject indoor air studies
and results of the U S Environmental Protection Agency (EPA) meta-analysis
Measurement error in exposure is potentially one of the most important methodological
problems in epidemiological studies of NO2 (as discussed in more detail in Chapter 14)
Thus measured NO2 concentrations are not exposure values per se, rather, estimating actual
exposure requires knowledge of both pollutant levels and related human activity patterns
The effects studied may be related to peak exposures, average exposures, or a combination of
the two To the extent that health effects depend on peak exposures rather than average
exposures, the exposure estimates used in the above studies and meta-analyses introduce
exposure measurement error These studies cannot distinguish between the relative
contributions of peak and average exposures and their relationship with the observed health
16-4
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effects Additionally, a by-product of NO2, nitrous acid (HONO), may be a factor in
observed effects, however, only very limited health and aerometnc data are available that
examine such possibilities Also, although the level of similarity and common elements
between the outcome measures in the NO2 studies provide some confidence in their use in
the quantitative analysis, the symptoms and illnesses combined are to some extent different
and could indeed reflect different underlying processes Thus, caution is necessary in
interpreting the meta-analysis results
Although there is evidence that suggests that increased estimated NO2 exposure is
associated with increased respiratory symptoms in children aged 5 to 12 years, the exposure
estimates may be inadequate to determine a quantitative relationship between estimated
exposure and symptoms The studies that measured NO^ exposure did so only for periods of
1 to 2 weeks and reported the values as averages None of the studies attempted to relate the
effects seen to the pattern of exposure, such as short-term peaks Furthermore, the
extrapolation to possible patterns of ambient exposure is difficult
As for possible mechanisms underlying NC>2-induction of respiratory morbidity effects
of the types observed to be increased in the above epidemiological studies, animal studies
discussed in Chapter 13 show that NO2 exposure can (1) impaii components of the
respiratory host defense system and (2) increase susceptibility to respiratory infection
Increases in respiratory symptoms and disease among children observed in epidemiologic
studies of NO2 exposure may, therefore, reflect increased susceptibility to respiratory
infection due to NO2 impacts on respiratory defenses The animal toxicology data, as
discussed below, provide a biologically plausible basis for hypothesizing such a relationship,
but the hypothesis requires further testing (see Section 16 2 3)
16.2.3 Biological Bases Relating Nitrogen Dioxide Exposure to Respiratory
Morbidity: Effects of Nitrogen Dioxide on the Respiratory Host
Defense System
The lung is one of the common sites of attack of microorganisms Although many
types of microorganisms are implicated in respiratory infection, viruses represent a major
cause, particularly for infants and children In a viral respiratory infection, viral replication
and altered immune responses to viral infections produce signs and symptoms of respiratory
illness (Douglas, 1986) The respiratory system has several defense mechanisms against
16-5
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inhaled infectious and chemical agents Host defense mechanisms comprise a complex,
cooperative response system of several cell types, cell products, tissues, and organs Two
major approaches (discussed below) have been used to demonstrate the effects of NO2 on
host defenses* (1) evaluation of effects on selected mechanisms of host defenses, and (2) use
of infectivity models, which reflect the overall functioning of all host defense mechanisms
against the infectious agent used
Animal studies provide important evidence indicating that several defense system
components are targets for inhaled NO2, including key elements of host defenses such as
alveolar macrophages (AMs) and the humoral and cell-mediated immune system Animal
toxicological studies (Chapter 13) further show that NO2 exposure can impair the respiratory
host defense system sufficiently so as to result in the host being more susceptible to
respiratory infection Human clinical studies (Chapter 15) of host defenses are rare and their
results are equivocal, but suggestive of the potential for NO2 effects
Although the ciliated epithelial cells involved in mucociliary transport in the conducting
airways exhibit morphological changes at NO2 concentrations as low as 0 5 ppm for 7 mo of
exposure (Yamamoto and Takahashi, 1984), mucociliary clearance is not affected by NO2
q
exposures at <5.0 ppm (9,400 jwg/m ) (Schlesinger et al, 1987) As a foreign agent
deposits below the mucociliary region in the gaseous exchange region of the lung, host
defenses are provided primarily by the AM, which acts to remove or kill viable particles, to
remove nonviable particles, and to process and present antigens to lymphocytes for antibody
production Exposure to NO2 has produced a variety of effects on AMs in several animal
species. For example, Schlesinger et al (1987) and Schlesinger (1987a,b) observed a
decrease in the phagocytic ability of rabbit AMs after a 13-day (2 h/day) exposure to
0.3 ppm (560 jwg/m3) and an increase in phagocytosis after 2 days of exposure to 1 0 ppm
*2
(1,880 /ig/m ) Additional effects observed at higher concentrations (e g , between 0 5 and
5 ppm) include decreased pulmonary bactericidal activity, altered metabolism, increases in
numbers of macrophages, and morphological changes (Rombout et al, 1986, Aranyi et al ,
1976; Goldstein et al, 1974, Suzuki et al, 1986, Chang et al, 1986, Mochitate et al, 1986,
Robison et al, 1990) Decreases in the ability of AMs to engulf foreign particles
(phagocytosis) and bactericidal activity are likely highly related to increased susceptibility to
pulmonary infections Controlled human exposure studies (0 6 ppm for 3 h) have also
16-6
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examined AM function and show that these cells, when exposed to NO2, tended (p = 0 07)
to inactivate influenza virus in vitro less effectively than cells collected after air exposure
(Frampton et al , 1989a) Also Devlin et al (1992) reported that macrophages recovered
from the predominantly alveolar fraction of bronchoalveolar lavage fluid showed a 42 %
decrease in ability to phagocytose Candida albicans
Together, the humoral and cell-mediated immune systems are essential for antibody
production and the secretion of cellular products that (1) regulate normal defense responses
and/or (2) are lethal to certain invading organisms Although the pulmonary immune system
would better reflect defenses against respiratory infection, it has not been adequately studied
after NO2 exposure However, there is some indication that exposure to NO2 suppresses
some of the systemic immune responses and that the effects are both concentration- and tune-
dependent For example, a significant suppression of antibody production by spleen cells has
been reported in experimental animals exposed for 1 mo to NO2 concentrations as low as
0 4 ppm (Fujunaki et al, 1982) Subchronic exposure (7 weeks) to NO2 also resulted in
decreased numbers of circulating T lymphocytes, T-helper/inducer lymphocytes, and
3
T-cytotoxic/suppressor lymphocytes in mice at NO2 levels as low as 0 25 ppm (470 jwg/m )
(Richters and Damji, 1988) The cause of this suppression is not clear
Animal infectivity studies present key data relating NO2 exposure to effects on the
overall functioning of host defense mechanisms In these studies, animals were exposed to
varying concentrations and durations of NO2, followed by exposure to an aerosol containing
an infectious agent Microbially induced mortality was used as the health end point
Exposure to NO2 mcreased both bacteria- and influenza-induced mortality after subchronic
exposures to levels as low as 0 5 to 1 0 ppm NO2 (Ehrlich and Henry, 1968, Ito, 1971,
Ehrkch et al, 1977) After acute (2-h) exposure, 2 0 ppm NO2 has been the lowest effective
concentration measured using the bacterial infectivity model (Ehrlich et al , 1977) Nitrogen
dioxide increases microbially induced mortality by impairing the host's ability to defend the
respiratory tract from infectious agents, thereby increasing susceptibility to viral,
mycoplasma, and bacterial infections (Ehrlich and Henry, 1968, Ito, 1971, Ehrlich et al,
1977, Parker et al, 1989, Gardner et al, 1977a,b, 1979, 1980, 1982, Graham et al, 1987,
Jakab, 1987a,b, Motomiya et al , 1973, Miller et al , 1987) Using an animal model
designed to evaluate the effects of NO2 on nonfatal respiratory infection, NO2 decreased the
16-7
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intrapulmonary bactericidal activity in mice in a concentration-related manner, without a
change hi mucociliary clearance (Goldstein et al, 1973) Exposure to NO2 was found to
increase the seventy of mycoplasma-induced lesions within the lung, but did not increase the
susceptibility of the mice to the infection (Parker et al , 1989) Animal studies have also
shown that influenza infection is exacerbated with NO2 exposure (Ito, 1971) In studies with
cytomegalovirus and paramyxovirus (Jakab, 1987a,b, Rose et al, 1988), the pathogenesis of
these infections was enhanced
The animal toxicology literature also provides evidence that the host's response to
inhaled NO2 can be significantly influenced by the exposure duration, concentration, and
temporal pattern of exposure The relationship of concentration (C) times duration (T, time)
to susceptibility to respiratory infections indicates that, when the product of C x T is held
constant and the individual C's and T's are varied, a difference in response occurs The
incidence of mortality was significantly more influenced by the concentration of NO2 than by
the duration of the exposure (Gardner et al, 1977a,b) The exposure pattern of NO2 is also
important when comparing and determining the effects of continuous versus intermittent
exposure. When such data were adjusted for differences in C x T, the incidence of
respiratory infection was essentially the same for both groups (Gardner et al , 1979) When
animal studies were designed to mimic a typical urban outdoor exposure environment having
periodic spikes of NO2 superimposed on a lower continuous background level of NO2, the
evidence indicates that the animals exposed to the baseline plus short-term spikes were
significantly more susceptible to a laboratory-induced infection than either the control or the
background-NO2-exposed mice (Miller et al , 1987, Gardner et al , 1982, Graham et al ,
1987). It should be noted that the exposure patterns tested in animals are likely to be
different from indoor NO2 exposure patterns This body of work for host defenses in mice
shows that an average exposure value (C x T) is not an exact index or predictor of effects,
rather, actual patterns of exposure more accurately represent the causative exposure
It is also of interest that morphological studies, too, indicate that NO2 concentrations
play a more important role in inducing lung lesions than do exposure durations when the
product of C X T is constant (Rombout et al, 1986) The influence of concentration was
greater with intermittent NO2 exposure than with continuous exposure
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Recent controlled human exposure studies examining the effects of NO2 on pulmonary
host defense systems have reported a trend (not statistically significant) toward an elevated
rate of infection by a laboratory-induced, live attenuated influenza (A/Korea/reassortment)
virus (Goings et al , 1989) Frampton et al (1989a) also reported a trend (p < 0 07) for
less effective inactivation of virus by AMs obtained from human subjects exposed
continuously to 0 60 ppm NO2, but no effects of virus inactivation were seen in subjects
exposed continuously to 0 05 ppm with 2 0-ppm spikes Exposure to NO2 may transiently
increase levels of antiprotease alpha-2-macroglobulin (o^M) in lung lavage fluid Although
serving as an indicator of changes in the protease-antiprotease balance, alterations in a2M in
alveoli may have significance for local immunoregulation and may alter AM defenses against
infection (Frampton et al, 1989b) These findings suggest, but do not prove, that NO2 may
play a role in increasing the susceptibility of adults to respiratory virus infections
There is a hypothesis that the epidemiological associations between NO2 exposure and
respiratory symptoms/disease represent increased risk for respiratory infection The weight
of the evidence from several animal toxicological and human clinical studies, as summarized
above, shows that NO2 decreases host defense mechanisms against bacterial and viral
infections Some of these host defense studies are on mechanisms active in humans (e g ,
AM functions) Other studies are on net defense functioning using outcome measures not
valid for "humans (i e , mortality in the infectivity model), but nonetheless involving
mechanisms shared with humans Thus, these animal and human clinical studies provide a
biologically plausible basis for the hypothesis However, to test the hypothesis, it would be
necessary to perform additional animal toxicological studies, to conduct more diagnostic tests
for infection in future epidemiological studies, and to apply additional approaches in
controlled human exposure studies
16.2.4 Emphysema and Exposure to Nitrogen Dioxide
Studies on several animal species have shown that chronic exposure to high NO2 levels
(relative to ambient) can cause emphysema Because emphysema is an irreversible disease,
representing an important public health concern, whether NO2 creates a risk for this disease
in humans is a major question Although this question cannot be definitely answered yet, the
potential for risk warrants discussion here The definition of emphysema as used in the
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United States is an anatomic one best characterized by National Institutes of Health (NIH)
(1985) criteria "An animal model of emphysema is defined as an abnormal state of the
lungs in which there is enlargement of the airspace distal to the terminal bronchiole
Airspace enlargement should be determined qualitatively in appropriate specimens and
quantitatively by stereologic methods " An additional essential criterion for human
emphysema is the destruction of alveolar walls
Several studies (Haydon et al, 1967, Freeman et al, 1972, Port et al, 1977) relate
long-term (1- to more than 30-mo) exposure of rats and rabbits to high concentrations of
NO2 (> 8 ppm, much greater than ambient levels) with morphologic lung lesions that meet
the 1985 NIH workshop criteria for a human model of emphysema (i e , alveolar wall
destruction occurred in addition to other characteristic changes) One study (Hyde et al,
1978) reported on dogs exposed to a mixture of 0 64 ppm NO2 and 0 25 ppm nitric oxide
(NO) for 68 mo Upon examination 32 to 36 mo after exposure ceased, the dogs had
morphologic lesions that meet the 1985 NIH workshop criteria for human emphysema
In the same dogs, pulmonary function was also measured Pulmonary function decrements
observed at the end of exposure progressed postexposure This suggests that the
morphological effects may also have been progressive Another group of dogs in the same
study was exposed to a mixture of "low" NO2 (0 14 ppm) and "high" NO (1 1 ppm), but
emphysema was not observed Because the study did not include an NO2-only group, it is
not possible to discern the effects of NO2 in the mixture However, the presence of
emphysema in the "high" NO2-"low" NO group and its absence in the "low" NO2-"high"
NO group implies that NO2 was a significant etiologic factor
Emphysema was reportedly observed in numerous other NO2 studies with several
species of animals, but either the reports lacked sufficient detail for independent conclusions
to be drawn or only the criteria for animal (not human) emphysema were met Several other
studies discussed in Chapter 13 were negative for emphysema Various factors such as the
exposure protocol and morphologic methods may also play a role in the outcome of studies
Potential differences may relate to the animal species used, age of the animals during
exposure, concentration and duration of exposure, and the duration after exposure ceases
before the animals are evaluated for emphysematous pathology
16-10
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In spite of the fact that there is a fairly extensive toxicologic data base concerning
morphologic effects of NO2, it is still not possible to establish a reasonably accurate
"no-observed-effect" level for emphysema This is likely due to a combination of factors.
the complexity of changes occurring with NO2 exposure, the lack of published papers
utilizing highly sensitive morphometnc techniques, interspecies differences in response, and
inadequate description of methods and findings in some published reports Qualitatively,
then, it is clear that NO2 can cause emphysema in animals. However, although the lowest
effective NO2 concentrations/exposure durations that induce emphysematous lung lesions can
not yet be determined from available studies, the NO2 exposures that have been found to
cause emphysema (according to the NIH criteria) are fai higher than those currently reported
in ambient air
16.3 CONCENTRATION-RESPONSE RELATIONSHIPS: HEALTH
EFFECTS OF EXPOSURE TO NITROGEN DIOXIDE
16.3.1 Clinical Studies
Table 16-1 summarizes key health effects observed in controlled human exposure
(clinical) studies with NO2 exposure durations of 0 5 to 3 h The physiological end point
that, to date, appears to be the most sensitive indicator of response is a change in airway
responsiveness to bronchoconstactors in asthmatics This increase in airway responsiveness
has been observed in some, but not all studies, and only at relatively low NO2 concentrations
within the range 0 2 to 0 3 ppm Additionally, small decreases in functional expiratory
volume in 1 s (FEVj) or forced vital capacity (FVC) in adult or adolescent asthmatics have
been observed in response to the same levels of NO2 However, NO2 concentration-response
relationships are not evident for either airway responsiveness or pulmonary function changes
A second category of sensitive subjects are patients with chronic obstructive pulmonary
disease (COPD) Although small decreases have been observed in FVC and FEV1 in COPD
patients exposed to 0 3 ppm in one study, no effects were seen in other studies at higher
exposure levels At higher exposure levels (more than 1 5 ppm), NO2 exposure results in
increased airway responsiveness and increased airway resistance in healthy adults However,
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TABLE 16-1. KEY HUMAN HEALTH EFFECTS OF EXPOSURE TO
NITROGEN DIOXIDE—CLINICAL STUDIES8
NO2 (ppm)
(Exposure Duration) Observed Effects References
0 2-0 3 (0 5-2 0 h)
0 3 (3 75 h)
1 5-2 0 (2-3 h)
5:2 00 (1-3 h)
Trend toward increased airway
responsiveness to challenges in asthmatics
(Fohnsbee, 1992) However, no
significant effects observed by same or
other investigators at NC>2 levels up to
4 ppm Small (4-6%) decreases in FEVj
or FVC in adult or adolescent asthmatics,
in response to NO2 alone
Small decreases (5-9%) in FVC and
FEVt in COPD patients with mild
exercise No effects seen by other
investigators for COPD patients at
0 5-2 0 ppm NO2
Increased airway responsiveness to
bronchoconstnctors in healthy adults
However, effects not detected by other
investigators at 2-4 ppm
Lung function changes (e g , increased
airway resistance) in healthy subjects
Effects not found by others at 2-4 ppm
Klemman et al (1983)
Bauer et al (1986a,b)
Koemgetal (1988)
Bylin et al (1985, 1988)
Mohsenin (1987a)
Torres and Magnussen (1990)
Morrow and Utell (1989)
Mohsenin (1987b)
Framptonetal (1991)
Bed and Ulmer (1976)
Von Nieding et al (1979)
Von Nieding and Wagner (1977)
Von Nieding et al (1980)
NO2 = Nitrogen dioxide
FEVj = Functional expiratory volume in 1 s
FVC = Forced vital capacity
COPD — Chronic obstructive pulmonary disease
some researchers have not observed any NO2-induced changes in airway resistance at NO2
levels between 2 and 4 ppm
16.3.2 Epidemiological Studies
The collective, combined evidence from epidemiology studies examining relationships
between estimates of exposure to NO2 and lower respiratory symptoms and disease in
children aged 5 to 12 years (as evaluated by an EPA meta-analysis yielding quantitative
16-12
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estimates of effects) tends to demonstrate that increased risk for respiratory illness among
children is associated with exposure to NO2s as summarized in Table 16-2 In individual
indoor studies of infants 2 years of age and younger, no consistent relationship was found
between estimates of NO2 exposure and the prevalence of respiratory symptoms and disease
Based on a meta-analyses of these infant studies, the combined odds ratio for the increase in
respiratory disease per increase of 0 015 ppm NO2 was 1 09 with a 95 % confidence interval
of 0 95 to 1 26 Thus, although the overall combined estimate is positive, it clearly contains
the no-effect value of 1 0, (i e , is not statistically significant), and so we cannot conclude
that the evidence suggests an effect in infants comparable to that seen in older children (see
Table 16-2) Higher levels (>0 3 ppm during a shift at work) in an occupational setting
were related to an elevated prevalence of acute respiratory symptoms in adults Also,
episodic exposures occurring over a period of 1-h or longer at levels possibly as high as
1 5 ppm or higher have resulted in the occurrence of acute respiratory symptoms Lastly,
exceptionally high acute occupational exposures of 25 to 100 ppm NO2 result in bronchial
pneumonia, bronchitis, or bronchiolitis, and very extreme occupational NO2 exposures
(>200 ppm) have been associated with effects that range from hypoxemia and transient
obstruction of the airways to death
16.3.3 Animal Toxicological Studies
Numerous concentration-response studies have been conducted with animals using a
wide range of exposure durations and end points, all of which influence the outcome The
major classes of effects observed at concentrations less than 1 0 ppm include decrements in
host defenses, alterations in lung metabolism (e g , increased lipid peroxidation and
antioxidant metabolism), epithelial remodeling of the lower respiratory tract, thickening of
the centnacinar interstitium, and a variety of extrapulmonary changes Such findings can be
qualitatively extrapolated to humans, but major uncertainties in respiratory tract dosimetry
and species sensitivity currently preclude a quantitative extrapolation Substantially higher
NO2 concentrations ( > 12 ppm) have caused emphysema as defined by NTH criteria
In infectivity studies examining C x T and pattern of exposure, concentration had more
influence than tune of exposure in increasing susceptibility to respiratory bacterial infection
in mice Furthermore, the exact pattern of exposure played a major role in experimental
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TABLE 16-2. KEY HUMAN HEALTH EFFECTS OF EXPOSURE TO
NITROGEN DIOXIDE—EPIDEMIOLOGICAL STUDIES
NO2 (ppm)
(Exposure Duration)
Observed Effects
References
0 015-ppm increase, where mean
weekly concentrations in
bedrooms in studies reporting
levels were predominately
between 0 008 and 0 065 ppm
NO2 (in 1- and 2-week integrated
average NO2 concentration
estimating an unspecified long-
term average)
0 015-ppm increase in annual
average of 2-week NO2 levels,
where mean weekly
concentrations in bedrooms were
predominately between 0 005 and
0 050 ppm NO2
A meta-analysis shows increased risk of lower
respiratory symptoms/disease m children 5 to
12 years old associated with exposure estimates
of NO2 levels The 95 % confidence interval of
the odds ratio estimated by Hasselblad et al
(1992) was 1 1 to 1 3 Predominant source of
exposure contrast is homes with gas stoves vs
homes with electric stoves
In individual indoor studies of infants 2 years of
age and younger, no consistent relationship was
found between estimates of NO2 exposure and
the prevalence of respiratory symptoms and
disease Based on a meta-analyses of these
infant studies, the combined odds ratio for the
increase in respiratory disease per increase of
0 015 ppm NO2 was 1 09 with a 95% confidence
interval of 0 95 to 1 26 Thus, although the
overall combined estimate is positive, it clearly
contains the no-effect value of 1 0, (i e , is not
statistically significant), and so we cannot
conclude that the evidence suggests an effect in
infants comparable to that seen in older children
(see Chapter 14)
Meliaetal (1977,
1979, 1980, 1982)
Wareetal (1984)
Neas et al (1991)
Ekwoetal (1983)
Dykstraetal (1990)
Keller et al (1979)
Samet et al (1993)
Margohsetal (1992)
Dockeryetal (1989)
Ogstonetal (1985)
Wareetal (1984)
Ekwoetal (1983)
Meliaetal (1983)
>0.3 ppm (average exposure
during work shift)
Elevated prevalence of acute respiratory
symptoms
Gamble et al (1987)
Episodic exposure during hockey
game to NO2 levels of 1 5 ppm
or higher
Occurrence of acute respiratory symptoms
(cough, chest pain, dyspnea)
Smith etal (1992)
Hedbergetal (1989)
25 to 100 ppm (episodic
occupational exposure)
Bronchial pneumonia, bronchitis, and
bronchiolitis induced by exceptionally high NO2
exposure
Grayson (1956)
>200 ppm (extreme episodic
exposures)
Extreme exposure health outcomes range from
hypoxerma/transient airway obstruction to death
Douglas etal (1989)
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outcomes Even so, duration is still important For example, as exposure proceeds from
weeks to months at a given concentration, structural changes in the lung become more
severe Also, at longer exposure durations, lower NO2 concentrations cause effects Due to
the large number of animal toxicological studies and the variety of exposure regimes, it is not
possible to succinctly display the full range of concentration-responses Therefore
Table 16-3 lists a few key studies showing the lowest concentrations that caused several types
of effects.
