EPA
United States
Environmental Protection
Agency
Environmental Criteria and
Assessment Office
Washington DC 20460
EPA/600/8-84/020dP
August 1986
Research and Development
Air Quality Criteria for
Ozone and Other
Photochemical
Oxidants
Volume IV of V
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EPA-600/8-84-020dF
August 1986
Air Quality Criteria
for Ozone and Other
Photochemical Oxidants
Volume IV of V
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade
names or commercial products does not constitute.endorsement or
recommendation.
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ABSTRACT
Scientific information is presented and evaluated relative to the health
and welfare effects associated with exposure to ozone and other photochemical
oxidants. Although it is not intended as a complete and detailed literature
review, the document covers pertinent literature through early 1985.
Data on health and welfare effects are emphasized, but additional infor-
mation is provided for understanding the nature of the oxidant pollution pro-
blem and for evaluating the reliability of effects data as well as their
relevance to potential exposures to ozone and other oxidants at concentrations
occurring in ambient air. Information is presented on the following exposure-
related topics: nature, source, measurement, and concentrations of precursors
to ozone and other photochemical oxidants; the formation of ozone and other
photochemical oxidants and their transport once formed; the properties, chem-
istry, and measurement of ozone and other photochemical oxidants; and the
concentrations of ozone and other photochemical oxidants that are typically
found in ambient air.
The specific areas addressed by chapters on health and welfare effects
are the toxicological appraisal of effects of ozone and other oxidants; effects
observed in controlled human exposures; effects observed in field and epidemic-
logical studies; effects on vegetation seen in field and controlled exposures;
effects on natural and agroecosystems; and effects on nonbiological materials
observed in field and chamber studies.
111
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AIR QUALITY CRITERIA FOR OZONE
AND OTHER PHOTOCHEMICAL OXIDANTS
Page
VOLUME I
Chapter 1. Summary and Conclusions 1-1
VOLUME II
Chapter 2. Introduction .. . 2-1
Chapter 3. Properties, Chemistry, and Transport of Ozone and
Other Photochemical Qxidants and Their Precursors 3-1
Chapter 4. Sampling and Measurement of Ozone and Other
Photochemical Oxidants and Their Precursors .» 4-1
Chapter 5. ' Concentrations of Ozone and Other Photochemical
^Oxidants in Ambient Air ,. 5-1
VOLUME III
Chapter 6. Effects of Ozone and Other Photochemical Oxidants
on Vegetation 6-1
Chapter 7. Effects of Ozone on Natural Ecosystems and Their
Components 7-1
Chapter 8. Effects of Ozone and Other Photochemical Oxidants
on Nonbiological Materials 8-1
VOLUME IV
Chapter 9. Toxicological Effects of Ozone and Other
Photochemical Oxidants 9-1
VOLUME V
Chapter 10. Controlled Human Studies of the Effects of Ozone
and Other Photochemical Oxidants 10-1
Chapter 11. Field and Epidemiological Studies of the Effects
of Ozone and Other Photochemical Oxidants 11-1
Chapter 12. Evaluation of Health Effects Data for Ozone and
Other Photochemical Oxidants 12-1
IV
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TABLE OF CONTENTS
Page
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS xiii
AUTHORS, CONTRIBUTORS, AND REVIEWERS xiv
9. TOXICOLOGICAL EFFECTS OF OZONE AND OTHER PHOTOCHEMICAL OKIDANTS ... 9-1
9.1 INTRODUCTION 9-1
9.2 REGIONAL DOSIMETRY IN THE RESPIRATORY TRACT 9-3
9.2.1 Absorption in Experimental Animals 9-4
9.2.1.1 Nasopharyngeal Absorption ..... ., 9-4
9.2.1.2 Lower Respiratory Tract Absorption 9-5
9.2.2 Ozone Dosimetry Models J 9-6
9.2.2.1 Modeling Nasal Uptake .. ............/;.; 9-6
9.2.2.2 Lower Respiratory Tract Dosimetry
Models 9-6
9.2.3 Predictions of Lower Respiratory Tract Ozone
Dosimetry Modeling 9-10
9.2.3.1 Illustration of Dosimetry Simulations 9-11
9.2.3.2 Comparison of Simulations to Experimental
Data .............. 9-14
9.2.3.3 Uses of Predicted Dose . 9-15
9.3 EFFECTS OF OZONE ON THE RESPIRATORY TRACT 9-16
9.3.1 Morphological Effects 9-16
9.3.1.1 Sites Affected 9-16
9.3.1.2 Sequence in which Sites are Affected
as a Function of Concentration and
Duration of Exposure 9-41
9.3.1.3 Structural Elements Affected ... ...... 9-42
9.3.1.4 Considerations of Degree of Suscepti-
bility to Morphological Changes 9-46
9.3.2 Pulmonary Function Effects ........ 9-52
9.3.2.1 Short-Term Exposure 9-52
9.3.2.2 Long-Term Exposure 9-57
9.3.2.3 Airway Reactivity 9-62
9.3.3 Biochemically Detected Effects 9-73
9.3.3.1 Introduction 9-73
9.3.3.2 Antioxidant Metabol ism 9-73
9.3.3.3 Oxidative and Energy Metabolism ... ... 9-88
9.3.3.4 Monooxygenases , 9-92
9.3.3.5 Lactate Dehydrogenase and Lysosomal
Enzymes 9-95
9.3.3.6 Protein Synthesis 9-98
9.3.3.7 Lipid Metabolism and Content of the
Lung 9-108
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TABLE OF CONTENTS (continued)
9.5
9.6
9.3.4
9.3.5
9.3.3.8 Lung Permeability
9.3.3.9 Proposed Molecular Mechanisms of
Effects
Effects on Host Defense Mechanisms
Mucociliary Clearance
Alveolar Macrophages
Interaction with Infectious Agents
Immunology•
9.3.4.1
9.3.4.2
9.3.4.3
9.3.4.4
Tolerance
9.4 EXTRAPULMONARY EFFECTS OF OZONE
9.
9.
9.
9.4.4
9.4.5
9.4.6
Central Nervous System and Behavioral Effects
Cardiovascular Effects
Hematological