16.4 SUBPOPULATIONS POTENTIALLY AT RISK FOR NITROGEN
DIOXIDE HEALTH EFFECTS
Certain groups within the population may be more susceptible to the effects of NO2
exposure, including persons with preexisting respiratory disease, children, and the elderly
The reasons for paying special attention to these groups is that (1) they may be affected by
lower levels of NO2 than other subpopulations and (2) the impact of an effect of given
magnitude may be greater Some causes of heightened susceptibility are better understood
than others Subpopulations that already have reduced ventilatory reserves (e g , the elderly
and persons with asthma, emphysema, and chronic bronchitis) will be more impacted than
other groups by decrements in pulmonary function For example, a healthy young person
may not even notice a small percentage change in pulmonary function, but a person whose
activities are already limited by reduced lung function may not have the reserve to
compensate for the same percentage change
The National Institutes of Health (1991) estimates that approximately 10 million persons
in the United States have asthma In the general population, asthma prevalence rates
increased by 29% from 1980 to 1987 For those under 20 years old, asthma rates increased
from approximately 35 to 50 per 1,000 persons, a 45% increase The airways of asthmatics
may be hyperresponsive to a variety of inhaled materials, including pollens, cold-dry air,
allergens, and air pollutants Asthmatics have the potential to be among the most susceptible
members of the population with regard to respiratory responses to NO2 (Section 15 3 1)
On the average, asthmatics are much more sensitive to inhaled bronchoconstrictors such as
histamine, methacholine, or carbachol The potential addition of an NO2-induced increase in
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TABLE 16-3. KEY ANIMAL TOXICOLOGICAL EFFECTS OF EXPOSURE TO
NITROGEN DIOXIDE3
NO2 (ppm)
(Exposure Duration) Species Observed Effects References
Rat
Mouse
Rabbit
0.04 ppm
(continuous, 9 mo)
0 2 ppm (continuous Mouse
base for 1 year) plus
0 8 ppm (1-h peak,
2X/day,
5 days/week)
0 25 ppm
(7 h/day,
5 days/week,
7 weeks)
0.3 ppm
(2 h/day, 2 days)
0 4 ppm (continuous, Mouse
4 weeks)
0.4 ppm (continuous, Rat
9 mo)
0 4 ppm (continuous, Rat
up to 27 mo)
0.5 ppm (continuous, Mouse
3 mo)
0 5-28 ppm (6 mm Mouse
to 1 year)
Increased lipid peroxidation (ethane
in exhaled breath)
Increased susceptibility to respiratory
infection and decreased vital capacity
and respiratory system compliance,
compared to control or baseline only
Systemic effect on cell-mediated
immunity
Sagai et al (1984)
Miller etal (1987)
Richters and Damji (1988, 1990)
Decreased phagocytosis of alveolar Schlesinger (1987a,b)
macrophages
Decreased systemic humoral immunity Fujimaki et al , (1982)
Increased antioxidants and antioxidant Sagai et al (1984)
metabolism
Slight increase in thickness of air-blood Kubota et al (1987)
barrier at 18 mo, becoming significant
by 27 mo, also alterations in bronchiolar
and alveolar epithelium by 27 mo
Increased susceptibility to respiratory Ehrlich and Henry (1968)
infection
0 5 ppm (continuous
base, 6 weeks) plus
1 5 ppm (1-h peak,
2X/day,
5 days/week)
Rat
Linear increase in susceptibility to
respiratory infection with time,
increased slope of curve with increased
concentration, C more important than T
Alterations in Type 2 cells and increased Crapo et al
Gardner et al (1977a,b)
Coffin etal (1977)
(1984)
interstitial matrix of proximal alveolar
region, no changes in terminal
bronchiolar region of adults
Chang et al (1986, 1988)
NO2 = Nitrogen dioxide
C = Concentration of exposure
T = Duration (time) of exposure
airway response to the already heightened responsiveness to other substances raises the
possibility of exacerbation of this pulmonary disease by NO2, as discussed in Section 15 4
Other potentially susceptible groups include patients with COPD, such as emphysema
and chronic bronchitis Some of these patients have airway hyperresponsiveness to physical
and chemical stimuli A major concern with COPD patients is the absence of an adequate
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ventilatory reserve, a susceptibility factor described abo\e In addition, the poor distribution
of respiratory tract ventilation in COPD may lead to a greater delivery of NO2 to the
segment of the lung that is well ventilated, thus resulting in a greater regional tissue dose
Also, NO2 exposure may alter already unpaired defense mechanisms, making this population
potentially susceptible to respiratory infection It is estimated (U S Department of Health
and Human Services, 1990, Collins, 1988) that 14 million persons («6%) suffer from
COPD in the United States
Because more than 2 million Americans have emphysema, it would be important to
know whether NO2 has the potential to exacerbate the disease Lafuma et al (1987) exposed
both normal hamsters and hamsters with laboratory-induced (with elastase) emphysema to
2 0 ppm NO2 for 8 h/day, 5 days/week for 8 weeks Nitrogen dioxide exposure appeared to
aggravate the elastase-induced emphysematous lesion The investigators suggested that the
results may imply a role for NO2 in enhancing preexisting emphysema In contrast,
Mauderly et al (1989, 1990) found that when elastase-induced emphysematous rats were
chronically (7 h/day, 5 days/week, 2 years) exposed to 9 5 ppm NO2, they were not more
susceptible to NO2 in terms of exacerbation of the elastase-induced lesions Therefore, it is
not clear what the potential would be for exacerbation oi emphysema in humans at ambient
concentrations
Based upon epidemiology studies, children aged 5 to 12 years constitute a subpopulation
potentially susceptible to an increase in respiratory morbidity associated with NO2 exposure
(Chapter 14) Data on the resident population of the United States provide information on
the number of children in various age ranges (Table 16-4) Approximately 18 million
children are in the age group 5 to 9 years, whereas around 17 million children are in the age
group 10 to 14 years However, the fraction of the numbers of potentially-at-nsk children in
various age groups that are actually exposed to NO2 concentrations/patterns sufficient to
induce respiratory morbidity has not been determined
Another potential susceptible subpopulation group is immunocompromised individuals,
who would have an increased susceptibility for infectious pulmonary disease as well as other
health effects Such people would hypothetically be more susceptible to agents, such as
NO2, that further compromise host defenses Immunocompromised groups could include
those people with abnormalities in polymorphonuclear leukocyte (PMN) number or function
16-17
-------
TABLE 16-4. ESTIMATES OF THE RESIDENT POPULATION OF
CHILDREN AND YOUNG ADULTS OF THE UNITED STATES,
BY AGE AND SEX, JULY 1, 1989
Age
All Ages
<1
Ito4
5 to 9
10 to 14
15 to 19
Total
248,239,000
3,945,000
14,808,000
18,212,000
16,950,000
17,812,000
Population
Male
120,982,000
2,020,000
7,578,000
9,321,000
8,689,000
9,091,000
Female
127,258,000
1,925,000
7,229,000
8,891,000
8,260,000
8,721,000
Source Centers for Disease Control (1990)
and those with humoral and/or cell-mediated immunity dysfunctions Hopewell (1989)
discusses potentially immunocompromised groups in general, without regard to pollutant
exposures Reduction in the number of circulating PMNs is particularly common in patients
undergoing chemotherapy for malignancies, in patients with acute leukemia, and in patients
who have had bone marrow transplantation Also, the use of corticosteroids, antmeoplastic
drugs, irradiation, and alcohol can decrease the effectiveness of PMNs by decreasing
chemotaxis or adherence In addition, alterations in PMN chemotaxis have been described in
cirrhosis, renal failure, and Hodgkm's disease
Antibody production is primarily a function of B lymphocytes, thus decreased antibody
production occurs when numbers of B cells are critically reduced or when the ability to
respond to specific antigens is unpaired Conditions associated with defective antibody
production include patients (1) infected with the human immunodeficiency virus (HIV),
(2) with multiple myeloma, (3) with chronic lymphocytic leukemia, and (4) in the
splenectomized state
Cell-mediated immunity is mainly responsible for fending off intracellular pathogens
and neoplastic disease Killing of microbes within the lung is accomplished primarily by
AMs and secondarily by various T lymphocytes Abnormalities of cell-mediated immunity
are produced by various diseases and include acquired immune deficiency syndrome (AIDS)
and untreated Hodgkm's disease Also, therapeutic interventions that cause defects of cell-
16-18
-------
mediated immunity are radiation treatment, corticosteroids, azathiopnne, and
cytotoxic/immunosuppressive drugs
The number of people with reduced immune function related to kidney transplants,
AIDS, and chemotherapy can be estimated The U S Bureau of the Census (1991) indicates
that, based on reports of procurement programs and transplant centers m the United States,
approximately 8,890 kidney transplant procedures were done in 1989 Also, Karon et al
(1990) reported that the Centers for Disease Control (CDC) estimated in 1990 that
(1) approximately 1 million persons m the United States were then currently infected with
HIV and (2) an estimated 52,000 to 57,000 cases of AIDS were expected to be diagnosed
during 1990 As of October 1991, state and local health departments had reported to the
CDC 196,034 AIDS cases among persons of all ages in the United States (Centers for
Disease Control, 1992) As for potentially at-nsk chemotherapy patients, in 1990,
approximately 1 million new cases of cancer occurred m the United States (U S. Bureau of
the Census, 1991) Steele et al (1991) estimate that about 25% of the total number of
patients diagnosed with cancer m 1 year are prescribed chemotherapy as a first course of
treatment In addition, many cancer patients are treated by radiation
Although the above immunocompromised groups represent potentially at-nsk
susceptible populations for NO2 effects, no human research has examined NO2 exposure m
these groups Thus, there only now exists a hypothesized association with increased
susceptibility to NO2 Although it is clear that NO2 can affect AMs, humoral immunity, and
cell mediated immunity in otherwise normal animals (Chapter 13), the animal-to-human
extrapolation cannot yet be made quantitatively Nevertheless, it may be prudent to consider
including such reduced immune function groups as susceptible subpopulations at potentially
increased risk for NO2-induced health effects
16.5 NITROGEN DIOXIDE LEVELS, EXPOSURES, AND ESTIMATES
16.5.1 Ambient and Indoor Nitrogen Dioxide Levels
In urban areas, hourly NO2 patterns at fixed-site, ambient air monitors often show a
bunodal pattern of morning and evening peaks, related to motor vehicular traffic patterns,
superimposed on a lower baseline level Sites affected by large stationary sources of NO2
16-19
-------
(or NO that rapidly converts to NO2) are often characterized by short episodes at relatively
high concentrations Electric generating stations provide a source of NO2 in such rural and
urban areas Occurrences of hourly average NO2 concentrations >0 10 ppm are very
infrequent In fact, only about 5% of hourly average NO2 concentrations exceed
^:0 05 ppm (Aerometnc Information Retrieval System, 1992, U S Environmental Protection
Agency, 1991a,b)
The highest hourly and annual ambient NO2 levels reported are from monitoring
stations in California The seasonal patterns at California stations are usually quite marked
and reach their highest levels through the fall and winter months, whereas stations elsewhere
in the United States usually have less prominent seasonal patterns and may peak in the
winter, in the summer, or contain little discernable variation One-hour NO2 values can
exceed 0.2 ppm, but, in 1988, only 16 stations (12 in California) reported an apparently
credible second high 1-h value greater than 0 2 ppm Because at least 98 % of 1-h values at
most stations are below 0 1 ppm, such values above 0 2 ppm are quite rare excursions
Thus, ambient 1-h values of 0 1 ppm are more typical 1-h maximums (Aerometnc
Information Retrieval System, 1992, U S Environmental Protection Agency, 1991a,b)
Since 1980, the U S nationwide mean annual-average level among reporting NO2
stations has been consistently below 0 03 ppm For the period 1980 to 1990, there were
indications of a downward trend for the composite annual-average NO2 concentration
(U.S. Environmental Protection Agency, 1991b) The 1990 composite NO2 average was 8%
less than the 1981 level, a statistically significant difference For 103 Metropolitan Statistical
Areas reporting a valid year's data for at least one station in 1988, 1989, and 1990, annual
averages ranged from 0 007 to 0 061 ppm The collective mode for the peak annual average
hi the 1988 and 1989 period was approximately 0 02 ppm The only recently measured
exceedances of the current 0 053 ppm NO2 annual National Ambient Air Quality Standard
occurred at stations in Southern California (Aerometnc Information Retrieval System, 1992,
U.S. Environmental Protection Agency, 1991a,b)
Most people, however, spend a significant portion of their time indoors This can
result in increased NO2 exposure, depending on the presence and use of indoor sources
(e.g., gas stoves, kerosene heaters, and unvented gas space heaters) or reduced exposure,
depending on the absence of such sources and on the tightness of home construction and the
16-20
-------
use of air conditioning and other building features that affect the degree of penetration of
outdoor NO2 into buildings Harlos et al (1987a) report maximum kitchen NO2 values for
1-h of 0 419 ppm during gas stove use, and a mean maximum of 0 182 ppm averaged over
the 4-h sampling period
Several studies have examined the issue of human exposure to NO2 and the relationship
of indoor/outdoor air quality for occupants of homes with and without significant indoor
sources of NO2 (Quackenboss et al , 1986, Sexton et al , 1983, Colome et al, 1987,
Leaderer et al , 1987) Quackenboss et al (1986) found that in the winter in Portage, WI,
indoor weekly average NO2 concentrations were 3 2 times higher than outdoor NO2 levels in
gas stove homes, while indoor levels were 0 6 tunes outdoor NO2 levels in electric stove
homes during the same period The fact that indoor NO2 levels in electric stove homes were
below measured outdoor levels may be due to chemical reactions of NO2 with indoor
surfaces Given the large amount of tune spent at home by most subjects, Quackenboss
et al (1986) found relatively high correlations between weekly measurements of indoor NO2
concentrations and total personal exposure in gas stove homes (r = 0 85 for summer and
0 87 for winter) and to a lesser extent in electric stove homes (r = 0 68 for summer and
0 61 for winter) Correlation between outdoor NO2 levels and total personal exposure is less
in the summer (r = 0 55 for gas stove homes and r = 0 68 for electric stove homes) and
much lower in the winter (r = 0 20 and 0 28, respectively) Another factor that would
affect total exposure is respiratory ventilation rate, whic h may differ indoors and outdoors
depending on levels and patterns of human activities
Colome et al (1987), in a study of over 600 randomly sampled residences in Southern
California, report that outdoor concentrations of NO2 are found to be the single most
important determinant of average indoor levels of NO2 in Southern California Outdoor NO2
levels accounted for between 15 to 40% of the variation in indoor concentrations in this
study Based on the regression analysis of data from multiple homes, indoor/outdoor ratios
varied from 0 46 to 1 00, depending on the season of the year
It is remarkable that the contribution of gas cooking to indoor NO2 levels is as highly
consistent as it is among studies for locations (kitchens, bedrooms, activity rooms) within the
residences and by season, given the great variability of the factors that govern the emissions
(source type, source condition, source use, and source venting) and dilution and removal of
16-21
-------
NO2 indoors (house volume, infiltration, etc ) This consistency is not observed until the
impact of outdoor concentrations is corrected for because background levels can vary
considerably over tune and geographic area The impact of gas cooking and possibly other
unvented or improperly vented combustion sources on indoor NO2 levels is superimposed
upon the indoor background level resulting from outdoor levels In areas where outdoor
levels are low, concentrations indoors from gas appliances will be higher than, and in many
cases, much higher than outdoor levels (e g , Marbury et al, 1988, Quackenboss et al ,
1987, 1988, Spengler et al, 1983, Leaderer et al, 1986, Ryan et al, 1988a,b) If outdoor
concentrations are high, then indoor levels in homes with gas appliances can be closer to or
even lower than the outdoor levels (Wilson et al, 1986)
A by-product of NO2, HONO, may be a factor contributing to observed health effects,
however, no health data and limited exposure data are available to enable further evaluation
of this possibility (see Chapter 7) Brauer et al (1991) suggested that HONO is potentially
less likely to be neutralized by ammonia than other acids Additionally, HONO may be
absorbed at a higher rate in the pulmonary tract than is NO2 due to its greater solubility
The contrast in both mean and hourly peak values between NO2 and HONO (e g , respective
peak and mean levels of 0 280 ppm and 0 069 ppm for NO2 versus 0 029 ppm and
0.019 ppm for HONO), as indicated by data from Brauer et al (1990), suggests that for
HONO to be a significant risk factor, it would have to be more toxicologically potent than
NO2 (Neas et al, 1991).
16.5.2 Patterns of Potential Exposure to Nitrogen Dioxide and Related
Health Effects
Relationships between estimates of NO2 exposure and increased respiratory symptoms
and diseases have been observed in epidemiological studies of children aged 5 to 12 years
(see Chapter 14) Actual exposure would optimally be measured with personal exposure
monitors with rather bnef averaging tunes, but this is not yet technically feasible Even so,
delivered dose is responsible for effects Although dose has an obvious broad relationship to
exposure, it is very significantly influenced by ventilation rates/patterns and respiratory tract
anatomy For example, even in the same microenvironment, a sedentary child would receive
a different dose than a sedentary adult, and exercise would result in still different doses
16-22
-------
Lacking such precise estimates of dose, it is necessary 1o use available exposure estimates
Here exposure is defined as the combination of an individual's average NO2 exposure
concentration over tune for all settings or environments Average indoor residential
concentrations (e g , whole-house average or bedroom level) tend to be the best predictors of
personal NO2 exposure (see Chapter 8) Exposure was estimated in the NO2 epidemiological
studies by either direct measure of pollutant levels or by use of the surrogate measure
provided by differences in NO2 levels observed in homes with gas cookers and homes with
electric stoves The concentration measures for both aie typically derived from 1- to 2-week
averages as determined by Palmes tubes
Measuring NO2 levels averaged over 1 to 2 weeks presents two concepts that require
discussion First, how does such a measure provide data on discerning the hypothesized
difference between two patterns of exposure and their potentially different relationships to
observed health effects, and, second, how adequately does such a measure estimate long-term
exposures
16.5.2.1 Patterns of Exposure
Exposure to NO2 can be characterized by different patterns For example, one
exposure pattern, best typified by a home without an NO2 emission source, is a cumulative
average level that estimates exposure resulting from a mildly fluctuating background level
without transient higher levels being experienced A second exposure pattern is usually
associated with the use of a gas cooking stove in the home It consists both of a cumulative
average fluctuating background level and short-term (1 to 2-h) peak concentrations
Although hour-by-hour characterization of exposure would be most helpful in examining
exposure-response data, in both cases, cumulative averages are the available human exposure
data, typically 2-week averages
It is important to consider evidence useful in evaluating the relationship between NO2
exposure patterns and health effects to discern the potential effects of ambient air patterns
The epidemiology studies reviewed in Chapter 14 predominantly contrast exposures between
electric stove homes and gas stove homes, except for those studies examining ambient
exposures Spengler et al (1979) and several key studies reviewed in Chapters 7, 8, and 14
show that a striking difference in NO2 levels exists between homes using gas versus electric
16-23
-------
cooking Short-term NO2 levels in homes with emission sources have been characterized for
1-h tune penods (see Chapter 7) Harlos et al (1987a) report maximum 1-h kitchen NO2
levels of 0 419 ppm during gas stove use, and mean maximums of 0 182 ppm averaged over
the 4-h sampling period As discussed in Chapter 7, both short-term peak concentrations and
longer term mean concentrations are lower in activity rooms and bedrooms than in kitchens
Being out of the kitchen or house during gas stove use would thusly limit exposure to high
short-term levels House and bedroom averages correlate better than kitchen averages with
personal exposure data (see Chapter 7) This mainly reflects the small amount of time spent
in the kitchen (with high peaks) as opposed to other indoor locations and the smaller
contribution that peaks make to the total exposure
Again, as discussed above, ambient levels are typically characterized by 1-h and annual
averages. Annual averages in 1988 and 1989 in United States Metropolitan Statistical Areas
ranged from 0 007 to 0 061 ppm, with a collective mode representing a U S annual average
of approximately 0 02 ppm Typical 1-h values range from zero to 0 1 ppm (188 /*g/m3),
with a mode of 0 05 ppm (see Figure 7-8) Thus, outdoor 1-h values more typically peak
near 0 1 ppm
Available data do not adequately examine the relative contributions of peak and average
exposures and their relationship with observed health effects Advances in monitoring and in
the determination of health outcome measures are needed to better examine the potential
relationship between peak exposures and health outcomes as contrasted to longer term
pollutant measures These needs represent important areas for future research
16.5.2.2 Long-Term Exposure Estimates
Several of the epidemiology studies in the quantitative analysis in Chapter 14 used a
single 2-week NO2 average or used two 2-week NO2 averages to characterize long-term
exposure The representativeness of such estimates of long-term exposure (e g , 1 year) is a
consideration in generalizing the results of these studies to the long-term ambient situation
To roughly estimate the possible divergence from an actual annual ambient mean resulting
from selecting one or two 2-week averages, data from the U S Environmental Protection
Agency's Aerometnc Information and Retrieval System data bank (AIRS, 1991) for
16-24
-------
10 stations, selected from among those having fairly complete records for 1988 or 1989
(see Chapter 7), have been analyzed for the variability m their 2-week averages
(Table 16-5) These stations represent various regions of the United States but emphasize
California, where high levels most frequently occur For each station, 2-week averages were
calculated week by week, these were then ranked and the 10th and 90th percentiles were
determined These two percentiles were then divided by the station's annual mean to provide
a common frame of reference For example, the 10th and 90th percentile fractions for a
single 2-week average for Los Angeles in 1988 were 0 77 and 1 27 tunes the annual mean,
respectively This would indicate that 80% of the 2-week averages were between 77 and
127% of the annual average of 0 061 ppm (0 047 to 0 078 ppni, respectively) In a similar
manner, percentiles were calculated from two 2-week averages, using the means of 2-week
averages that were 26 weeks apart, thus providing data for two opposite seasons such as
winter and summer The 26 possible averages were raniked as before Because these
averages represent more weeks of data taken at two different tunes, it is expected that they
would come closer to the true annual average, this is generally the case
The results suggest that most (80%) of the single 2-week averages are within 80 and
125 % of the mean, except possibly for cities (such as Dallas) with very low means The two
2-week averages produce a better estimate, with most tying between 85 and 120% of the
mean The reader is cautioned that these numbers and .analyses may not be representative of
actual exposure situations Also, the use of a single outdoor monitor may not produce a
representative exposure estimate These selected data and analyses are offered only to
describe potential relationships between ambient NO2 annual averages and 2-week data
periods
Further, forty sites were chosen (for their availability of data) to try to roughly estimate
the uncertainty of these estimates The sites were split into three categones depending on the
value of the annual average, that is, (1) 0 001 to 0 020 ppm, (2) 0 020 to 0 040 ppm, or
(3) > 0 040 ppm NO2 For each site, up to 52 2-week averages (starting at the beginning of
each week) were calculated, along with the overall annual average Each 2-week average
can be thought of as an estimate of the annual average, and the standard error of this
estimate was calculated across all sites within a particular exposure range The standard
error was estimated from the absolute deviations instead of the deviations squared to avoid
16-25
-------
TABLE 16-5. U.S. ENVIRONMENTAL PROTECTION AGENCY
ANALYSIS OF VARIABILITY IN TWO-WEEK AMBIENT AVERAGES OF
ONE-HOUR NITROGEN DIOXIDE DATA AT 10 SELECTED LOCATIONS
Fraction of Annual Average
City
Los Angeles, CA
Azusa, CA
Upland, CA
Anaheim, CA
New York, NY
Chicago, IL
Cincinnati, OH
Worcester, MA
Miami, FL
Dallas, TX
Year
1988
1989
1988
1988
1988
1989
1989
1988
1989
1989
Annual
Average
(ppm)
0061
0051
0047
0046
0041
0034
0030
0029
0018
0011
2-Week Average
10th
Percentile
077
081
079
071
084
086
084
081
077
045
90th
1 27
127
138
123
1 19
1 18
123
1 19
126
146
Two 2-Week Average
10th
087
083
087
086
091
093
088
083
090
075
90th
Percentile
120
1 17
122
1 10
1 16
1 10
1 11
1 23
1 18
1 31
Source Aerometric Information Retrieval System (1991)
possible problems from nonnormally distributed data In a similar manner, standard errors
were estimated from two 2-week averages, using the average of 2-week averages that were
26 weeks apart The number of sites, ranges of annual means, and average standard errors
of the one 2-week and two 2-week estimates are given in Table 16-6
Table 16-6 shows that the standard error of the estimate goes up with the annual
average itself In general, one 2-week estimate has a standard error of about 25 % of the
mean, whereas the two 2-week estimate has a standard error of about 15 % of the mean
A similar analysis was made for indoor sites using data from homes in the Albuquerque
area supplied by Lambert (1991) One hundred sites were selected (by Lambert), including
25 electric stove homes and 75 gas stove homes The extensive data set consisted of
26 2-week averages for each home Standard errors were calculated for three different
estimates. (1) one 2-week average, (2) two 2-week averages, and (3) a fixed value estimate
depending on whether the home used a gas stove versus an electric stove When all the
electric stove homes were combined, the overall average NO2 for the electric stove homes
was 6.51 /tg/m3 (0 003 ppm) The gas stove homes averaged 32 67 /tg/m3 (0 017 ppm)
16-26
-------
TABLE 16-6. AVERAGE ANNUAL NITROGEN DIOXIDE MEANS (ppm) AND
STANDARD ERRORS OF ESTIMATES FOR 40 SITES IN THE
UNITED STATES BY ANNUAL AVERAGE AS DERIVED BY THE
U.S. ENVIRONMENTAL PROTECTION AGENCY FROM
THE AEROMETRIC INFORMATION RETRIEVAL SYSTEM (1991)
Ranges of
Annual Averages
0 001-0 020
0 020-0 040
>0040
Number of Sites
7
16
17
Average Mean
00159
00313
00495
Average Standard
One 2-Week
Average
00046
00080
00111
Error of Estimate
Two 2-Week
Average
00028
00041
00056
These two overall averages were used as the fixed estimate of each home's annual NO2
level The estimated standard errors were calculated in the same manner as was done for the
outdoor data The results are presented in Table 16-7.
TABLE 16-7. AVERAGE ANNUAL NITROGEN DIOXIDE MEANS (ppm)
AND STANDARD ERRORS OF ESTIMATES FOR 100 HOMES
BASED ON DATA OF LAMBERT (1991) AS DERIVED BY THE
U.S. ENVIRONMENTAL PROTECTION AGENCY
Source of
Exposure
Electric Stove
Gas Stove
Number of
Homes
25
75
Overall
Average
0003
0017
Average Standard Error of the Estimate
One 2-Week Two 2- Week Fixed
0 001 0 0008 0 0014
0 010 0 005 0 012
The standard errors from the estimates of indoor exposure are somewhat higher than
the standard errors of the outdoor estimates The standard error of the one 2-week average
is about 50% of the mean, and the standard error of the two 2-week average is about 25% of
the mean The fixed estimate (using an overall average for electric homes and a separate
overall average for gas stove homes) had a slightly larger standard error than did the one
2-week average This suggests that measured 2-week averages are better for characterizing
exposure than a fixed average based only on the presence or absence of a gas stove
16-27
-------
However, a single 2-week average is only slightly better Although the standard errors are
quite large (compared with the mean), the estimates are adequate to distinguish a high
exposure (usually with a gas stove present) from a low exposure (usually with an electnc
stove present) household
16.5.3 Nitrogen Dioxide Exposure Estimates
The following exposure analysis estimates the potential impact of outdoor NO2
concentrations on exposure in children and adults This analysis is based on a simple
approach that does not adequately take into account the interaction of activity or location and
the NO2 level in that location during the tune period in that microenvironment Instead,
averages are used Further efforts are beyond the scope of this document, but would be
necessary to produce a more appropriate analysis This limited exposure estimate provides a
perspective for this document Two factors are needed for this analysis The first factor is
the fraction of tune the children spent outdoors This, of course, will vary by age, season,
and locality The second factor is the increase in indoor concentrations of NO2 (e g , in the
bedroom) as a function of the outdoor concentration, which also vanes by season and
locality.
The fraction of time spent outdoors is estimated by several studies (see Table 16-8),
and the estimates are highly dependent on age In general, the fractions ranged from about
0.3 to 20% of the tune being spent outdoors Rather than assume that a single value applies,
several different percents (0 3, 1, 3, 5, 10, 15, and 20) are evaluated in the analysis
The contribution of outdoor NO2 concentrations to indoor levels must be estimated
To start, average outdoor values for the whole season are used The analysis assumes no
indoor sources The model used is based on a physical mass balance equation (Butler et al,
1990; Drye et al., 1989) The model (see Chapter 8) states that the indoor concentration
(Cm) will be a linear function of the outdoor concentration (Cout), with a constant term (A)
For purposes of estimating the increase in indoor NO2 from outdoor NO2, only the
regression coefficient for the indoor/outdoor (I/O) ratio itself is needed An indoor/outdoor
ratio of 0 59 may be an appropriate value (see Chapter 7) Combining the estimates derived
by Drye et al. (1989) and Butler et al (1990) for NO2 concentration in a bedroom (Cbed)
for summer and winter using a simple average into a single equation yields
16-28
-------
TABLE 16-8. NITROGEN DIOXIDE EXPOSURE ESTIMATES IN PARTS PER
MILLION (ppm) DERIVED BY THE U.S. ENVIRONMENTAL
PROTECTION AGENCY AS FUNCTION OF OUTDOOR NITROGEN DIOXIDE
CONCENTRATION AND PERCENT TIME OUTDOORS, WHERE
INDOOR/OUTDOOR RATIO EQUALS C .59 AND ASSUMING A
BASELINE CONCENTRATION OF 0.005 ppm
Outdoor NO2
ppm
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
Percent of Time Outdoors
03a
0005
0008
0011
0014
0017
0020
0023
0026
0029
0032
lb
0005
0008
0011
0014
0017
0020
0023
0026
0029
0032
3C
0005
0008
0011
0014
0017
0020
0023
0026
0029
0032
5d
0005
0008
0011
0014
0017
0020
0023
0026
0029
0032
10e
0005
0008
0011
0014
0018
0021
0024
0027
0030
0033
15f
0005
0008
0012
0015
0018
0021
0025
0028
0031
0034
20g'h
0005
0008
0012
0015
0018
0022
0025
0029
0032
0035
Harlos et al (1987b) Children < 1 year old
Dorre et al (1990) Children 2 to 3 years old, minimum time
cDorre et al (1990) Children 2 to 3 years old, mean time
dNoy et al (1986) Housewives
6Clausing et al (1986) High school students
Quackenboss et al (1982) Family members
8Schwab et al (1990) Fourth through sixth graders
Adair and Spengler (1989) Children can spend as much as 50% of their time outdoors in the summer
Cbed = 0 59 Cout + A! (16-1)
From Equation 16-1, the total NO2 exposure (Ctotaj) can then be estimated for a child who
spends Pout fraction of his time outdoors, assuming a baseline indoor concentration of Cm
+ 0 59 x (Cout - CJ} X (1 - Pout) (16-2)
-4- f y P
^ ^out A out
16-29
-------
The increase in NO2 exposure (Cmcj) is estimated by
Cmcr = 0 59 X (Cout - CJ X (1 - P0J (16-3)
X ?
Table 16-8 presents values for this exposure estimate as a function of outdoor NO2
concentration and percent of tune outdoors for the I/O ratio 0 59 As long as the regression
coefficient is high (0 59 in this case), the fraction of time outdoors will have little impact on
the exposure For smaller values of the coefficient (e g , near 0 3), the activity pattern
would have a much larger impact
Table 16-9 shows changes in the exposure estimate in a nest of subtables where the
percent tune outdoors and the outdoor NO2 level vary for specific I/O ratios of 0 1 to 0 8
Indoor/outdoor ratios depend on several factors, such as season, use of air conditioning, tight
versus loose homes, and location in the United States (see Chapter 7)
16-30
-------
TABLE 16-9. NITROGEN DIOXIDE EXPOSURE ESTIMATES IN PARTS PER
MILLION (ppm) DERIVED BY THE U.S. ENVIRONMENTAL
PROTECTION AGENCY AT SELECTED OUTDOOR NITROGEN DIOXIDE
CONCENTRATIONS, INDOOR/OUTDOOR CONCENTRATION RATIOS,
AND PERCENTAGES OF TIME SPENT OUTDOORS, COMPARED
WITH A NITROGEN DIOXIDE EXPOSURE OF 0.005 ppma
Outdoor
NO2 (ppm)
0020
0025
0030
0035
0040
0045
0050
0055
0060
0020
0025
0030
0035
0040
0045
0050
0055
0060
0020
0025
0030
0035
0040
0045
0050
0055
0060
0020
0025
0030
0035
0040
0045
0050
0055
0060
Percentage of Time Outdoors
' 03
0007
0007
0008
0008
0009
0009
0010
0010
0011
0008
0009
0010
0011
0012
0013
0014
0015
0016
0010
0011
0013
0014
0016
0017
0019
0020
0022
0011
0013
0015
0017
0019
0021
0023
0025
0027
1
0007
0007
0008
0008
0009
0009
0010
0010
0011
0008
0009
0010
0011
0012
0013
0014
0015
0016
0010
0011
0013
0014
0016
0017
0019
0020
0022
0011
0013
0015
0017
0019
0021
0023
0025
0027
3 5 10
I/OR of 0 1
0 007 0 007 0 008
0 008 0 008 0 009
0 008 0 009 0 010
0 009 0 009 0 Oil
0 009 0 010 0 012
0 010 0 Oil 0 013
0011 0012 0014
0011 0012 0014
0 012 0 013 0 015
I/OR of 02
0 008 0 009 0 009
0 009 0 010 0 Oil
0011 0011 0012
0 012 0 012 0 013
0 013 0 013 0 015
0 014 0 015 0 016
0 015 0 016 0 018
0 016 0 017 0 019
0 017 0 018 0 020
I/OR of 03
0 010 0 010 0 Oil
0011 0012 0012
0 013 0 013 0 014
0 015 0 015 0 016
0 016 0 017 0 018
0 018 0 018 0 020
0 019 0 020 0 022
0 021 0 022 0 023
0 023 0 023 0 025
I/OR of 04
0011 0011 0012
0 013 0 014 0 014
0 015 0 016 0 016
0 018 0 018 0 019
0 020 0 020 0 021
0 022 0 022 0 023
0 024 0 024 0 026
0 026 0 026 0 028
0 028 0 029 0 030
15
20
Outdoor
Percentage of Time Outdoors
03
1
3 5
10
15 20
I/OR of 05
0009
0010
0011
0012
0013
0014
0016
0017
0018
0010
0011
0013
0015
0016
0018
0019
0021
0023
0009
0011
0012
0013
0015
0016
0018
0019
0020
0010
0012
0014
0016
0018
0019
0021
0023
0025
0020
002-5
0030
003-5
0040
0045
0050
005'5
0060
0020
0025
0030
0035
0040
0045
0050
0055
0060
0013
0015
0018
0020
0023
0025
0028
0030
0033
0014
0017
0020
0023
0026
0029
0032
0035
0038
0013
0015
0018
0020
0023
0025
0028
0030
0033
0014
0017
0020
0023
0026
0029
0032
0035
0038
0013 0013
0015 0015
0018 0018
0020 0021
0023 0023
0026 0026
0028 0029
0031 0031
0033 0034
I/OR of 0
0014 0014
0017 0017
0020 0020
0023 0024
0026 0027
0029 0030
0033 0033
0036 0036
0039 0039
0013
0016
0019
0021
0024
0027
0030
0032
0035
6
0015
0018
0021
0024
0027
0031
0034
0037
0040
0014 0014
0016 0017
0019 0020
0022 0023
0025 0026
0028 0029
0031 0032
0034 0035
0037 0038
0015 0015
0018 0019
0021 0022
0025 0025
0028 0029
0031 0032
0035 0036
0038 0039
0041 0042
I/OR of 07
0011
0013
0015
0017
0019
0021
0023
0025
0027
0012
0015
0017
0020
0022
0025
0027
0029
0032
0012
0014
0016
0018
0020
0023
0025
0027
0029
0013
0015
0018
0021
0023
0026
0028
0031
0034
0020
0025
0030
0035
0040
0045
0050
0055
0060
0020
0025
0030
0035
0040
0045
0050
0055
0060
0016
0019
0023
0026
0030
0033
0037
0040
0044
0017
0021
0025
0029
0033
0037
0041
0045
0049
0016
0019
0023
0026
0030
0033
0037
0040
0044
0017
0021
0025
0029
0033
0037
0041
0045
0049
0016 0016
0019 0019
0023 0023
0026 0026
0030 0030
0033 0034
0037 0037
0040 0041
0044 0044
I/OR of 0
0017 0017
0021 0021
0025 0025
0029 0029
0033 0033
0037 0037
0041 0041
0045 0045
0049 0050
0016
0020
0023
0027
0031
0034
0038
0041
0045
8
0017
0021
0025
0030
0034
0038
0042
0046
0050
0016 0016
0020 0020
0024 0024
0027 0028
0031 0032
0035 0035
0039 0039
0042 0043
0046 0047
0017 0018
0022 0022
0026 0026
0030 0030
0034 0034
0038 0039
0042 0043
0046 0047
0051 0051
aNC>2 = Nitrogen dioxide
I/O R = Indoor/outdoor ratio
16-31
-------
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Richters, A , Damji, K S (1988) Changes in T-lymphocyte subpopulations and natural killer cells following
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Richters, A , Damji, K S (1990) The relationship between inhalation of nitrogen dioxide, the immune system,
and progression of a spontaneously occurring lymphoma in AKR mice J Environ Pathol Toxicol
Oncol 10 225-230
Robison, T W , Duncan, D P , Forroan, H J (1990) Chemoattractant and leukotnene B4 production from rat
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Rombout, P J A , Dormans, JAMA, Marra, M , Van Esch, G J (1986) Influence of exposure regimen
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Rose, R M , Fuglestad, J M , Skormk, W A , Hammer, S M , Wolfthal, S F , Beck, B D , Brain, J D
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16-39
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16-41
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APPENDIX A.
GLOSSARY OF TERMS AND SYMBOLS
A
"A" strain
9-AA
AaDO2
AAS
AATCC
ACH
Ad
AD
AICHE
AIDS
AIRS
Al
A13+
A12(S04)3
AM
a2-M
AMP
AMT
ANC
ANOVA
ANSA
ANSI
APCD
ABBREVIATIONS, ACRONYMS, AND SYMBOLS
Angstrom (10" meter)
A particular type of influenza virus
9-Amino-acndme
Difference between alveolar and artenabzed partial pressure of oxygen
Atomic absorption spectroscopy
American Association of Textile Chemists and Colonsts
Air changes per hour
A particular strain of laboratory mouse
Annular denuder
American Institute of Chemical Engineers
Acquired immune deficiency syndrome
Aerometnc Information Retrieval System
Aluminum
Aluminum ion
Aluminum sulfate
Alveolar macrophage
Alpha-2-macroglobulin
Adenosine monophosphate, adenosine 5' phosphate
Arithmetic mean thickness
Acid-neutralizing capacity
Analysis of variance
8-amino-l-naphthalene-sulfonic acid
American National Standards Institute
Air Pollution Control District
A-l
-------
APHA
GJjPI
A/PR/8
A/PR/8/34
AQCR
AQIRP
AQSM
ASIM
atm
ATP
avg
BAKI
BAJL
BaSO4
BHA
BHPN
BHR
BHT
BMRC
BP
Br
BrNO3
BrO
scat
°C
13C
14C
Ca
Ca2+
CAA
cAMP
American Pubhc Health Association
Alpha-1-protease inhibitor
A particular strain of influenza virus
A particular strain of influenza virus
Air Quality Control Region
Air Quality Improvement Research Program
Air Quality Simulation Model
American Society for Testing and Materials
One atmosphere, a unit of pressure
Adenosine tnphosphate
Average
Potassium iodide solution acidified with bone acid
Bronchoalveolar lavage
Barium sulfate
Butylated hydroxyamsole
Af-bis(2-hydroxypropyl)nitrosamine
Bronchial or airways hyperresponsiveness
Butylated hydroxytoluene
British Medical Research Council
Blood pressure
Bromine
Bromine nitrate
Bromine monoxide
Extinction coefficient due to scatter by aerosols
Degrees Celsius (Centigrade)
Carbon-13
Carbon-14, a radioactive form of carbon
Calcium
Calcium ion
Clean Air Act
Cyclic adenosine monophosphate, adenosine 5'-phosphate
A-2
-------
CAMP
CASAC
C57BL
C57BL/6
CD-I
CDC
cGMP
CH4
C3H
C3H6
ChE
CI
Cl
cr
C12
CL
CLdyn
CLM
CLM-PC
C1N03
CIO
cm
CNG
CNS
CO
C02
13C02
CoA
COH
COPD
Community Air Monitoring Program
Clean Air Scientific Advisory Committee
A particular strain of laboratory mouse
A particular strain of laboratory mouse
A particular strain of laboratory mouse
Centers for Disease Control
Cyclic guanosine monophosphate, guanosine 5'-phosphate
Methane
A particular strain of laboratory mouse
Propylene
Cholinesterase
Confidence interval
Chlorine
Chloride ion
Chlorine molecule
Lung compliance
Dynamic lung compliance
Chemiluminescence
Chemiluminescence with photolytic converter
Chlorine nitrate
Chlorine monoxide
Static lung compliance
Centimeter
Compressed natural gas
Central nervous system, the brain and spinal cord
Carbon monoxide
Carbon dioxide
Carbon-13 labeled carbon dioxide
Coenzyme A
Coefficient of haze
Chronic obstructive pulmonary disease
A-3
-------
CPK
CR-1
CRD
CRD
CSTR
C X T
Cu-Cd
CV
CV
CVM
D10
D = CT
DD
DEN
DIAL
DIFK3N
DIN
DMA
DMN
DNA
DOAS
DON
DPPD
EC
EDF
EGR
EKG
EPA
EPRI
Creatine phosphokinase
A particular strain of laboratory mouse
Chronic respiratory disease
Completely randomized design
Continuously stirred tank reactor
Exposure concentration in ppm multiplied by time of exposure in hours or
other tune measurement
Copper-cadmium
Closing volume
Coefficient of variance
Contingent valuation method
Dose that would cause a 10% decrease in functional expiratory volume
in 1 second
Dose equals concentration multiplied by tune
Denuder difference
Diethylnitrosamine (also DENA)
Differential absorption lidar
Diffusion Kinetics Model
Total inorganic nitrogen
Diffusion capacity of the lung for carbon monoxide
Drmethylamine
Dimethylnitrosamine
Deoxynbonucleic acid
Differential optical absorption spectroscopy
Dissolved organic nitrogen
Af,2V-chphenylphenylenediamine
Prefix of International Commission on Enzymes' identification numbers
Environmental Defense Fund
Exhaust-gas recrrculation
Electrocardiogram
U S Environmental Protection Agency
Electric Power Research Institute
A-4
-------
EV
°F
FAV
FCV
Fe
FEF
FeSO4
FET
FEV
75
FEV10
FEV25-75%
FP
FRM
ft
FT
FTIR
FVC
FW
g
Gaw
GC
GC
GC-ECD
GC-FID
GC-MS
GDH
GM
GMP
GOGAT
G-6-P
Electee vehicle
Degrees Fahrenheit
Final acute value
Final chronic value
Iron
Forced expiratory flow
Iron sulfate
First-edge tune
Forced expiratory volume
0 75-Second forced expiratory volume
One-second forced expiratory volume
Forced expiratory volume at 25 to 75 % of vital capacity
Filter pack
Federal Reference Method for air quality measurement
Foot
Fourier transform spectroscopy (also FS)
Fourier transform infrared spectroscopy
Forced vital capacity
Fresh weight of plant material
Gram
Airway conductance
Gas chromatography
Guanylate cyclase
Gas chromatography with electron capture detection
Gas chromatography with flame lonization detection
Gas chromatograph in combination with mass spectrometry
Glutamase dehydrogenase
General Motors Corporation
Guanosine 5'-phosphate, guanosine nionophosphate
Glutamine oxoglutarate aminotransferase (or glutamate synthase)
Glucose-6-phosphate
A-5
-------
GP-CLM
GPT
GS
GSH
GSSG
GTE/CITE
h
H*
H+
3
H
ha
HBEF
HbO2
HC
HC1
HCN
HDV
HP
5-HLAA
HIV
hi>
HNO2
HNO3
HO*
HO2*
H2O
H2O2
HONO
HO2NO2
Gas-phase cheimluminescence
Gas-phase titration
Glutamine synthetase
A tnpeptide, glutathione (reduced form)
The disulfide (oxidized) form of GSH
Global Troposphenc Experiment/Chemical Instrumentation Test and
Evaluation
Hour
Hydrogen (free radical)
Hydrogen ion
Tritium, a radioactive form of hydrogen
Hectare
Hubbard Brook Experimental Forest
Oxyhemoglobin
Hydrocarbon
Hydrochloric acid
Hydrogen cyanide
Heavy-duty vehicle
Hydrogen fluoride
5-Hydroxyindoleacetic acid
Human immunodeficiency virus
Planck's constant (h) tunes the frequency of radiated energy (?) = Quanta
of energy (E)
Nitrous acid (liquid form)
Nitric acid (also HONO^
Hydroxyl free radical (also OH)
Hydroperoxyl free radical
Water
Hydrogen peroxide
Nitrous acid (gaseous form)
Peroxymtnc acid
A-6
-------
HPIC
HPLC
HR
H2S
H2S04
5-HT
3H-thymidine
IARC
1C
IF
IPS
Ig
IgA
IgG
IgG2
IgM
IL-1
in
IR
IRGA
k
K
K+
kg
km
L
LAR
LC50
LD50
High performance ion chromatograph
High performace liquid chromatograph
Heart rate
Hydrogen sulfide
Sulfuric acid
Serotomn
Tntiated thymiduie
International Agency for Research on Cancer
Ion chomatography
Irrigation and fertilization
Integrated Forest Study
Immunoglobulins
Immunoglobuhn A fraction
Immunoglobulin G fraction
Immunoglobulin GI fraction
Immunoglobulin G2 fraction
Immunoglobulin M fraction
Interleukin-1
Inch
Infrared
Infrared gas analysis
Rate constant or dissociation constants
Potassium
Potassium ion
Kilogram
Kilometer
Liter (also K)
Leaf area ratio
Lethal concentration 50%, that concentration which is lethal to 50% of test
subjects
Lethal dose 50%, dose which is lethal to 50% of the subjects
A-7
-------
LDH
LDV
UDF
IM
LNG
logEF
LPG
LPS
LT50
LTB4
m
M
M
M85
M100
MAK
max
MDL
MERL
MFR
Mg
Mg
2+
mg/m3
MgO
MgSO4
mm
MIT
mL
Lactic acid (lactate) dehydrogenase
Light-duty vehicle
Laser-induced fluorescence
Light microscope
Liquefied natural gas
Base 20 logarithm of the emission factor
Liquefied petroleum gas
Bacterial hpopolysacchande
The tune required for 50% of the test animals to die when given a lethal
dose
Leukotnene B4
Meter
Molar
Third body (in a reaction)
Fuel blended from 85 % methanol and 15 % gasoline
Methanol
Maximum permissible concentration (in Germany)
Maximum
Minimum detection limit
Marine Ecosystem Research Laboratory
Maximal flow rate
Magnesium
Magnesium ion
Microgram
Micrograms per cubic meter
Milligrams per cubic meter
Magnesium oxide
Magnesium sulfate
Minute
Massachusetts Institute of Technology
MilMiter
Microliter
A-8
-------
mM
MMD
MMEF
MMFR
mo
MFC
MSA
MSCET
MT
N
N
N2
13
N
15N
Na+
NA
NAAQS
NaCl
Na2CO3
NAD+
NADB
NADH
NADP
NADP+
NADPH
NaF
NAMS
NaNO2
NaNO3
NaOH
Micrometer
Millunolar fcjf
Mass median diameter
Maximal midexpiratory flow
Mid-maximal flow rate
»
Month ;
Maxunum permissible concentration (in the U S S R)
1
Metropolitan Statistical Area
Month and State current emission trends
f
Metric ton
Atomic nitrogen
Normal
Molecular nitrogen
Nitrogen-13, a radioactive form of nitrogen
Nitrogen-15
Sodium ion
Not applicable
National Ambient Air Quality Standard
Sodium chloride, common table salt
Sodium carbonate
Nicotinamide-adenine dinucleotide (+ indicates oxidized form)
National Air Data Bank
Nicotinanude-adenine dinucleotide (reduced form)
National Acid Deposition Program
Nicotinamide ademne dinucleotide phosphate
Nicotinamide-adenine dinucleotide phosphate (reduced form)
Sodium fluoride
National Air Monitoring Station
Sodium nitrite
Sodium nitrate
Sodium hydroxide
A-9
-------
NAPAP
NaR
NAR
NAS
NASA
NASN
NCF
NDIR
NDMA
NEDA
NEDS
NEIC
ng
NGV
NH3
NH4+
15NH4+
NHLBI
NH4NO3
(NH4)2S04
NIH
NiR
NIST
NK
nm
NMHC
N-6-MI
NMOR
NO
NO2
15
NOo
National Acid Precipitation Assessment Program
Nitrate reductase
Net assimilation ratio
National Academy of Sciences
National Aeronautics and Space Administration
National Air Surveillance Network
Neutrophil chemotactic factor
Nondispersive infrared
Nitrosodimethylamine
2V-(l-Naphthyl)-ethylenediaminedihydrochlonde
National Emissions Data System
National Enforcement Investigations Center
Nanogram
Natural gas vehicle
Ammonia
Ammonium ion
Nitrogen-15 labeled ammonium ion
National Heart, Lung, and Blood Institute
Ammonium nitrate
Ammonium sulfate
National Institutes of Health
Nitrite reductase
National Institute of Standards and Technology
Natural killer cell
Nanometer
Nonmethane hydrocarbon
A^rntrosoheptamethyleneimine
W-nitrosomorpholine
Nitnc oxide
Nitrogen dioxide
Nitrogen-15 labeled nitrogen dioxide
A-10
-------
NO2"
NO3
NO3"
15N03-
N2O
N2O3
N204
N205
NOHb
NOX
NOy
NPN
4-NQO
NRA
NSA
NSF
NSS
NSWS
O
02
03
OAQPS
CK'D)
ODS
OH
OH'
ON+
[15O]-NO2
0(3P)
OR
P
Nitrite ion
Nitrate radical or nitrogen tnoxide
Nitrate ion
Nitrogen-15 labeled nitrate ion
Nitrous oxide
Dinitrogen tnoxide
Dimtrogen tetroxide
Dinitrogen pentoxide
Nitrosylhemoglobui
Nitrogen oxides
Sum of oxides of nitrogen and other oxidized nitrogen compounds,
excluding nitrous oxide
n-Propyl nitrate
4-Nitroquinohne-1 -oxide
Nitrate reductase activity
Nitrosating agent
National Science Foundation
National Stream Survey
National Surface Water Survey
Atomic oxygen
Molecular oxygen
Ozone
Office of Air Quality Planning and Standards
Excited atomic oxygen
Oxygen depletion sensor
Hydroxyl group
Hydroxide ion
Nitrosomum ion
Oxygen-15 labeled nitrogen dioxide
Ground state atomic oxygen
Odds ratio
Phosphorus
A-ll
-------
P Phosphorus-32, a radioactive form of phosphorus
PaCO2 Arterial partial pressure of carbon dioxide
PACO2 Alveolar partial pressure of carbon dioxide
PAH p-Ammiohippunc acid
PAN Peroxyacetyl nitrate
PaO2 Arterial partial pressure of oxygen
PAO2 Alveolar partial pressure of oxygen
PARS Precusion Accuracy Reporting System
PBzN Peroxybenzoyl nitrate
PD40 Dose required to reduce specific airway conductance by 40%
PD10Q Dose of methacholine required to double specific airway resistance
PD10RHE Provocative dose in respiratory heat exchange units needed to decrease
functional expiratory volume in 1 second by 10%
PD8uSO2 Concentration of sulfur dioxide required to increase specific airway
resistance by 8 units
PEF Peak expiratory flow
PEFR Peak expiratory flow rate
PEFV Partial expiratory flow volume
PEF40%VC Partial expiratory flow at 40% of vital capacity
PF Photofragmentation
PFC Plaque-forming cell
6-P-G 6-Phosphogluconate
pH Log of the reciprocal of the hydrogen ion concentration
PHA Phytohemagglutimn
P, Inorganic phosphate
PM Particulate Matter
PM Photomultiplier
PMN Polymorphonuclear leukocyte
PN Particulate nitrate
PO2 Partial oxygen pressure
ppb Parts per billion
pphm Parts per hundred million
A-12
-------
ppm
PPN
ppt
PSC
PSD
P value
PV100SRaw(S02)
Q
QRS
RAMS
RAPS
^aw
RBC
RCBD
RC(O)O2NO2
RD
RGR
RH
RHE
RIBD
RM
RNA
RO2*
RR
RSD
RSV
RP
RUBISCO
RV
S
Parts per million
Peroxypropionyl nitrate
Parts per trillion
Polar stratospheric cloud
Passive sampling device
Probability
Ventilation of sulfur dioxide required to produce a 100 % increase in
specific airway resistance
Cardiac output
A complex of three distinct electrocardiogram waves which represent the
beginning of ventricular contraction
Regional Air Monitoring System
Regional Air Pollution Study
Airway resistance
Red blood cell, erythrocyte
Randomized complete block diagram
Peroxyacylmtrate
Relative duration
Relative growth rate
Relative humidity
Respiratory heat exchange
Randomized incomplete block diagram
Reference method for air quality measurement
Ribonucleic acid
Organic peroxy radical (where R is an organic moiety)
Relative risk
Relative standard deviation
Respiratory syncytial virus
Total respiratory resistance
Ribulose-1,5-biphosphate carboxylase-oxygenase
Residual volume
Sulfur
A-13
-------
SAI
SD
SEM
SEM
SES
SF6
SGaw
SGOT
SGPT
SH-
SMSA
SN
SO2
SOD
SOX
SP
SPF
SRBC
SRM
SS
STP
TA
TEA
TOLAS
TEA
TEM
TFR
Tg
TGS-ANSA
Science Applications, Inc
Standard deviation
Scanning electron microscope
Standard error of the mean
Socioeconomic status
Sulfur hexafluonde
Specific airway conductance
Serum glutamic-oxaloacetic transaminase
Serum glutamic-pyruvic transaminase
Sulfhydryl group
Standard Metropolitan Statistical Area
Suspended nitrates
Sulfur dioxide
Sulfate ion (also SO4~)
Superoxide dismutase
Sulfur oxides
Single photon
Specific pathogen free
Specific airway resistance
Sheep red blood cell
Standard reference material
Suspended sulfates
Standard temperature and pressure
Tungstic acid
Thiobarbitunc acid
Tunable-diode laser spectroscopy
Triethanolamine
Transmission electron microscope
Transition flow reactor
f\ 10
Terragram, 10 metric tons or 10 grams
A 24-hour method for the detection of analysis of NQ2 in ambient air
A-14
-------
TGV
TLC
T-NH3
TNT
TP
TP
TPTT
TSP
TTFMS
UV
UVGSH
VC
VE
VE
VEE
V/FFV
Vmax
VOC
VT
v/v
V50%VC
WBC
WTP
Xe
ZEV
Zn
Thoracic gas volume
Total lung capacity
Total ammonia
Trinitrotoluene
Total phosphate
Two photon
20% transport tune
Total suspended particulate
Two-tone frequency modulated spectroscopy
Ultraviolet
Unvented gas space heater
Vital capacity
Ventilatory volume
Minute ventilation
Venezuelan equine encephalomyelitis (virus)
Variable- or flexible-fuel vehicle
Maximum expiratory flow rate
Volatile organic compound
Total volume
Volume per volume
Ventilation at 50 % vital capacity
White blood cell
Willingness to pay
Xenon
Zero Emission Vehicle
Zinc
Greater than
Less than
Approximately
A-15
-------
GLOSSARY
AaDO2 Alveolar-arterial difference or gradient of the partial pressure of oxygen An overall
measure of the efficiency of the lung as a gas exchanger In healthy subjects, the
gradient is 5 to 15 millimeters of mercury (torr)
A/PR/8 virus* A type of virus capable of causing influenza in laboratory animals, also,
A/PR/8/34
Abscission The process whereby leaves, leaflets, fruits, or other plant parts become detached
from the plant
Absorption coefficient A quantity which characterizes the attenuation with distance of a beam
of electromagnetic radiation (like light) in a substance
Absorption spectrum The spectrum that results after any radiation has passed through an
absorbing substance
Abstraction Removal of some constituent of a substance or molecule
Acetaldehyde* CH3CHO, an intermediate in yeast fermentation of carbohydrate and in alcohol
metabolism, also called acetic aldehyde, ethaldehyde, ethanal
Acetate rayon A staple or filament fiber made by extrusion of cellulose acetate It is
saponified by dilute alkali, whereas viscose rayon remains unchanged
Acetylcholine A naturally occurring substance in the body that can cause constriction of the
bronchi ui the lungs
Acid: A substance that can donate hydrogen ions
Acid dyes' A large group of synthetic coal-tar-derived dyes that produce bright shades in a
wide color range Low cost and ease of application are features that make them the
most widely used dyes for wool Also used on nylon The term acid dye is derived
from their precipitation in an acid bath
Acid mucopolysacchande A class of compounds composed of protein and polysacchande
Mucopolysacchandes comprise much of the substance of connective tissue
Acid phosphatase An enzyme (EC 3132) that catalyzes the disassociation of phosphate
(PC>4) from a wide range of monoesters of orthophosphonc acid Acid phosphatase is
active in an acidic pH range
Acid rain' Rain having a pH less than 5 6, the minimum expected from atmospheric carbon
dioxide
A-16
-------
Acrolein CH2=CHCHO, a volatile, flammable, oily liquid giving off irritant vapor Strong
irritant of skin and mucuous membranes Also called acrylic aldehyde or 2-propenal
Acrylics (plastics) Plastics that are made from acrylic acid and are light in weight, have
great breakage resistance, and lack odor and taste Not resistant to scratching, burns,
hot water, alcohol, or cleaning fluids Examples include Lucite and Plexiglas Acrylics
are thermoplastics and are softened by heat and hardened into definite shapes by
cooling
Acrylic fiber The generic name of manufactured fibers derived from acrylic resins
(minimum of 85 % acrylonitnte units)
Actinic A term applied to wavelengths of light too small to affect one's sense of sight, such
as ultraviolet
Actinomycetes Members of the genus Actinomyces, nonmotile, nonsporeforming, anaerobic
bacteria, including both soil-dwelling saprophytes and disease-producing parasites
Activation energy The energy required to bring about a chemical reaction
Acute respiratory disease Respiratory infection, usually with rapid onset and of short
duration
Acute toxicity Any poisonous effect produced by a single short-term exposure that results in
severe biological harm or death
Acyl Any organic radical or group that remains intact when an organic acid forms an ester
Adenoma An ordinarily benign neoplasm (tumor) of epithelial tissue, usually well
circumscribed, tending to compress adjacent tissue rather than infiltrating or invading
Adenosine monophosphate (AMP) A nucleotide found among the hydrolysis products of all
nucleic acids, also called adenylic acid
Adenosine tnphosphatase (ATPase) An enzyme (EC 3 6 1 3) in muscle and elsewhere that
catalyzes the release of the high-energy, terminal phosphate group of adenosine
tnphosphate
Adrenalectomy Removal of an adrenal gland This gland is located near or upon the kidney
and is the site of origin of a number of hormones
Adsorption Adhesion of a thin layer of molecules to a liquid or solid surface
Advection Horizontal flow of air at the surface or aloft, one of the means by which heat is
transferred from one region of the earth to another
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Aerodynamic diameter The diameter of a unit density sphere having the same settling speed
(under gravity) as the particle in question of whatever shape and density
Aerosol* Solid particles or liquid droplets that are dispersed or suspended in a gas
Agglutination The process by which suspended bacteria, cells, or similar particles adhere
and form into clamps.
Airborne pathogen A disease-causing microorganism that travels in the air or on particles in
the air.
Air pollutant A substance present in the ambient atmosphere, resulting from human activity
or from natural processes, which may cause damage to human health or welfare, the
natural environment, or materials or objects
Air spaces: All alveolar ducts, alveolar sacs, and alveoli To be contrasted with airways
Airway conductance (Gaw)- Reciprocal of airway resistance Gaw =
Airway resistance (RaW) The (factional) resistance to airflow afforded by the airways
between the airway opening at the mouth and the alveoli
Airways. All passageways of the respiratory tract from mouth or nares down to and including
respiratory bronchioles To be contrasted with air spaces
Alanine aminotransferase An enzyme (EC 2612) transferring amino groups from L-alamne
to 2-ketoglutarate Also known as alanine transaminase
AlbumuT A type of simple, water-soluble protein widely distributed throughout animal tissues
and fluids, particularly serum
O
Aldehyde: An organic compound characterized by the group -GH
Aldolase An enzyme (EC 4127) involved in metabolism of fructose that catalyzes the
formation of two three-carbon intermediates in the major pathway of carbohydrate
metabolism
Algal bloom: Sudden spurt in growth of algae that can adversely affect water quality
Alkali: A salt of sodium or potassium capable of neutralizing acids
Alkaline phosphatase A phosphatase (EC 3131) with an optimum pH of 8 6, present
ubiquitously
Allergen: A material that, as a result of commg into contact with appropriate tissues of an
animal body, induces a state of sensitivity resulting in various reactions, generally
associated with idiosyncratic hypersensitivities
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Alpha-hydroxybutyrate dehydrogenase An enzyme (EC 1 1 1 30), present mainly in
mitochondria, that catalyzes the conversion of hydroxybutyrate to acetoacetate
intermediate biochemical pathways
Alpha rhythm A rhythmic pulsation obtained in brain waves exhibited in the sleeping state of
an individual
Alveolar capillary membrane Finest portion of alveolar capillaries, where gas transfer to and
from blood takes place
Alveolar macrophage (AM) A large, mononuclear, phagocytic cell found on the alveolar
surface, responsible for particle clearance from the deep lung and for viral and bacterial
killing
Alveolar oxygen partial pressure (PAO2) Partial pressure of oxygen in the air contained in
the air sacs of the lungs
Alveolar septa The tissue between two adjacent pulmonary alveoli, consisting of a
close-meshed capillary network covered on both surfaces by thin alveolar epithelial
cells
Alveolus An air cell, a terminal, sac-like dilation in the- lung Gas exchange (oxygen/carbon
dioxide) occurs here
Ambient The atmosphere to which the general population may be exposed Construed here
not to include atmospheric conditions indoors, or in the workplace
Amine A substance that may be derived from ammonia (NH3) by the replacement of one,
two or three of the hydrogen (H) atoms by hydrocarbons or other radicals (primary,
secondary, or tertiary amines, respectively)
Ammo acids Molecules consisting of a carboxyl group, a basic ammo group, and a residue
group attached to a central carbon atom Serve as the building blocks of proteins
/7-Aminohippuric acid (PAH) A compound used to determine renal plasma flow
Aminotriazole A systemic herbicide (C2H4N4) used in areas other than croplands, that also
possesses some antithyroid activity, also called amitrole
Ammonification Decomposition with production of ammonia or ammonium compounds,
especially by the action of bacteria on nitrogenous organic matter
Ammonium Anion (NH4+) or radical (NH4) derived from ammonia by combination with
hydrogen Present in rainwater, soils and many commercial fertilizers
Amnestic Pertains to immunologic memory upon receiving a second dose of antigen, the
host "remembers" the first dose and responds faster to the challenge
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Anaerobic* Living, active or occurring in the absence of free oxygen
Anaerobic bacteria A type of microscopic organism that can live in an environment not
containing free oxygen
Anaphylactic dyspneic attack Difficulty in breathing associated with a systemic allergic
response
Anaphylaxis' A term commonly used to denote the immediate, transient kind of
immunological (allergic) reaction characterized by contraction of smooth muscle and
dilation of capillaries due to release of pharmacologically active substances
Angiospernr A plant having seeds enclosed in an ovary, a flowering plant
Angina pectons* Severe constricting pain in the chest that may be caused by depletion of
oxygen delivery to the heart muscle, usually caused by coronary disease
Angstrom (A): A unit (10" centimeter) used in the measurement of the wavelength of light
Anhydride- A compound resulting from removal of water from two molecules of a carboxykc
(-COOH) acid Also, may refer to those substances (anhydrous) that do not contain
water in chemical combination
Anion. A negatively charged atom, radical, or ion
Anorexia. Diminished appetite, aversion to food
Anoxic' Without or deprived of oxygen
Antagonism' When the effects of a mixture are less than the sum of the effects of each
individual chemical
Anthraquinone. A yellow crystalline ketone (C^HgO^ derived from anthracene and used in
the manufacture of dyes
Anthropogenic Of, relating to, or influenced by humans An anthropogenic source of
pollution is one caused by human actions
Antibody Any body or substance evoked by the stimulus of an antigen and that reacts
specifically with an antigen in some demonstrable way
Antigen' A material such as a foreign protein that, as a result of coming in contact with
appropriate tissues of an animal, after a latent penod, induces a state of sensitivity
and/or the production of an antibody
Antistatic agent A chemical compound applied to fabrics to reduce or eliminate accumulation
of static electricity
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Arachidomc acid Long-chain fatty-acid that serves as a precursor of prostaglandins
Area source In air pollution, any small individual fuel combustion or other pollutant source,
also, all such sources grouped over a specific area
Aromatic Belonging to that senes of carbon-hydrogen compounds in which the carbon atoms
form closed rings containing unsaturated bonds (as in benzene)
Arterial partial pressure of oxygen (PaO2) Portion of total pressure of dissolved gases in
arterial blood as measured directly from arterial blood
Arterialized partial pressure of oxygen The portion of total pressure of dissolved gases in
arterial blood attributed to oxygen, as measured fiom nonartenal (e.g , ear-prick)
blood
Arteriosclerosis Commonly called hardening of the arteries A condition that exists when
the walls of the blood vessels thicken and become infiltrated with excessive amounts of
minerals and fatty materials
Artifact A spurious measurement produced by the sampling 01 analysis process
Ascorbic acid Vitamin C, a strong reducing agent with antioxidant properties
Aspartate transaminase Also known as aspartate aminolransferase (EC 2611) An enzyme
catalyzing the transfer of an amine group from glutamic acid to oxaloacetic acid,
forming aspartic acid in the process Serum level of the enzyme is increased in
myocardial infarction and in diseases involving destruction of liver cells
Asphyxia Impaired exchange of oxygen and carbon dioxide, excess of carbon dioxide,
and/or lack of oxygen, usually caused by ventilatory problems
Asthma A disease characterized by an increased responsiveness of the airways to various
stimuli and manifested by slowing of forced expiration that changes in seventy either
spontaneously or as a result of therapy The term asthma may be modified by words or
phrases indicating its etiology, factors provoking attacks, or duration
Asymptomatic Presenting no subjective evidence of disease
Atmosphere The body of air surrounding the earth Also, a measure of pressure (atm) equal
to the pressure of air at sea level, 14 7 pounds per square inch
Atmospheric deposition Removal of pollutants from the atmosphere onto land, vegetation,
water bodies, or other objects by absorption, sedimentation, Brownian diffusion,
impaction, or precipitation in rain
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Atomic absorption spectrometry A measurement method based on the absorption of radiant
energy by gaseous ground-state atoms The amount of absorption depends on the
population of the ground state, which is related to the concentration of the sample being
analyzed
Atopic Clinical hyperreactivity of the airways associated with asthma and allergies
Atropine: A poisonous, white crystalline alkaloid (C17H23NO3) derived from belladonna and
related plants, used to relieve spasms of smooth muscles It is an anticholinergic agent
that blocks the parasympathetic actions of acetylcholine and other cholinergic agents
Autocorrelation Statistical interdependence of variables being analyzed, produces problems,
for example, when observations may be related to previous measurements or other
conditions
Autoimmune disease A condition in which antibodies are produced against the subject's own
tissues.
Autologous. A term referring to cellular elements, such as red blood cells and alveolar
macrophages, from the same organism, also, something natually and normally
occurring in some part of the body
Autotrophic A term applied to those microorganisms that are able to maintain life without an
exogenous organic supply of energy, or which only need carbon dioxide or carbonates
and simple inorganic nitrogen
Autotrophic bacteria A class of microorganisms that require only carbon dioxide or
carbonates and a simple inorganic nitrogen compound for carrying on life processes
Auxin* An organic substance that causes lengthening of the stem when applied in low
concentrations to shoots of growing plants
Awn: One of the slender bristles that terminate the glumes of the spikelet in some cereals and
other grasses
Azo dye: Dyes in which the azo group is the chromophore and joins benzene or naphthalene
rings
Background measurement A measurement of pollutants in ambient air due to natural sources,
usually taken in remote areas
Bactericidal activity The process of killing bacteria
Barre. Bars or stapes in a fabric, caused by uneven weaving, irregular yarn, or uneven dye
distribution
Basal cell* One of the innermost cells of the deeper epidermis of the skin
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Base cation Ca2+, Mg2+, K+, or Na+
Base saturation The degree to which soil cation exchange capacity is occupied by base
cations This is expressed as a percent, using charge-equivalents
Benzenethiol A compound of benzene and a hydrosulfide group
Beta (j3)-hpoprotem A biochemical complex or compound containing both kpid and protein
and characterized by having a large molecular weight, rich in cholesterol Found in
certain fractions of human plasma
Bilateral renal sclerosis A hardening of both kidneys of chronic inflammatory origin
Biomass That part of a given habitat consisting of living matter
Biosphere The part of the earth's crust, waters, and atmosphere where living organisms can
subsist
Biphasic Having two distinct successive stages
Bleb A collection of fluid beneath the skin, usually smaller than bullae or blisters
Blood urea The chief end product of nitrogen metabolism in mammals, excreted in human
urine in the amount of about 32 grams (1 ounce) a day
Bloom A greenish-gray appearance imparted to silk and pile fabncs either by nature of the
weave or by the finish, also, the creamy white color observed on some good cottons
Blue-green algae A group of simple plants that are the only nitiogen-fixing organisms that
photosynthesize as do higher plants
Bnghtener A compound, such as a dye, that adheres to fabncs in order to provide better
brightness or whiteness by converting ultraviolet radiation to visible light Sometimes
called optical bleach or whitening agent The dyes used are of the florescent type
Broad bean The large flat edible seed of an Old World upright vetch (Viaafabd), or the
plant itself, widely grown for its seeds and for fodder
Bronchi. The first subdivisions of the trachea, which conduct air to and from the bronchioles
of the lungs
Bronchiole One of the finer subdivisions of the bronchial (trachea) tubes, less than
1 millimeter in diameter, and having no cartilage in its wall
Bronchiolitis Inflammation of the bronchioles, which may be acute or chronic If the
etiology is known, it should be stated If permanent occlusion of the lumens is present,
the term bronchiohtis obliterans may be used
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Bronchiohtis fibrosa obliterans syndrome Obstruction of the bronchioles by fibrous
granulation arising from an ulcerated mucosa, the condition may follow inhalation of
irritant gases
Bronchitis A nonneoplastic disorder of structure or function of the bronchi resulting from
infectious or noninfectious irritation The term bronchitis should be modified by
appropriate words or phrases to indicate its etiology, its chromcity, the presence of
associated airways dysfunction, or the type of anatomic change The term chronic
bronchitis, when unqualified, refers to a condition associated with prolonged exposure
to nonspecific bronchial irritants and accompanied by mucus hypersecretion and certain
structural alterations in the bronchi Anatomic changes may include hypertrophy of the
mucous-secreting apparatus and epithelial metaplasia, as well as more classic evidences
of inflammation In epidemiologic studies, the presence of cough or sputum production
on most days for at least three months of the year for at least two consecutive years has
sometimes been accepted as a criterion for the diagnosis
Bronchoconstrictor An agent that causes a reduction in the caliber (diameter) of a bronchial
tube
Bronchodilator An agent that causes an increase in the caliber (diameter) of a bronchus or
bronchial tube
Bronchopneumoma Acute inflammation of the walls of the smaller bronchial tubes, with
irregular area of consolidation due to spread of the inflammation into penbronchiolar
alveoli and the alveolar ducts
Bronchospasm Temporary narrowing of the bronchi due to a violent, involuntary contraction
of the smooth muscle of the bronchi
Bronchus. One of the subdivisions of the trachea serving to convey air to and from the lungs
The trachea divides into right and left main bronchi, which in turn form lobar,
segmental, and subsegmental bronchi
Browman diffusion Diffusion by random movement of particles suspended in liquid or gas,
resulting from the impact of molecules of the fluid surrounding the particles
BTPS conditions (BTPS) Body temperature, barometric pressure, and saturated with water
vapor These are the conditions existing in the gas phase of the lungs For humans,
the normal temperature is 37 °C, the pressure is based on the barometric pressure, and
the partial pressure of water vapor is 47 torr
Buffer A substance in solution capable of neutralizing both acids and bases and thereby
maintaining the original pH of the solution
Buffering In reference to soil acidification, this is resistance to change resultmg from
reserves of acid or base cations on the soil cation-exchange sites
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Buffering capacity Ability of a body of water and its watershed to neutralize introduced acid
Butanol A four-carbon, straight-chain alcohol, C4H9OH, also known as butyl alcohol
Butylated hydroxytoluene (BHT) A crystalline phenolic antioxidant
Butylated hydroxyamsol (BHA) An antioxidant
C labeling Use of a radioactive form of carbon as a (racer, often in metabolic studies
C-proline An ammo acid that has been labeled with radioactive carbon
Calcareous Resembling or consisting of calcium carbonate (lime), or growing on limestone
or lime-containing soils
Calorie Amount of heat required to raise temperature of 1 gram of water at 15 °C by 1 °C
Cannula A tube that is inserted into a body cavity, or other tube or vessel, usually to remove
fluid
Capillary The smallest type of vessel, resembles a hair Usually in reference to a blood or
lymphatic capillary vessel
Carbachol A cholinergic parasympathetic stimulant, carbamoylcholine chloride
(C6H15CIN2O2), that produces constriction of the bronchial smooth muscles similar to
acetylchohne
Carbon monoxide An odorless, colorless, toxic gas with a strong affinity for hemoglobin and
cytochrome, it reduces oxygen absorption capacity, transport, and utilization
Carboxyhemoglobin A fairly stable union of carbon monoxide with hemoglobin that
interferes with the normal transfer of carbon dioxide and oxygen during circulation of
blood Increasing levels of carboxyhemoglobin result in various degrees of
asphyxiation, including death
Carcinogen Any agent producing or playing a stimulatory role m the formation of a
malignancy
Carcinoma Malignant new growth made up of epithelial cells tending to infiltrate the
surrounding tissues and giving nse to metastases
Cardiac output The volume of blood passing through the heart per unit time
Cardiovascular Relating to the heart and the blood vessels or the circulation
Carotene Lipid-soluble yellow-to-orange-red pigments universally present the photosynthetic
tissues of higher plants, algae, and the photosynthetic bacteria ,
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Cascade impactor A device for measuring the size distribution of particulates and/or
aerosols, consisting of a series of plates with orifices of graduated size that separate the
sample into a number of fractions of decreasing aerodynamic diameter
Catabolisnr Destructive metabolism involving the release of energy and resulting in
breakdown of complex materials in the organism
Catalase An enzyme (EC 11116) catalyzing the decomposition of hydrogen peroxide to
water and oxygen
Catalysis* A modification of the rate of a chemical reaction by some material, which is
unchanged at the end of the reaction
Catalytic converter' An air pollution abatement device that removes organic contaminants by
oxidizing them into carbon dioxide and water
Catecholamine A pyrocatechol with an alkalamine side chain, functioning as a hormone or
neurotransmitter, such as epmephnne, norepinephrine, or dopamine
Cathepsins: Enzymes that have the ability to hydrolyze certain proteins and peptides, occur in
cellular structures known as lysosomes
Cation A positively charged ion
Cation exchange capacity. The ability of a soil to absorb positively charged ions by
electostatic forces This absorption occurs on negatively charged sites on clays and
organic matter in soils
Cellular permeability Ability of gases to enter and leave cells, a sensitive indicator of injury
to deep-lung cells
Cellulose: The basic substance that is contained in all vegetable fibers It is a carbohydrate
and constitutes the major substance in plant life Used to make cellulose acetate and
rayon
Cellulose acetate Commonly refers to fibers or fabncs in which the cellulose is only partially
acetylated with acetate groups An ester made by reacting cellulose with acetic
anhydride with sulfate as a catalyst
Cellulose rayon A regenerated cellulose that is chemically the same as cellulose except for
physical differences in molecular weight and crystallmity
Cellulose triacetate A cellulose fiber that is completely acetylated Fabncs of triacetate have
higher heat resistance than acetate and may be safely ironed at higher temperature
Such fabncs have improved ease-of-care characteristics because after heat treatment
during manufacture, a change in the crystalline structure of the fiber occurs
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Cellulosics Cotton, viscose rayon, and other fibers made of natural-fiber raw materials
Celsius scale The thermometnc scale in which the freezing point of water is 0 and the
boiling point is 100
Central hepatic necrosis The pathologic death of one or more cells, or of a portion of the
liver, involving the cells adjacent to the central veins
Central nervous system (CNS) The brain and the spinal cord
Centroacinar area The center portion of a gland shaped as a bunch of grapes
Cerebellum The large posterior brain-mass lying above Ihe pons and medulla and beneath the
posterior portion of the cerebrum
Cerebral cortex The layer of gray matter covering the entire surface of the cerebral
hemisphere of mammals
Chain reaction A reaction that stimulates its own repetition
Challenge Exposure of a test organism to a virus, bacteria, or other stress-causing agent,
used in conjunction with exposure to a pollutant of interest, to explore possible
susceptibility brought on by the pollutant
Chamber study Research conducted using a closed vessel in which pollutants are reacted or
substances are exposed to pollutants
Chemiluminescence A measurement technique in which radiation is produced as a result of
chemical reaction
Chemotactic' Relating to attraction or repulsion of living protoplasm by chemical stimuli
Chlorophyll A group of closely related green photosynthetic pigments occurring in leaves,
bactena, and organisms
Chloroplast A plant cell inclusion body containing chlorophyll
Chlorosis Discoloration of normally green plant parts that can be caused by disease, lack of
nutrients, or various air pollutants, resulting in the failure of chlorophyll to develop
Cholesterol A steroid alcohol (C2H45OH), the most abundant steroid in animal cells and
body fluids
Chobnesterase (CHE) One (EC 3 1 1 8) of a family of enzymes capable of catalyzing the
hydrolysis of acylcholines
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Chondrosarcoma' A malignant neoplasm derived from cartilage cells, occurring most
frequently near the ends of long bones
Chromatid Each of the two strands formed by longitudinal duplication of a chromosome that
becomes visible during an early stage of cell division
Chromophore A chemical group that produces color in a molecule by absorbing near
ultraviolet or visible radiation when bonded to a nonabsorbing, saturated residue that
possesses no unshared, nonbonding valence electrons
Chromosome. One of the bodies (46 in humans) in the cell nucleus that is the bearer and
earner of genetic information
Chronic obstructive pulmonary disease (COPD) This term refers to diseases of uncertain
etiology characterized by persistent slowing of airflow during forced expiration It is
recommended that a more specific term, such as chronic obstructive bronchitis or
chronic obstructive emphysema, be used whenever possible Synonymous with chronic
obstructive lung disease (COLD)
Cilia: Motile, often hairlike extensions of a cell surface
Ciliary action' Movements of cilia in the upper respiratory tract, which move mucus and
foreign material upward
Cihogenesis. The formation of cilia
Citric acid (Krebs) cycle A major biochemical pathway in cells, involving terminal oxidation
of fatty acids and carbohydrates. It yields a major portion of energy needed for
essential body functions and is the major source of carbon dioxide It couples the
glycolytic breakdown of sugar in the cytoplasm with those reactions producing
adenosine tnphosphate in the mitochondria It also serves to regulate the synthesis of a
number of compounds required by a cell
Clara cell A nonciHated cell in the epithelium of the respiratory tract
Closing capacity (CC) Closmg volume plus residual volume, often expressed as a ratio of
total lung capacity (TLC) (i e , CC/TLC%)
Closing volume (CV) The volume exhaled after the expired gas concentration is inflected
from an alveolar plateau during a controlled breathing maneuver (Most commonly
obtained during a single-breath nitrogen washout test) Because the value obtained is
dependent on the specific test technique, the method used must be designated in the
text, and when necessary, specified by a qualifying symbol Closmg volume is often
expressed as a ratio of the vital capacity (VC) (i e , CV/VC%)
Codon A sequence of three nucleotides that encodes information required to direct the
synthesis of one or more ammo acids
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Coefficient of haze (COH) A measurement of visibility interference in the atmosphere
Cohort A group of individuals or vital statistics about them having a statistical factor in
common in a demographic study (e g , year of biith, sex, level of exposure to a
pollutant, etc)
Collagen The major protein of the white fibers of connective tissue, cartilage, and bond
Comprises over half the protein of the mammal.
Collisional deactivation Reduction in energy of excited molecules caused by collision with
other molecules or other objects such as the walls of a container
Colonmetnc A chemical analysis method relying on measurement of the degree of color
produced in a solution by reaction with the pollutant of interest
Community exposure A situation in which people in a sizeable area are subjected to ambient
pollutant concentrations
Compliance (CL,Cst) A measure of distensibility Pulmonary compliance is given by the
slope of a static volume-pressure curve at a point, or the linear approximation of a
nearly straight portion of such a curve, expressed as the change in volume per unit
change in distending pressure (liters per centimeter of water or milliliters per centimeter
of water) Because the static volume-pressure characteristics of lungs are nonlinear
(static compliance decreases as lung volume increases) and vary according to the
previous volume history (static compliance at a given volume increases immediately
after fuE inflation and decreases following deflation), careful specification of the
conditions of measurement are necessary Absolute values also depend on organ size
See also dynamic compliance
Complement Thermolabile substance present in serum that is destructive to certain bacteria
and other cells that have been sensitized by specific complement-fixing antibody
Compound A substance with its own distinct properties, formed by the chemical combination
of two or more elements in fixed proportion
Concanavalin-A One of two crystalline globulins occurring in the jack bean, a potent
hemagglutimn
Conductance (G) The reciprocal of resistance See airway conductance
Conifer A plant, generally evergreen, needle-leafed, bearing naked seeds singly or in cones
Converter See catalytic converter
Coordination number The number of bonds formed by the central atom in a complex
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Copolymer The product of the process of polymerization in which two or more monomenc
substances are mixed prior to polymerization Nylon is a copolymer
Coproporphynn One of two porphynn compounds found normally in feces as a
decomposition product of bilirubin (a bile pigment) Porphynn is a widely distributed
pigment consisting of four pyrrole nuclei joined in a ring
Cordage. A general term which includes banding, cable, cord, rope, string, and twine made
from fibers Synthetic fibers used in making cordage include nylon and dacron
Corrosion. Destruction or deterioration of a material because of reaction with its
environment
Corticosterone: A steroid obtained from the adrenal cortex It induces some deposition of
glycogen in the liver, sodium conservation, and potassium excretion
Cosmopolitan- In the biological sciences, a term denoting worldwide distribution
Coulometnc: Chemical analysis performed by determining the amount of a substance released
in electrolysis by measuring the number of coulombs used
Coumann A toxic white crystalline lactone (C9H6O^) found in plants
Coupler. A chemical used to combine two others in a reaction (e g , to produce the azo dye
in the Gness-Saltzman method for nitrogen dioxide)
Crevice corrosion Localized corrosion occurring within crevices on metal surfaces exposed
to corrosives
Critical Load A quantitative estimate of an exposure to one or more pollutants below which
significant harmful effects on specified sensitive elements of the ecosystem do not occur
according to present knowledge
Crosslink To connect, by an atom or molecule, parallel chains in a complex chemical
molecule, such as a polymer
Cryogenic trap. A pollutant sampling method in which a gaseous pollutant is condensed out
of sampled air by cooling (e g , traps in one method for mtrosamines are maintained
below —79 °C, using solvents maintained at their freezing points)
Cuboidal Resembling a cube in shape
Cultivar An organism produced by parents belonging to different species or to different
strains of the same species, originating and persisting under cultivation
Cuticle. A thin outer layer, such as the thin continuous fatty film on the surface of many
higher plants
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Cyanosis A dark bluish or purplish coloration of the skin and mucous membrane due to
deficient oxygenation of the blood
Cyclic GMP Guanosine 5'-phosphoric acid
Cytochrome A class of hemoprotein whose principal biological function is electron and/or
hydrogen transport
Cytology The anatomy, physiology, pathology, and chemistry of the cell
Cytoplasm The substance of a cell exclusive of the nucleus
Dacron The trade name for polyester fibers made by EI du Pont de Nemours and Co ,
Inc , made from dimethyl terephthalate and ethylene glycol
Dark adaptation The process by which the eye adjusts under reduced illumination and the
sensitivity of the eye to light is greatly increased
Dark respiration Metabolic activity of plants at night, consuming oxygen to use stored sugars
and releasing carbon dioxide
Deciduous plants Plants that drop their leaves at the end of the growing season
Degradation (textiles) The decomposition of fabric or its components or characteristics
(color, strength, elasticity) by means of light, heat, or air pollution
Denitnfication A bacterial process occurring in soils, or water, in which nitrate is used as
the terminal electron acceptor and is reduced primarily to molecular nitrogen It is
essentially an anaerobic process, it can occur in the presence of low levels of oxygen
only if the microorganisms are metabolizing in an anoxic microzone
De novo. Over again.
Deoxynbonucleic acid (DNA) A nucleic acid considered to be the earner of genetic
information coded in the sequence of purine and pynmidine bases (organic bases)
It has the form of a double-stranded helix of a linear polymer
Depauperate Falling short of natural development or size
Deposition
Acidic Removal of acidic pollutants from the atmosphere by dry and wet deposition
Dry Removal of pollutants from the atmosphere through interactions with various
surfaces of plants, land, and water
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Respiratory tract The depositing of inhaled pollutants within the respiratory tract,
which depends on breathing patterns, airway geometry, and the physical and chemical
properties of the inhaled pollutants
Wet Removal of pollutants from the atmosphere by precipitation (e g , rain or snow)
Derivative spectrophotometer An instrument with an increased capability for detecting
overlapping spectral lines and bands and also for suppressing instrumentally scattered
light
Desorb: To release a substance that has been taken into another substance or held on its
surface, the opposite of absorption or adsorption
Desquamation The sheading of the outer layer of any surface
Detection hmit A level below which an element or chemical compound cannot be reliably
detected by the method or measurement being used for analysis
Detritus- Loose material that results directly from disintegration
DeVarda alloy An alloy of 50% copper, 45% aluminum, and 5% zinc
Diastohc blood pressure The blood pressure as measured during the period of filling the
cavities of the heart with blood
Diazonium salt A chemical compound (usually colored) of the general structure ArN2 Cl",
where Ar refers to an aromatic group
Diazotizer A chemical that, when reacted with amines (RNH2 for example), produces a
diazonium salt (usually a colored compound)
Dichotomous sampler A device used to collect separately fine and coarse particles from an
aerosol and to measure gravimetrically the concentration of such different-sized
particles in the ambient air
Differentiation The process by which a cell, such as a fertilized egg, divides into specialized
cells, such as the embryonic types that eventually develop into an entire organism
Diffusion: The process by which molecules or other particles intermingle as a result of their
random thermal motion
Diffusing capacity of the lung (DL, DLO2, DLCO2, DLCO) Amount of gas (oxygen, carbon
monoxide, carbon dioxide) commonly expressed as milliliters of gas (standard
temperature and pressure, dry) diffusing between alveolar gas and pulmonary capillary
blood per torr mean gas pressure difference per minute (such as mL O2/min-torr)
Synonymous with transfer factor and diffusion factor
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Duner A compound formed by the union of two like radicals or molecules
Dimenze Formation of dimers t
1,6-diphosphofructose aldolase An enzyme (EC 4 1 1 13) cleaving fructose 1,6-bisphosphate
to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate
D-2,3-diphosphoglycerate A salt or ester of 2,3-diphosphoglyceiic acid, a major component
of certaui mammalian erythrocytes involved in the i elease of oxygen from
oxyhemoglobin Also a postulated intermediate in the biochemical pathway involving
the conversion of 3- to 2-phosphoglycenc acid
Diplococcus pneumomae A species of spherical-shaped bacteria belonging to the genus
Streptococcus May be a causal agent in pneumonia
Direct dye A dye with an affinity for most fibers; used mainly when color resistance to
washing is not important
Disperse dyes Also known as acetate dyes, these dyes were developed for use on acetate
fabrics, and are now also used on synthetic fibers
Distal Far from some reference point such as median line of the body, point of attachment,
or origin
Diurnal Having a repeating pattern or cycle 24 hours long
DLCO The diffusing capacity of the lungs for carbon monoxide The ability of the lungs to
transfer carbon monoxide from the alveolar air into the pulmonary capillary blood
Dorsal kyphosis Abnormal curvative of the spine, hunchback
Dose The quantity of a substance to betaken all at one tune or in fractional amounts within a
given period, also the total amount of a pollutant delivered or concentration per unit
tune tunes tune
Dose-response curve A curve on a graph based on responses occurring in a system as a
result of a series of stimuli intensities or doses
Dry deposition The processes by which matter is transferred to ground from the atmosphere,
other than precipitation, includes surface absorption of gases and sedimentation,
Browman diffusion, and impaction of particles
Dyemg A process of coloring fibers, yarns, or fabrics wnth either natural or synthetic dyes
Dynamic calibration Testing of a monitoring system using a continuous sample stream of
known concentration
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Dynamic compliance (Cdyn) the ratio of the tidal volume to the change in intrapleural
pressure between the points of zero flow at the extremes of tidal volume (L/cm H2O or
mL/cm H2O) Because at the points of zero airflow at the extremes of tidal volume,
volume acceleration is usually other than zero, and because, particularly in abnormal
states, flow may still be taking place within lungs between regions that are exchanging
volume, dynamic compliance may differ from static compliance, the latter pertaining to
condition of zero volume acceleration and zero gas flow throughout the lungs
In normal lungs at ordinary volumes and respiratory frequencies, static and dynamic
compliance are the same
Dynel. A trademark for a modacrylic staple fiber spun from a copolymer of acrylomtnle and
vinyl chloride It has high strength, quick-drying properties, and resistance to alkalies
and acids
Dyspepsia. Indigestion, upset stomach
Dyspnea* Shortness of breath, difficulty or distress in breathing, rapid breathing
Ecosystem The interacting system of a biological community and its environment
Eddy: A current of water or air running contrary to the main current
Edema Pressure of excess fluid in cells, intercellular tissue, or cavities of the body
Elastance (E) The reciprocal of compliance (expressed in centimeters of water per liter or
centimeters of water per milliliter)
Elastomer. A synthetic rubber product that has the physical properties of natural rubber
Electrocardiogram The graphic record of the electrical currents that initiate the heart's
contraction
Electrode One of the two extremities of an electric circuit
Electrolyte* A nonmetallic electric conductor in which current is earned by the movement of
ions; also a substance that displays these qualities when dissolved in water or another
solvent
Electronegativity Measure of affinity for negative charges or electrons
Electron microscopy A technique that utilizes a focused beam of electrons to produce a
high-resolution image of minute objects such as particulate matter, bacteria, viruses,
andDNA
Electronic excitation energy Energy associated in the transition of electrons from their
normal low-energy orbitals to orbitals of higher energy
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Electrophihc Having an affinity for electrons
Electrophoresis A technique by which compounds can be separated from a complex mixture
by their attraction to the positive or negative pole of an applied electric potential
Eluant A liquid used in the process of elution
Elute To perform an elution
Elution Separation of one material from another by washing or by dissolving one in a
solvent in which the other is not soluble
Elutriate To separate a coarse, insoluble powder from a finer one by suspending them
in water and pouring off the finer powder from the upper part of the fluid
Emission spectrometry A rapid analytical technique based on measurement of the
characteristic radiation emitted by thermally or electrically excited atoms or ions
Emphysema A condition of the lung characterized by abnormal, permanent enlargement
of airspaces distal to the terminal bronchiole, accompanied by the destruction of
their walls, and without obvious fibrosis
Emphysematous lesions A wound or injury to the lung as a result of emphysema
Empirical modeling Characterization and descnption of a phenomena based on expenence or
observation
i '
Encephalitis Inflammation of the brain
Endoplasmic reticulum An elaborate membrane structuie extending from the nuclear
membrane or eucaryotic cells to the cytoplasmic membrane
Endothekum A layer of flat cells lining especially blood and lymphatic vessels
Entropy A measure of disorder or randomness in a syslem Low entropy is associated with
highly ordered systems
Enzyme* Any of numerous proteins produced by living cells that catalyze biological
reactions
Enzyme Commission (EC) The International Commission on Enzymes, established in 1956,
developed a scheme of classification and nomenclature under which each enzyme is
assigned an EC number that identifies it by function
Eosinophils Leukocytes (white blood cells) that stain readily with the dye eosin
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Epidemiology. A study of the distribution and determinants of disease in human population
groups.
Epidermis. The outermost living layer of cells of any organism
Epididymal fat pads The fatty tissue located near the epididymis The epididymis is the first
convoluted portion of the excretory duct of the testis
Epiphyte' A plant growing on another plant but obtaining food from the atmosphere
Epithelial: Relating to epithelium, the membranous cellular layer that covers free surfaces or
lines tubes or cavities of an animal body, which encloses, protects, secretes, excretes
and/or assimilates
Erosion corrosion Acceleration or increase in rate of deterioration or attack on a metal
because of relative movement between a corrosive fluid and the metal surface
Characterized by grooves, gullies, or waves in the metal surface
Erythrocyte: A mature red blood cell
Eschenchia coll A short, gram-negative, rod-shaped bacteria common to the human
intestinal tract A frequent cause of infections in the urogemtal tract
Esophageal Relating to the portion of the digestive tract between the pharynx and the
stomach
Estrus. That portion or phase of the sexual cycle of female animals characterized by
willingness to permit coitus
Estrus cycle The series of physiologic uterine, ovarian, and other changes that occur in
higher animals
Etiolation Paleness and/or altered development resulting from the absence of light
Etiology The causes of a disease or condition, also, the study of causes
Eucaryotic: Pertaining to those cells having a well-defined nucleus surrounded by a
double-layered membrane
Eutrophication- Elevation of the level of nutrients in a body of water, which can contribute to
accelerated plant growth and filling
Excited state: A state of higher electronic energy than the ground state, usually a less stable
one
Expiratory (maximum) flow rate The maximum rate at which air can be expelled from the
lungs
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Exposure level Concentration of a contaminant to which an individual or a population is
exposed
Extinction coefficient A measure of the space rate of diminution, or extinction, of any
transmitted light, thus, it is the attenuation coefficient applied to visible radiation
Extramedullary hematopoiesis The process of formation and development of the various
types of blood cells and other formed elements not including that occurring in bone
marrow
Extravasate To exclude from or pass out of a vessel into the tissues, applies to urine, lymph,
blood, and similar fluids
Far ultraviolet Radiation in the range of wavelengths from 100 to 190 nanometers
Federal Reference Method (FRM) For nitrogen dioxide, the EPA-approved analyzers based
on the gas-phase chemiluminescent measurement principle and associated calibration
procedures, regulatory specifications prescribed in Title 40, Code of Federal
Regulations, Part 50, Appendix F
Fenestrae Anatomical aperatures often closed by a membrane
FEVt/FVC A ratio of tuned (t = 0 5, 1, 2, 3 seconds) forced expiratory volume (FEVt) to
forced vital capacity (FVC) The ratio is often expressed in percent
(100 X FEVt/FVC) It is an index of airway obstruction
Fiber- A fine, threadlike piece, as of cotton, jute, or asbestos
Fiber-reactive dye A water-soluble dyestuff that reacts chemically with the cellulose in fibers
under alkaline conditions; the dye contains two chlorine atoms that combine with the
hydroxyl groups of the cellulose
Fibrin A white insoluble elastic filamentous protein denved from fibnnogen by the action of
thrombin, especially in the clotting of blood
Fibroadenoma A benign neoplasm denved from glandular epithelium, involving proliferating
fibroblasts, cells found in connective tissue
Fibroblast An elongated cell with cytoplasmic processes present in connective tissue, capable
of forming collagen fibers
Fibrosis The formation of fibrous tissue, usually as a reparative or reactive process and not
as a normal constituent of an organ or tissue
Fine particles Airborne particles smaller than 2 to 3 micrometers in aerodynamic diameter
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Flocculation Separation of material from a solution or suspension by reaction with a
flocculant to create fluffy masses containing the material to be removed
Flow volume curve Graph of instantaneous forced expiratory flow recorded at the mouth,
against corresponding lung volume When recorded over the full vital capacity, the
curve includes maximum expiratory flow rates at all lung volumes in the vital capacity
range and is called a maximum expiratory flow-volume curve (MEFV) A partial
expiratory flow-volume curve (PEFV) is one which describes maximum expiratory flow
rate over a portion of the vital capacity only
Fly ash: Fine, solid particles of noncombustible ash earned out of a bed of solid fuel by a
draft.
Fogs: Suspension of liquid droplets formed by condensation of vapor or atomization, the
concentration of particles is sufficiently high to obscure visibility
Folded-path optical system A long (e g , 8 to 22 meters) chamber with multiple mirrors at
the ends which can be used to reflect an infrared beam through an ambient air sample
many times, a spectrometer can be used with such a system to detect trace pollutants at
very low levels
Forced expiratory flow (FEF) Related to some portion of the forced vital capacity (FVC)
curve Modifiers refer to the amount of the FVC already exhaled when the
measurement is made For example
FEF75% = Instantaneous forced exhaled flow after 75% of the forced vital capacity has
been exhaled
FEF2oo-i,200 = Mean forced expiratory flow between 200 milliliters and
1,200 mJHiters of the forced vital capacity (formerly called the maximum expiratory
flow rate [MEFR])
= Mean forced expiratory flow during the middle half of the forced vital
capacity (formerly called the maximum midexpiratory flow rate [MMFR])
FEFmax = The maximal forced expiratory flow achieved during an forced vital
capacity
Forced expiratory volume (FEV) Denotes the volume of gas that is exhaled in a given time
interval from the beginning of the execution of a forced vital capacity Conventionally,
the times used are 05,0 75, or 1 second, symbolized FEV0 5, FEV0 75, and FEVj 0,
respectively These values are often expressed as a percent of the forced vital capacity,
for example, (FEVl o/FVC) x 100
Forced inspiratory vital capacity (FIVC) The maximal volume of air inspired with a
maximally forced effort from a position of maximal expiration
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Forced vital capacity (FVC) The maximum volume of aiir that can be forcibly expelled from
the lungs after the deepest inspiration
Fractional threshold concentration The portion of the concentration at which an event or a
response begins to occur, expressed as a fraction
Free radical Any of a variety of highly reactive atoms or molecules characterized by having
an unpaired electron
Fritted bubbler A porous glass device used in air pollutant sampling systems to introduce
small bubbles into solution
Functional residual capacity (FRC) The volume of gas remaining in the lungs at the end of a
normal expiration It is the sum of expiratory reserve volume and residual volume (see
pulmonary measurements)
Gas chromatography (GC) A method of separating and analyzing mixtures of chemical
substances A flow of gas causes the components of a mixture to migrate differentially
from a narrow starting zone in a special porous, insoluble sorptive medium The
pattern formed by zones of separated pigments and of colorless substances in this
process is called a chromatogram, and can be analyzed to obtain the concentration of
identified pollutants
Gas exchange Movement of oxygen from the alveoli into the pulmonary capillary blood as
carbon dioxide enters the alveoli from the blood In broader terms, the exchange of
gases between alveoli and lung capillaries
Gas-liquid chromatography A method of separating and analyzing volatile organic
compounds in which a sample is vaporized and swept through a column filled with solid
support material covered with a nonvolatile liquid Components of the sample can be
identified and their concentrations can be determined by analysis of the characteristics
of their retention in the column because compound;, have varying degrees of solubility
in the liquid medium
Gas trapping Trapping of gas behind small airways that were opened during inspiration but
closed during forceful expiration It is a volume difference between forced vital
capacity and vital capacity
Gastric juice A thin watery digestive fluid secreted by glands in the mucous membrane of
the stomach
Gastroenteritis Inflammation of the mucous membrane of stomach and intestine
Genotype The type of genes possessed by an organism
Geometric mean An estimate of the average of a distribution Specifically, the nth root of
the product of n observations
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Geometric standard deviation A measure of variability of a distribution It is the
antiloganthm of the standard deviation of the logarithms of the observations
Globulins (a, b, q). A family of proteins precipitated from plasma (or serum) by
half-saturation with ammonium sulfate, or separable by electrophoresis The main
groups are the a, b, and q fractions, differing with respect to associated lipids and
carbohydrates and in their content of antibodies (immunoglobulins)
Glomerular nephrotic syndrome Dysfunction of the kidneys characterized by excessive
protein loss in the urine, accumulation of body fluids, and alteration in
albumin/globulin ratio
Glucose. A sugar that is a principal source of energy for humans and other organisms
Glucose-6-phosphate dehydrogenase An enzyme (EC 111 49) catalyzing the
dehydrogenation of glucose-6-phosphate to 6-phosphogluconolactone
Glutamic-oxaloacetic transaminase (SGOT) An enzyme (EC 2611) whose serum level
increases in myocardial infarction and in diseases involving destruction of liver cells
Also known as aspartate aminotransferase
Glutamic-pyruvic transaminase (SGPT) Now known as alanine aminotransferase
(EC 2612), the serum levels of this enzyme are used in liver function tests
Glutathione (GSH) A tnpeptide composed of glycine, cystine, and glutamic acid
Glutathione peroxidase An enzyme (EC 1111) that catalyzes the destruction of
hydroperoxides formed from fatty acids and other substances Protects tissues from
oxidative damage It is a selenium-containing protein
Glutathione reductase. The enzyme (EC 1642) that reduces the oxidized form of
glutathione
Glycolytic pathway The biochemical pathway by which glucose is converted to lactic acid in
various tissues, yielding energy as a result
Glycoside A type of chemical compound formed from the condensation of a sugar with
another chemical radical via a hemiacetal linkage
Goblet cells. Epithelial cells that have been distended with mucin and when this is discharged
as mucus, a goblet-shaped shell remains
Golgi apparatus: A membrane system involved with secretory functions and transport in a
cell Also known as a dictyosome
Grana The lamellar stacks of chlorophyll-containing material in plant chloroplasts
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Gnege carpet A carpet in its unfinished state (i e , beiore it has been scoured and dyed)
The term also is used for woven fabrics in the unbleached and unfinished state
Ground state The state of minimum electronic energy of a molecule or atom
Guanylate cyclase (GC) An enzyme (EC 4621) catalyzing the transformation of guanosine
tnphosphate to guanosine 3' 5'-cyclic phosphate
o
H-Thvmidine Thymine deoxynbonucleoside One of the four major nucleosides in DNA
H-thymiduie has been uniformly labeled with tritium, a radioactive form of hydrogen
Haze Fine dust, smoke, or fine vapor reducing transparency of air
Hemagglutination The agglutination of red blood cells Can be used as a measurement of
antibody concentration
Hematocnt The percentage of the volume of a blood sample occupied by cells
Hematology The medical specialty that pertains to the blood and blood-forming tissues
Hemochromatosis A disease characterized by pigmentation of the skin possibly due to
inherited excessive absorption of iron
Hemoglobin (Hb) The red, respiratory protein of the led blood cells, hemoglobin transports
oxygen from the lungs to the tissues as oxyhemoglobin (HbO2) and returns carbon
dioxide to the lungs as hemoglobin carbamate, completing the respiratory cycle
Hemolysis Alteration or destruction of red blood cells, causing hemoglobin to be released
into the medium in which the cells are suspended
Hepatectomy Complete removal of the liver in an experimental animal
Hepatic Relating to the liver
Hepatocyte A liver cell
Heterogeneous process A chemical reaction involving reactants of more than one phase or
state, such as one in which gases are absorbed into aerosol droplets, where the reaction
takes place
Heterologous A term referring to donor and recipient cellular elements from different
organisms, such as red blood cells from sheep and alveolar macrophage from rabbits
Heterotrophs Fungi and bacteria that rely on organic matter for their energy source
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Hexose monophosphate shunt Also called the phosphogluconate oxidative pathway of glucose
metabolism, which affords a total combustion of glucose independent of the citric acid
cycle. It is the important generator of nicotinamide-ademne dinucleotide phosphate
(reduced form) necessary for synthesis of fatty acids and the operation of various
enzymes It serves as a source of ribose and 4- and 7-carbon sugars
High volume (hi-vol) sampler A high flow-rate device used to collect particles from the
atmosphere and to gravimetncally measure the concentration of particles across a broad
range of sizes in ambient air
Histamine A depressor amine derived from the ammo acid histidine and found in all body
tissues, with the highest concentration in the lung, a powerful stimulant of gastric
secretion, a constrictor of bronchial smooth muscle, and a vasodilator that causes a fall
in blood pressure
Homogenate Commonly refers to tissue ground into a creamy consistency in which the cell
structure is disintegrated
Host defense mechanism Inherent means by which a biologic organism protects itself against
infection, such as antibody formation, macrophage action, ciliary action, etc
Host resistance The resistance exhibited by an organism, such as a human, to an infecting
agent, such as a virus or bacteria
Humoral: Relating to the extracellular fluids of the body, blood and lymph
Hybrid: An organism descended from parents belonging to different varieties or species
Hydrocarbons' A vast family of compounds containing carbon and hydrogen in various
combinations, found especially in fossil fuels Some contribute to photochemical smog
Hydrolysis* Decomposition involving splitting of a bond and addition of the H and OH parts
of water to the two sides of the split bond
Hydrometeor A product of the condensation of atmospheric water vapor (e g , fog, rain,
hail, snow)
Hydroxyprolme An amino acid found among the hydrolysis products of collagen
Hygroscopic* Pertaining to a marked ability to accelerate the condensation of water vapor
Hygroscopic growth Growth induced by moisture, often applied in reference to the growth in
size of inhaled particles within the respiratory tract in combination with resident
moisture
Hyperplasia Increase in the number of cells in a tissue or organ excluding tumor formation
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Hyperplastic Relating to hyperplasia, an increase in the number of cells
Hypertrophy Increase in the size of a tissue element, excluding tumor formation
Hypertension Abnormally elevated blood pressure
Hypolimma Portions of a lake below the thermocline in which water is stagnant and uniform
in temperature.
Hypoxia A lower than normal amount of oxygen in the air, blood, or tissues
Immunoglobulin (Ig) A class of structurally related proteins consisting of two pairs of
polypeptide chains Antibodies are immunoglobins and all immunoglobulins probably
function as antibodies
Immunoglobulin A (IgA) A type of antibody that comprises approximately 10 to 15 % of the
total amount of antibodies present in normal serum
Immunoglobulin G (IgG) A type of antibody that compnses approximately 80 % of the total
amount of antibodies present in normal serum Subtractions of IgG are fractions
Gj and 62
Immunoglobulin M (IgM) A type of antibody that compnses approximately 5 to 10% of the
total amount of antibodies present in normal serum
Impaction An impinging or striking of one object against another, also, the force transmitted
by this act
Impactor An instrument which collects samples of suspended particulates by directing a
stream of the suspension against a surface, or into a liquid or a void
Index of proliferation Ratio of promonocytes to polymorphic monocytes in the blood
Infarction Sudden insufficiency of arterial or venous blood supply due to emboh, thrombi, or
pressure
Infectivity model A testing system in which the susceptibility of animals to airborne
infectious agents with and without exposure to air pollutants is investigated to produce
information related to the possible effects of the pollutant on humans
Inflorescence The arrangement and development of flowers on an axis, also, a flower cluster
or a single flower
Influenza A2/Taiwan Virus An infectious-viral disease, believed to have originated in
Taiwan, characterized by sudden onset, chills, fevers, headache, and cough
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l-j
Infrared Light invisible to the human eye, between the wavelengths of 7 x 10" and
10~3 meter (7,000 and 10,000,000 Angstroms)
Infrared laser A device that utilizes the natural oscillations of atoms or molecules to generate
coherent electromagnetic radiation in the infrared region of the spectrum
Infrared spectrometer An instrument for measuring the relative amounts of radiant energy in
the infrared region of the spectrum as a function of wavelength
Ingestion To take in for digestion
In situ- In the natural or original position
Instrumental averaging tune The tune over which a single example or measurement is taken,
resulting in a measurement that is an average of the actual concentrations over that
period
Insult An injury or trauma
Intercostal Between the nbs, especially of a leaf
Interferant A substance that a measurement method cannot distinguish completely from the
one being measured, which therefore can cause some degree of false response or error
Interferon* A macromolecular substance produced in response to infection with active or
inactivated virus, capable of inducing a state of resistance
Intergranular corrosion A type of corrosion that takes place at and adjacent to grain
boundaries, with relatively little corrosion of the grains
Interstitial edema An accumulation of an excessive amount of fluids in a space within
tissues
Interstitial pneumonia A chronic inflammation of the interstitial tissue of the lung, resulting
in compression of air cells
Intralummal mucus Mucus that collects within any tubule
Intraperitoneal injection An injection of material into the serous sac that lines the abdominal
cavity
In utero Within the womb, not yet born
In vitro* Refers to experiments conducted outside the living organism
In vivo. Refers to experiments conducted within the living organism
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Irradiation Exposure to any form of radiation
Ischemia Local anemia due to mechanical obstruction (mainly arterial narrowing) of the
blood supply
Isoenzymes Also called isozymes One of a group of enzymes that are very similar in
catalytic properties, but may be differentiated by variations in physical properties, such
as isoelectnc point or electrophoretic mobility Lactic acid dehydrogenase is an
example of an enzyme having many isomenc forms
Isopleth A line on a map or chart connecting points of equal value
Jacobs-Hochheiser method The original Federal Reference Method for nitrogen dioxide,
currently unacceptable for air pollution work
Klebsiella pneumomae A species of rod-shaped bacteria found in soil, water, and in the
intestinal tract of humans and other animals Certain types may be causative agents in
pneumonia
Kyphosis An abnormal curvature of the spine, with convexity backward
Lactate A salt or ester of lactic acid
Lactic acid (lactate) dehydrogenase (LDH) An enzyme (EC 111 27) with many isomenc
forms that catalyzes the oxidation of lactate to pyruvate via transfer of hydrogen to
nicotinamide-adenine dinucleotide Isomenc forms of lactic acid dehydrogenase in the
blood are indicators of heart damage
Lamellar bodies Arranged in plates or scales One of the characteristics of Type n alveolar
cells
Lavage fluid Any fluid used to wash out hollow organs, such as the lung
Leaching The removal of elements from soil, litter, or plant foliage by water
Lecithin Any of several waxy hygroscopic phosphatides that are widely distributed in
animals and plants, they form colloidal solutions in water and have emulsifying,
wetting, and hygroscopic properties
Legume A plant with root nodules containing nitrogen-fixing bactena
Lesion A wound, injury, or other more or less circumsc nbed pathologic change in the
tissues
Leukocyte. Any of the white blood cells
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Lewis base A base, defined in the Lewis acid-base concept, is a substance that can donate an
electron pair
Lichens Perennial plants which are a combination of two plants, an alga and a fungus,
growing together in an association so intimate that they appear as one
Ligand: Those molecules or amons attached to the central atom in a complex
Light-fastness The ability of a dye to maintain its original color under natural or indoor
light
Linolemc acid An unsaturated fatty acid essential in nutation
Lipase. An enzyme that accelerates the hydrolysis or synthesis of fats or the breakdown of
hpoproteins
Lipids: A heterogeneous group of substances that occur widely in biological materials They
are characterized as a group by their extractability in nonpolar organic solvents
Lipofuscin Brown pigment granules representing lipid-contaimng residues of lysosomal
digestion Proposed to be an end product of kpid oxidation that accumulates in tissue
Lipoprotein Complex or protein containing lipid and protein
Loading rate The amount of a nutrient available to a unit area of body of water over a given
period of tune
Locomotor activity Movement of an organism from one place to another of its own volition
Long path length infrared absorption A measurement technique in which a system of mirrors
in a chamber is used to direct an infrared beam through a sample of air for a long
distance (up to 2 kilometers), the amount of infrared light absorbed is measured to
obtain the concentrations of pollutants present
Lung compliance (CI) The volume change produced by an increase in a unit change in
pressure across the lung (i e , between the pleura! surface and the mouth)
Lycra* A spandex textile fiber created by E I du Pont de Nemours & Co , Inc , with
excellent tensile strength, a long flex life and high resistance to abrasion and heat
degradation Used in brassieres, foundation garments, surgical hosiery, swim suits, and
military and industrial applications
Lymphocytes White blood cells formed in lymphoid tissue throughout the body, they
comprise about 22 to 28 % of the total number of leukocytes in the circulating blood
and function in immunity
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Lymphocytogram The ratio, in the blood, of lymphocytes with narrow cytoplasm to those
with broad cytoplasm
Lysosomes Organelles found in cells of higher organisms that contain high concentrations of
degradative enzymes and are known to destroy foreign substances that cells engulf by
pinocytosis and phagocytosis Believed to be a major site where proteins are broken
down
Lysozymes Lytic enzymes destructive to cell walls of certain bacteria Present in some body
fluids, including tears and serum
Macaco, specwsa A species of monkeys used in research
Macrophage Any large, ameboid, phagocytic cell having a nucleus without many lobes,
regardless of origin
Malaise A feeling of general discomfort or uneasiness, often the first indication of an
infection or disease
Malate dehydrogenase An enzyme (EC 1 1 1 37) with at least six isomenc forms that
catalyze the dehydrogenation of malate to oxaloacetate or its decarboxylation (removal
of a carbon dioxide group) to pyruvate Malate, oxaloacetate, and pyruvate are
intermediate components of biochemical pathways
Manmtol An alcohol derived from reduction of the sugar fructose Used in renal function
testing to measure glomerular (capillary) filtration
Manometer An instrument for the measurement of pressure of gases or vapors
Mass median diameter (MMD) Geometric median size of a distribution of particles based on
weight
Mass spectrometry (MS) A procedure for identifying the various kinds of particles present in
a given substance by ionizing the particles and subjecting a beam of the ionized
particles to an electee or magnetic field such that the field deflects the particles in
angles directly proportional to the masses of the particles
Maximal expiratory flow (Vmax x) Forced expiratory flow, related to the total lung capacity
or the actual volume of the lung at which the measurement is made Modifiers refer to
the amount of lung volume remaining when the measurement is made For example
Vmax75% = Instantaneous forced expiratory flow when the lung is at 75% of its total
lung capacity
Vmax3 o = Instantaneous forced expiratory flow when the lung volume is 3 0 liters
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Maximal expiratory flow rate (MEFR) Obsolete terminology See FEF2oo-i,200
forced expiratory flow
Maximal midexpiratory flow rate (MMFR or MMEF) See FEF25_75% under forced
expiratory flow
Maximal ventilation (max VE) The volume of air breathed in 1 minute during repetitive
maximal respiratory effort Synonymous with maximum ventilatory minute volume
Maximal voluntary ventilation (MW) The volume (liters per minute at body temperature
and pressure, saturated) of air breathed by a subject during voluntary maximum
hyperventilation (rapid deep breathing) lastmg a specific period of time Replaces
maximal breathing capacity
Mean (arithmetic) The sum of observations divided by sample size
Mechanical clearance See mucociliary action
Median A value in a collection of data values that is exceeded in magnitude by one-half the
entries in the collection
MEFR' See FEF2oo-i,200 under forced expiratory flow
Mesoscale Of or relating to meteorological phenomena from 1 to 100 kilometers in
horizontal extent
Messenger RNA A type of RNA that conveys genetic information encoded in the DNA to
direct protein synthesis
Metaplasia The abnormal transformation of an adult, fully differentiated tissue of one land
into a differentiated tissue of another kind
Metaproterenol A bronchodilator used for the treatment of bronchial asthma
Metastases The shifting of a disease from one part of the body to another, the appearance of
neoplasms in parts of the body remote from the seat of the primary tumor
Meteorology The science that deals with the atmosphere and its phenomena
Methachohne A parasympathomimetic bronchoconstnctor drug with similarities to carbachol
and acetylcholine
91
Methemoglobin: A form of hemoglobin in which the normal reduced state of iron (Fe ) has
been oxidized to ferric iron (Fe ) It contains oxygen in firm union with Fe and is
not capable of exchanging oxygen in normal respiratory processes
Methimazole' An antithyroid drug similar ui action to propylthiouracil
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Methyltransferase Any enzyme transferring methyl groups from one compound to another
Microcoulometnc Capable of measuring millionths of coulombs used in electrolysis of a
substance, to determine the amount of a substance in a sample
Microflora A small or strictly localized plant
Micron One-millionth of a meter
Microphage A small phagocyte, a polymorphonuclear leukocyte that is phagocytic
Millimolar One-thousandth of a molar solution A solution of one-thousandth of a mole
(in grams) per liter
Mineral acid anion An amon associated with strong, or mineral acids such as sulfuric, nitric,
or hydrochloric acid These anions include nitrate (NO3~), sulfate (SO4 "), and chloride
(CO
Minute ventilation (Vg) See pulmonary measurements
Minute volume The minute volume of breathing, a product of tidal volume times the
respiratory frequency in one minute, synonymous with minute ventilation
Mitochondria Organelles of the cell cytoplasm that contain enzymes active in the
conservation of energy obtained in the aerobic part of the breakdown of carbohydrates
and fats, in a process called respiration
MMFR Maximal midexpiratory flow See FEF25_75% under forced expiratory flow
Mobile sources. Automobiles, trucks, and other pollution sources that are not fixed in one
location
Modacrylic fiber A manufactured fiber in which the fiber-forming substance is any long
chain synthetic polymer composed of less than 85 % but at least 35 % by weight of
acrylomtnte units
Moiety One of two or more parts into which something is divided
Mole The mass, in grams, numerically equal to the molecular weight of a substance
Molecular correlation spectrometry A spectrophotometric technique that is used to identify
unknown absorbing materials and measure their concentrations by using preset
wavelengths
Molecular weight The weight of one molecule of a substance obtained by adding the
gram-atomic weights of each of the individual atoms in the substance
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Monocyte A relatively large mononuclear leukocyte, normally constituting 3 to 7% of the
leukocytes of the circulating blood
Morbidity. The quantity or state of being diseased, also, used in reference to the ratio of the
number of sick individuals to the total population of a community (i e , morbidity rate)
Mordant A substance that acts to bind dyes to a textile fiber of fabric
Morphological. Relating to the form and structure of an organism or any of its parts
Morphology Structure and form of an organism at any stage of its life history
Morphometry The quantitative measurement of structure (morphology)
Mortality rate For a given period of time, the ratio of the number of deaths occurring per
1,000 population Also known as death rate
Moving average: A procedure involving taking averages over a specific period prior to and
including a year in question, so that successive averaging periods overlap (e g , a
three-year moving average would include data from 1967 through 1969 for the 1969
average and from 1968 through 1970 for 1970)
Mucociliary action Ciliary action of the mucous membranes lining respiratory tract airways
that aids in removing particles from the lungs
Mucociliary clearance Removal of materials from the upper respiratory tract via ciliary
action
Mucociliary transport The process by which mucus is transported, by ciliary action, from the
lungs
Mucosa* The mucous membrane It consists of epithelium, lamina propria, and, in the
digestive tract, a layer of smooth muscle
Mucous membrane A mucus-secreting membrane that lines passages and cavities
communicating with the exterior of the body
Mucus The clear, viscid secretion of mucous membranes, consisting of mucin, epithelial
cells, leukocytes, and various inorganic salts suspended in water
Murine Relating to mice
Mutagen' A substance capable of causing, within an organism, biological changes that affect
potential offspring through genetic mutation
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Mutagemc Having the power to cause mutations A mutation is a change in the character of
a gene (a sequence of base pairs in DNA) that is perpetuated in subsequent divisions of
the cell in which it occurs
Myocardial infarction Infarction of any area of the heart muscle usually as a result of
occlusion of a coronary artery
Mycorrhizae Fungi that live in association with plant roots and assist in the uptake of water
and nutrients in exchange for carbohydrates
Nares The nostrils
Nasopharyngeal Relating to the nasal cavity and the phairynx (throat)
National Air Surveillance Network (NASN) Network of monitoring stations for sampling au-
to determine extent of air pollution, established jointly by federal and state
governments
Near ultraviolet Radiation of the wavelengths 2,000 to 4,000 Angstroms
Necrosis Death of cells that can discolor areas of a plant or kill the entire plant
Necrotic Pertaining to the pathologic death of one or more cells, or of a portion of tissue or
organ, resulting from irreversible damage
Neonate A newborn
Neoplasm An abnormal tissue that grows more rapidly than normal, synonymous with
tumor
Neoplasm The pathologic process that results in the formation and growth of a tumor
Neutrophil A mature white blood cell formed in bone marrow and released into the
circulating blood Neutrophils normally account for 54 to 65 % of the total number of
leukocytes
Ninhydrm An organic reagent used to identify amino acids
Nitramine A compound consisting of a nitrogen attached to the nitrogen of amine
Nitrate A salt or ester of nitric acid (NO3~ is used to symbolize the ionic form, NO3 is used
for the radical)
Nitrification The principal natural source of nitrate in which ammonium ions (NH4 ) are
oxidized to nitrites by specialized microorganisms Other organisms oxidize nitrites to
nitrates
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Nitnfiers' Soil microorganisms that convert ammonium ions (NH4+) or organic nitrogen to
nitrate ions (NO3"), a process referred to as nitrification Organisms that convert
NH4+ to NO3" are referred to as autotrophic nitnfiers, and organisms that convert
organic nitrogen to NO3" are referred to as heterotrophic nitnfiers
Nitrite A salt or ester of nitrous acid (NO2")
Nitrocellulose Any of several esters of nitric acid formed by its action on cellulose, used in
explosives, plastics, varnishes, and rayon, also called cellulose nitrate
Nitrogen cycle: Refers to the complex pathways by which mtrogen-contaming compounds are
moved from the atmosphere into organic life, into the soil, and back to the atmosphere
Nitrogen fixation. The metabolic assimilation of atmospheric nitrogen by soil
microorganisms, which becomes available for plant use when the microorganisms die,
also, industrial conversion of free nitrogen into combined forms used in production of
fertilizers and other products
Nitrogen oxide- A compound composed of only nitrogen and oxygen Components of
photochemical smog
Nitrogen saturation A condition in which ecosystems are unable to accumulate any more
nitrogen
Nitrogen washout The multiple breath curve obtained by plotting the fractional concentration
of nitrogen in expired alveolar gas versus tune for a subject switched from breathing
ambient air to an inspired mixture of pure oxygen A progressive decrease of nitrogen
concentration ensues, which may be analyzed into two or more exponential
components
Nitrosamine: A compound consisting of a nitrosyl group connected to the nitrogen of an
amine
Nitrosation. Addition of a nitrosyl group
W-Nitroso compounds Compounds carrying the functional nitrosyl group
Nitrosyl: A group composed of one oxygen and one nitrogen atom (-N=O)
Nitrosylhemoglobin (NOHb) The red, respiratory protein of erythrocytes to which a nitrosyl
group is attached
N/P Ratio. Ratio of nitrogen to phosphorous dissolved in lake water, important due to its
effect on plant growth
Nucleolus A small spherical mass of material within the substance of the nucleus of a cell
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Nucleophilic Having an affinity for atomic nuclei, electron-donating
Nucleoside A compound that consists of a punne or pynmidine base combined with
deoxynbose or nbose and found in RNA and DNA
5'-Nucleotidase An enzyme (EC 3135) that hydrolyzes nucleoside 5'-phosphates mto
phosphoric acid (H3PO4) and nucleosides
Nucleotide A compound consistmg of a sugar (nbose or deoxynbose), a base (a punne or a
pynmidine), and a phosphate, a basic structural unit of RNA and DNA
Nylon A genenc name chosen by E I du Pont de Nemours & Co , Inc for a group of
protein-like chemical products classed as synthetic linear polymers, two main types are
Nylon 6 and Nylon 66
Occlusion A point at which an opening is closed or obstructed
Olefin An open-chain hydrocarbon having at least one double bond
Olfactory Relating to the sense of smell
Olfactory epithelium • Hie inner lining of the nose and mouth that contains neural tissue
sensitive to smell
Oligotrophic A body of water deficient in plant nutrients, also generally having abundant
dissolved oxygen and no marked stratification
Orbitals Areas of high electron density in an atom or molecule
Orion An acrylic fiber produced by E I du Pont de Nemours and Co , Inc , based on a
polymer of acrylomtnte, used extensively for outdoor uses, it is resistant to chemicals
and withstands high temperatures.
Oronasal breathing Breathing through the nose and mouth simultaneously, typical human
breathing pattern at moderate to high levels of exercise versus normally predominant
nasal breathing while at rest
Osteogenic osteosarcoma The most common and malignant of bone sarcomas (tumors)
It arises from bone-forming cells and affects chiefly the ends of long bones
Ovarian primordial follicle A spheroidal cell aggregation in the ovary in which the
primordial oocyte (miniature female sex cell) is surrounded by a smgle layer of
flattened follicular cells
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Oxidant' A chemical compound that has the ability to remove electrons from another
chemical species, thereby oxidizing it, also, a substance containing oxygen that reacts in
air to produce a new substance, or one formed by the action of sunlight on oxides of
nitrogen and hydrocarbons
Oxidation' An ion or molecule undergoes oxidation by donating electrons
Oxidative deamination Removal of the anune (NH2) group from an amino compound by
reaction with oxygen
Oxidative phosphorylation The mitochondnal process by which "high-energy" phosphate
bonds form from the energy released as a result of the oxidation of various substrates
Principally occurs in the tncarboxylic acid pathway
Oxyhemoglobin. Hemoglobin in combination with oxygen It is the form of hemoglobin
present in arterial blood
Ozone layer A layer of the stratosphere from 20 to 50 kilometers above the earth's surface
characterized by high ozone content produced by ultraviolet radiation
Ozone scavenging Removal of ozone from ambient air or plumes by reaction with nitric
oxide, producing nitrogen dioxide and molecular oxygen
Paired electrons Electrons having opposite intrinsic spins about their own axes
Parenchyma: The essential and distinctive tissue of an organ or an abnormal growth, as
distinguished from its supportive framework
Parenchymal Referring to the distinguishing or specific cells of a gland or organ
Partial pressure The pressure exerted by a single component in a mixture of gases
Particle Any object, solid or liquid, having definite physical boundaries in all directions,
includes, for example, fine solid particles, such as dust, smoke, fumes, or smog, found
in the air or in emissions
Particulate matter (PMX) Matter in the form of small airborne liquid or solid particles
In the abbreviation, the subscript "x" indicates the particulate mean aerodynamic
diameter
Particulates Fine liquid or solid particles, such as dust, smoke, mist, fumes, or smog, found
in the air or in emissions
Pascal. A unit of pressure in the International System of Units One pascal is equal to
7.4 X 10" torr The pascal is equivalent to 1 N per square meter
Pathogen1 Any virus, microorganism, or other substance causing disease
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Pathophysiological Derangement of function seen in disease, alteration in function as
distinguished from structural defects
Peak expiratory flow (PEF) The highest forced expiratory flow measured with a peak flow
meter
Peptide bond The bond formed when two ammo acids react with each other
Percentiles The percentage of all observations exceeding or preceding some point, thus,
90th percentile is a level below which 90 % of the observations will fall
Perennial Trees and other plants that live more than one year are called perennials
Perfusate A liquid, solution or colloidal suspension that has been passed over a special
surface or through an appropriate structure
Perfusion Artificial passage of fluid through blood vessels
Permanent-press fabrics Fabrics in which applied resins contnbute to the easy care and
appearance of the fabric and to the crease and seaim flatness by reacting with the
cellulose on pressing after garment manufacture
Permeation tube A tube that is selectively porous to specific gases
Peroxidation Refers to the process by which certain organic compounds are converted to
peroxides
Peroxyacetyl nitrate (PAN) Pollutant created by action of sunlight on hydrocarbons and
nitrogen oxides in the air, an ingredient of photochemical smog
pH A measure of the effective acidity or alkalinity of a solution It is expressed as the
negative logarithm of the hydrogen ion concentration Pure water has a hydrogen ion
concentration equal to 10~ M per liter at standard conditions (25 °C) The negative
logarithm of this quantity is 7 Thus, pure water has a pH value of 7 (neutral) The
pH scale is usually considered as extending from 0 to 14 A pH less than 7 denotes
acidity, and a pH more than 7 denotes alkalinity
Phagocytosis A mechanism by which alveolar macrophages and polymorphonuclear
leukocytes engulf particles, one of several lung defense mechanisms by which foreign
agents (biological and nonbiological) are removed from the respiratory tract
Phenotype. The observable characteristics of an organism, resulting from the interaction
between an individual genetic structure and the environment m which development
takes place
Phenylthiourea A crystalline compound (C7H8N2S) thait is bitter or tasteless depending on a
single dominant gene in the taster
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Phlegm: Viscid mucus secreted in abnormal quantity in the respiratory passages
Phosphatase Any of a group of enzymes that liberate inorganic phosphate from phosphoric
esters (EC sub-subclass 313)
Phosphocreatine kinase An enzyme (EC 2732) catalyzing the formation of creatine and
adenosine tnphosphate, its breakdown is a source of energy in the contraction of
muscle; also called creatine phosphate
Phospholipid A molecule consisting of kpid and phosphoric acid group(s) An example is
lecithin Serves as an important structural factor in biological membranes
Photochemical oxidants Primary ozone, nitrogen dioxide, and peroxyacetyl nitrate, with
lesser amounts of other compounds, formed as products of atmosphenc reactions
involving organic pollutants, nitrogen oxides, oxygen, and sunlight
Photochemical smog Air pollution caused by chemical reaction of various airborne chemicals
in sunlight
Photodissociation The process by which a chemical compound breaks down into simpler
components under the influence of sunlight or other radiant energy
Photolysis' Decomposition upon irradiation by sunlight
Photomultiplier tube An electron multiplier in which electrons released by photoelectric
emission are multiplied in successive stages by dynodes that produce secondary
emissions
Photon A quantum of electromagnetic energy
Photostationary A substance or reaction that reaches and maintains a steady state in the
presence of light
Photosynthesis The process in which green parts of plants, when exposed to light under
suitable conditions of temperature and water supply, produce carbohydrates using
atmosphenc carbon dioxide and release oxygen
Phyllosphere Usually refers to the leaf surface of plants
Phytotoxic- Poisonous to plants
Phytoplankton. Minute aquatic plant life
Pi (El) bonds Bonds in which electron density is not symmetrical about a line joining the
bonded atoms
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Pinocytotic Refers to the cellular process (pinocytosis) in which the cytoplasmic membrane
forms imaginations m the form of narrow channels leading into the cell Liquids can
flow into these channels and the membrane pinches off pockets that are incorporated
into the cytoplasm and digested
Pitting A form of extremely localized corrosion that results in holes m the metal One of the
most destructive forms of corrosion
Pituitary A stalk-Like gland near the base of the brain that is attached to the hypothalmus
The anterior portion is a major repository for hormones that control growth, stimulate
other glands, and regulate the reproductive cycle
Placenta The organ in the uterus that provides metabolic interchange between the fetus and
mother
Plasmid Replicating unit, other than a nucleus gene, that contains nucleoprotein and is
involved in various aspects of metabolism in organisms, also called paragenes
Plasmolysis The dissolution of cellular components, or the shrinking of plant cells by
osmotic loss of cytoplasmic water
Plastic A plastic is one of a large group of organic compounds synthesized from cellulose,
hydrocarbons, proteins, or resins and capable of being cast, extruded, or molded into
various shapes
Plasticizer A chemical added to plastics to soften, increase malleability, or make them more
readily deformable
Platelet (blood) An irregularly-shaped disk with no definite nucleus, about one-third to one-
half the size of an erythrocyte and containing no hemoglobin Platelets are more
numerous than leukocytes, numbering from 200,000 to 300,000 per cubic millimeter of
blood
Plethysmograph A device for measuring and recording changes in volume of a part, organ,
or the whole body, a body plethysmograph is a chamber apparatus surrounding the
entire body
Pleura The serous membrane enveloping the lungs and lining the walls of the chest cavity
Plume Emission from a flue or chimney, usually m a stream-like distribution downwind of
the source, which can be distinguished from the surrounding air by appearance or
chemical characteristics
Pneumonia (interstitial) A chronic inflammation of the interstitial tissue of the lung, resulting
in compression of the air cells An acute, infectious disease
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Pneumonocytes A nonspecific term sometimes used in referring to types of cells
characteristic of the respiratory part of the lung
Podzol: Any of a group of zonal soils that develop in a moist climate, especially under
coniferous or mixed forests
Point source A single stationary location of pollutant discharge
Polarography A method of quantitative or qualitative analysis based on current-voltage
curves obtained by electrolysis of a solution with steadily increasing voltage
Pollution gradient' A series of exposure situations in which pollutant concentrations range
from high to low
Polyacrylonitnle. A polymer made by reacting ethylene oxide and hydrocyanic acid Dynel
and Orion are examples
Polyamides Polymerization products of chemical compounds which contain ammo (-NH2)
and carboxyl (-COOH) groups Condensation reactions between the groups form
amides (-CONEy Nylon is an example of a polyamide
Polycarbonate Any of various tough transparent thermoplastics characterized by high impact
strength and high softening temperature
Polycythemia An increase above the normal in the number of red cells in the blood
Polyester fiber A manufactured fiber in which the fiber-forming substance is any long-chain
synthetic polymer composed of at least 85 % by weight of an ester of a dihydnc alcohol
and terephthalic acid Dacron is an example
Polymer. A large molecule produced by linking together many like molecules
Polymerization In fiber manufacture, converting a chemical monomer (simple molecule) into
a fiber-forming material by joining many like molecules into a stable, long-chain
structure
Polymorphic monocyte Type of leukocyte with a multilobed nucleus
Polymorphonuclear leukocytes Cells that represent a secondary nonspecific cellular defense
mechanism They are transported to the lungs from the bloodstream when the burden
handled by the alveolar macrophages is too large
Polysacchandes Polymers made up of sugars An example is glycogen, which consists of
repeating units of glucose
Polystyrene- A thermoplastic plastic that may be transparent, opaque, or translucent It is
light in weight, tasteless, and odorless, it also is resistant to ordinary chemicals
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Polyurethane Any of various polymers that contain NHCOO linkages and are used especially
in flexible and rigid foams, elastomers, and resins*
Pores of Kohn Also known as interalveolar pores, pores between air cells Assumed to be
pathways for collateral ventilation
Precipitation Any of the various forms of water particles that fall from the atmosphere to the
ground, rain, snow, etc
Precursor A substance from which another substance is formed, specifically, one of the
anthropogenic or natural emissions or atmospheric constituents that reacts under
sunlight to form secondary pollutants comprising photochemical smog
Probe In air pollution sampling, the tube or other conduit extending into the atmosphere to
be sampled, through which the sample passes to treatment, storage, and/or analytical
equipment
Proline An amino acid (C5H9NO2) that can be synthesized from glutamate by animals
Promonocyte An immature monocyte not normally seen in the circulating blood
Proteinuna The presence of more than 0 3 gram of urinary protein in a 24-hour urine
collection
Pulmonary Relating to the lungs
Pulmonary edema An accumulation of excessive amounts of fluid in the lungs
Pulmonary lumen The spaces in the interior of the tubular elements of the lung (bronchioles
and alveolar ducts)
Pulmonary measurements Measurements of the volume of air moved during a normal or
forced inspiration or expiration Specific lung volume measurements are defined
independently
Lung volume measurements = Tidal volume, inspiratory reserve volume, expiratory
reserve volume, residual volume (four basic independent volumes)
Capacities = Combinations of basic volumes
Total lung capacity (TLC) = Tidal volume + inspiratory reserve volume + expiratory
reserve volume + residual volume, the volume of gas in the lungs at the tune of
maximal inspiration or the sum of all volume compartments The method of
measurement should be indicated, as with residual volume
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Vital capacity (VC) = Tidal volume + inspiratoiy reserve volume + expiratory
reserve volume, the greatest volume of gas that can be expelled by voluntary effort
after maximal inspiration Also forced vital capacity and forced inspiratory vital
capacity
Functional residual capacity (FRC) = Residual volume + expiratory reserve volume,
the volume of gas remaining in the lungs at the resting, end-tidal expiratory position
Equivalent to the sum of residual volume and expiratory reserve volume The method
of measurement should be indicated as with residual volume
Inspiratory capacity (1C) = Tidal volume + inspiratory reserve volume
Inspiratory vital capacity (TVC) = The maximal volume that can be inspired from the
resting end-expiratory position, also forced inspiratory vital capacity
Expiratory reserve volume (ERV) = The maximal volume that can be exhaled from the
resting end-tidal expiratory position See also Functional residual capacity
Residual volume (RV) = That volume of air remaining in the lungs after maximal
exhalation The method of measurement should be indicated in the text or, when
necessary, by appropriate qualifying symbols
Residual volume to total lung capacity ratio (RV/TLC) = A ratio that expresses the
percentage of the total lung capacity occupied by residual volume, varies somewhat
with age, but ordinarily should be no more than 20 to 30%
Tidal volume = That volume of air inhaled or exhaled with each breath during quiet
breathing, used only to indicate a subdivision of lung volume When tidal volume is
used in gas exchange formulations, the symbol VT should be used
Minute ventilation (MV) = The volume of gas exchanged per minute at rest or during
any stated activity, it is the tidal volume times the number of respirations per minute
See ventilation
Pulmonary resistance Sum of airway resistance and viscous tissue resistance
Purine bases Organic bases that are constituents of DNA and RNA, including adenine and
guanine
Purulent. Containing or forming pus
Pynmidine bases Organic bases found in DNA and RNA Cytosine and thymine occur in
DNA and cytosine and uracil are found in RNA
QRS: Graphical representation on the electrocardiogram of a complex of three distinct waves
that represent the beginning of ventricular contraction
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Quasistatic compliance Time dependent component of elasticity, compliance is the reciprocal
of elasticity
Rain out Removal of particles and/or gases from the atmosphere by their involvement in
cloud formation (particles act as condensation nuclei, gases are absorbed by cloud
droplets), with subsequent precipitation
Rayleigh scattering Coherent scattering in which the intensity of the light of wavelength X
scattered in any direction, making an angle with the incident direction, is directly
proportional to 1 + cos and inversely proportion.il to X
Reactive dyes Dyes that react chemically with cellulose in fibers under alkaline conditions
Also called fiber reactive or chemically reactive dyes
Reduction Acceptance of electrons by an ion or molecule
Reference method (RM) For nitrogen dioxide, an EPA-approved gas-phase chemiluminescent
analyzer and associated calibration techniques, regulatory specifications are described in
Title 40, Code of Federal Regulations, Part 50, Appendix F Formerly, Federal
Reference Method
Residual capacity The volume of air remaining in the lungs after a maximum expiratory
effort, same as residual volume
Residual volume (RV)- The volume of air remaining in the lungs after a maximal expiration
The residual volume is equal to the total lung capacity minus the vital capacity
Resin Any of various solid or semisolid amorphous natural organic substances, usually
derived from plant secretions, which are soluble in organic solvents but not in water,
also any of many synthetic substances with similar properties used in finishing fabncs,
for permanent press shrinkage control or water rq)ellency
Resistance flow (R) The ratio of the flow-resistive components of pressure to simultaneous
flow (in centimeters of water per liter per second) Flow-resistive components of
pressure are obtained by subtracting any elastic or inertial components, proportional
respectively to volume and volume acceleration Most flow resistances in the
respiratory system are nonlinear, varying with the magnitude and direction of flow,
with lung volume and lung volume history, and possibly with volume acceleration
Accordingly, careful specification of the conditions of measurement is necessary, see
airway resistance and total pulmonary resistance
Ribosomal RNA The most abundant RNA in a cell and an integral constituent of nbosomes
Ribosomes Discrete units of RNA and protein that are mstrumental in the synthesis of
proteins in a cell Aggregates are called polysomes
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Runoff: Water from precipitation, irrigation, or other sources that flows over the ground
surface to streams
Sclerosis Pathological hardening of tissue, especially from overgrowth of fibrous tissue or
increase in interstitial tissue
Secondary particles (or secondary aerosols) Dispersion aerosols that form in the atmosphere
as a result of chemical reactions, often involving gases
Selective leaching The removal of one element from a solid alloy by corrosion processes
Septa. A thin wall dividing two cavities or masses of softer tissue
Seromucoid' Pertaining to a mixture of watery and mucinous material such as that of certain
glands
Serum antiprotease A substance, present in serum, that inhibits the activity of proteinases
(enzymes that destroy proteins)
Sigma (s) bonds. Bonds in which electron density is symmetrical about a line joining the
bonded atoms
Silo-filler's disease Pulmonary lesion produced by oxides of nitrogen produced by fresh
silage
Single breath nitrogen elimination rate Percentage rise in nitrogen fraction per unit of
volume expired
Single breath nitrogen technique A procedure in which a vital capacity inspiration of
100% oxygen is followed by examination of nitrogen in the vital capacity expired
Singlet state: The highly reactive energy state of an atom in which certain electrons have
unpaired spins
Sink: A reactant with or absorber of a substance
Sodium arsemte (Na3AsO3) A compound used with sodium hydroxide in the absorbing
solution of a 24-hour integrated manual method for nitrogen dioxide
Sodium dithiomte- A strong reducing agent (a supplier of electrons)
Sodium metabisulfite (Na2S2O5) A compound used in absorbing solutions of nitrogen dioxide
analysis methods
Sorb. To take up and hold by absorption or adsorption
Sorbent A substance that takes up and holds another by absorption or adsorption
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Sorbitol dehydrogenase An enzyme that interconverts the sugars sorbitol and fructose
Sorption The process of being sorbed
Spandex A manufactured fiber in which the fiber-forming substance is a long chain synthetic
elastomer composed of at least 85 % of a segmented polyurethane
Specific airway conductance (SGaw) Airway conductance divided by the lung volume at
which it was measured, that is, normalized airway conductance Airway conductance
(Gaw)/thoracic gas volume (TGV)
Specific airway resistance (SR^) Airway resistance multiplied by the volume at which it
was measured SR&W = airway resistance (R^) X thoracic gas volume (TGV), liter
(L) X centimeter of water per liter per second (cm H2O/L/s)
Spectrometer An instrument used to measure radiation spectra or to determine wavelengths
of the various radiations _
Spectrophotometry A technique in which visible, ultraviolet, 01 infrared radiation is passed
through a substance or solution and the intensity ol light transmitted at various
wavelengths is measured to determine the spectrum of light absorbed
Spectroscopy Use of the spectrometer to determine concentrations of an air pollutant
Spermatocytes A cell destined to give nse to spermatozoa (sperm)
Sphingomyelins A group of phospholipids found in brain, spinal cord, kidney, and egg yolk
Sphygmomanometer An apparatus, consisting of a cuff and a pressure gauge, that is used to
measure blood pressure
Spirometer A mechanical device, including bellows or other sealed, moving parts, that
collects and stores gases and provides a graphical record of lung volume changes over
time See breathing pattern and respiratory cycle
Spirometry The measurement, by a form of gas meter (spirometer), of volumes of air that
can be moved in and out of the lungs
Spleen A large vascular organ located on the upper left side of the abdominal cavity It is a
blood-forming organ in early life It is a storage organ for red corpuscles and because
of the large number of macrophages, acts as a blood filter
Sputum Expectorated matter, especially mucus or mucopurulent matter expectorated as a
result of diseases of the air passages
Squamous Scale-like, scaly
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Standard deviation Measure of the dispersion of values about a mean value It is calculated
as the positive square root of the average of the squares of the individual deviations
from the mean
Standard temperature and pressure 0 °C, 760 millimeters of mercury
Standard temperature and pressure, dry (STPD) conditions These are the conditions of a
volume of gas at 0 °C and 760 torr, without water vapor An STPD volume of a given
gas contains a known number of moles of that gas
Staphylococcus aureus A spherically shaped, infectious species of bacteria found especially
on nasal mucous membrane and skin
Static lung compliance (CLstat) Measure of lung's elastic recoil (volume change resulting
from change in pressure) with no or insignificant airflow
Steady state exposure Exposure to air pollutants whose concentration remains constant for a
period of time
Steroids. A large family of chemical substances comprising many hormones and vitamins and
having large ring structures
Stilbene- An aromatic hydrocarbon (C14H12) used as a phosphor and in making dyes
Stoichiometric factor Used to express the conversion efficiency of a nonquantitative reaction,
such as the reaction of nitrogen dioxide with azo dyes in air monitoring methods
Stoma A minute opening or pore (plural is stomata)
Stratosphere That region of the atmosphere extending from 11 kilometers above the surface
of the earth to 50 kilometers At 50 kilometers above the earth, temperature rises to a
maximum of 0 °C
Streptococcus pyogenes A species of bacteria found in the human mouth throat, and
respiratory tract and in inflammatory exudates, blood stream, and lesions in human
diseases It causes formation of pus or even fatal septicemias
Stress corrosion cracking Cracking caused by simultaneous presence of tensile stress and a
specific corrosive medium The metal or alloy is virtually unattached over most of its
surface, while fine cracks progress through it
Strong interactions Forces or bond energies holding molecules together Thermal energy
will not disrupt the formed bonds
Sublobular hepatic necrosis The pathologic death of one or more cells, or of a portion of the
liver, beneath one or more lobes
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Succession The progressive natural development of vegetation towards a climax, during
which one community is gradually replaced by others
Succinate A salt of succinic acid involved in energy production in the citnc acid cycle
Sulfadiazine One of a group of sulfa drugs Highly effective against pneumococcal,
staphylococcal, and streptococcal infections
Sulfamethazine An antibacterial agent of the sulfonamide group, active against homolytic
streptococci, staphylococci, pneumococci, and memngococci
Sulfamlimide A crysialline sulfonamide (C6H8N2O2S), the amide of sulfanilic acid and
parent compound of most sulfa drugs
group A chemical radical consisting of sulfur and hydrogen (-SH) that confers
reducing~~potential to the chemical compound to which it is attached
ir ---
" -=- ._ Sulfur dioxide (SO2) Colorless gas with pungent odor leleased prunanly from burmng of
fossil fuels, such as coal, containing sulfur
Sulfur dyes Used only on vegetable fibers, such as cottons They are insoluble in water and
must be converted chemically in order to be soluble They are resistant (fast) to
alkalies and washing and fairly fast to sunlight
Supernatant The clear or partially clear liquid layer that separates from the homogenate upon
centnfugation or standing
Surfactant A substance capable of altering the physiochemical nature of surfaces, such as one
used to reduce surface tension of a liquid
Symbiotic A close association between two organisms of different species in which at least
one of the two benefits
Synergistic A relationship in which the combined action or effect of two or more
components is greater than that of the components acting separately.
Systolic Relating to the rhythmical contraction of the heart
Tachypnea Very rapid breathing
Teragram (Tg) One million metric tons, 1012 grams
Teratogenesis The disturbed growth processes resulting in a deformed fetus
Teratogenic Causing or relating to abnormal development of the fetus
Threshold The level at which a physiological or psychological effect begins to be produced
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Thylakoid' A membranous lamella of protein and lipid in plant chloroplasts where the
photochemical reactions of photosynthesis take place
Thymidme: A nucleoside (C^gH^^C^) that is composed of thymrne and deoxynbose, occurs
as a structural part of DNA
Tidal volume (V-p). The volume of air that is inspired or expired in a single breath during
regular breathing.
Titer: The standard of strength of a volumetric test solution For example, the titration of a
volume of antibody-containing serum with another volume containing virus
Tocopherol a-^-Tocopherol is one form of Vitamin E prepared synthetically The a. form
exhibits the most biological activity It is an antioxidant and retards rancidity of fats
Torr A unit of pressure sufficient to support a 1 -millimeter column of mercury, 760 torr =
1 atmosphere
Total lung capacity (TLC) The sum of all the compartments of the lung, or the volume of air
in the lungs at maximum inspiration
Total pulmonary resistance (RL) Resistance measured by relating flow-dependent
transpuhnonary pressure to airflow at the mouth Represents the total (factional)
resistance of the lung tissue (R^) and the airways (RaW), RL = Raw + ^u
Total suspended particulates (TSP) Solid and liquid particles present in the atmosphere
Trachea' Commonly known as the windpipe, a cartilaginous air tube extending from the
lamyx (voice box) into the thorax (chest), where it divides, serving as the entrance to
each of the lungs
Tracheobronchial region The area encompassed by the trachea to the gas exchange region of
the lung; the conducting airways
Transaminase Armnotransferase, an enzyme transferring an ammo group from an a-amino
acid to the carbonyl carbon atom of an a-keto acid
Transmissivity (UV) The percent of ultraviolet radiation passing through a medium
Transmittance The fraction of the radiant energy entering an absorbing layer that reaches the
layer's further boundary
Transpiration The process of the loss of water vapor from plants
Triethanolamine An amine dHOCE^CEy^N) used in the absorbing solution of one
analytical method for nitrogen dioxide
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Troposphere That portion of the atmosphere in which temperature decreases rapidly with
altitude, clouds form, and mixing of air masses by convection takes place Generally
extends to about 7 to 10 miles above the earth's surface
Type 1 cells Thin, alveolar surface, epithelial cells across which gas exchange occurs
Type 2 cells Thicker, alveolar surface, epithelial cells that produce surfactant and serve as
progenitor cells for Type 1 cell replacement
n
Ultraviolet Light invisible to the human eye of wavelengths between 4 x 10" and
5 x 10 meter (4,000 to 50 Angstroms)
Urea-formaldehyde resin A compound composed of urea and formaldehyde in an
arrangement that conveys thermosetting properties
Urobilinogen' One of the products of destruction of blood cells, found in the liver, intestines,
and urine
Uterus The womb, the hollow muscular organ in which the impregnated ovum (egg)
develops into the fetus
Vacuole A minute space in any tissue
Vagal Refers to the vagus nerve This mixed nerve arises near the medulla oblongata and
passes down from the cranial cavity to supply the larynx, lungs, heart, esophagus,
stomach, and most of the abdominal viscera
Valence The number of electrons capable of being bonded or donated by an atom during
bonding
Van Slyke reactions Reaction of primary amines, including ammo acids, with nitrous acid,
yielding molecular nitrogen.
Variance A measure of dispersion or variation of a sample from its expected value, it is
usually calculated as the square root of a sum of squared deviations about a mean
divided by the sample size
Vat dyes Dyes that have a high degree of resistance to fading by light, nitrogen oxides, and
washing Widely used on cotton and viscose rayon Colors are brilliant and of almost
any shade The name was originally derived from their application in a vat
Venezuelan equine encephalomyelitis A form of equine encephalomyelitis found in parts of
South America, Panama, Trinidad, and the United States, and caused by a virus
Fever, diarrhea, and depression are common In humans, there is fever and severe
headache after an incubation period of 2 to 5 days
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Ventilation Physiological process by which gas is exchanged between the outside air and the
lungs The word ventilation sometimes designates ventilatory flow rate (or ventilatory
minute volume), which is the product of the tidal volume multiplied by the ventilatory
frequency Conditions are usually indicated as modifiers, for example
VE = Expired volume per minute (liters per minute, body temperature and pressure,
saturated [BTPS]),
Vj = Inspired volume per minute (liters per minute, BTPS)
Ventilation is often referred to as "total ventilation" to distinguish it from "alveolar
ventilation" (see ventilation, alveolar)
Ventilation, alveolar (VA) The portion of the total ventilation that is involved in gas
exchange with the blood, alveolar ventilation is less than total ventilation because when
a tidal volume of gas leaves the alveolar spaces, the last part does not get expelled from
the body but occupies the dead space, to be reinspired with the next inspiration Thus,
the volume of alveolar gas actually expelled completely is equal to the tidal volume
minus the volume of the dead space This truly complete expiration volume tunes the
ventilatory frequency constitutes the alveolar ventilation
Ventilation, dead-space (VD) Ventilation per minute of the physiologic dead space (volume
of gas not involved in gas exchange with the blood), at body temperature and pressure,
saturated conditions, defined by the following equation
VD = VE(PaCO2 - PECO2)/(PaCO2 - PiCO2)
Ventilation/perfusion ratio (VA/Q) Ratio of the alveolar ventilation to the blood perfusion
volume flow through the pulmonary parenchyma, such as pulmonary blood flow or
right heart cardia output, this ratio is a fundamental determinant of the oxygen and
carbon dioxide pressure of the alveolar gas and of the end-capillary blood Throughout
the lungs, the local ventilation/perfusion ratios vary, and, consequently, the local
alveolar gas and end-capillary blood compositions also vary
Villus. A projection from the surface, especially of a mucous membrane
Vinyl chloride A gaseous chemical suspected of causmg at least one type of cancer It is
used pnmanly in the manufacture of polyvinyl chloride, a plastic
Viscose rayon Filaments of regenerated cellulose coagulated from a solution of cellulose
xanthate Raw materials can be cotton linters or chips of spruce, pine, or hemlock
Visible region Light between the wavelengths of 4,000 and 8,000 Angstroms
Visual range The distance at which an object can be distinguished from background
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Vital capacity (VC) The greatest volume of air that can be exhaled from the lungs after a
maximum inspiration (see Pulmonary measurements)
Vitamin E Any of several fat-soluble vitamins (tocopherols) essential in nutrition of various
vertebrates
Washout The capture of gases and particles by falling raindrops
Weak interactions Forces, electrostatic in nature, that bind atoms and/or molecules to each
other Thermal energy will disrupt the interaction Also called Van der Waal's forces
Weathering In this context, weathering refers to the releases of base cations from soil
minerals to cationic forms, which can be taken up by plants, leached, or absorbed to
cation-exchange sites
Wet deposition The process by which atmospheric substances aie returned to earth in the
form of rain or other precipitation
Wheat germ lipase An enzyme, obtained from wheat germ, that is capable of cleaving a fatty
acid from a neutral fat, a lipolytic enzyme
X-ray fluorescence spectrometry A nondestructive technique that utilizes the principle that
every element emits characteristic x-ray emissions when excited by high-energy
radiation
Zeolites Hydrous silicates analogous to feldspars, occurring in lavas and various soils
Zooplankton Minute animal life floating or swimming weakly in a body of water
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* U S GOVERNMEHT PRINTING OFFICE 1993—550-001 / 80325
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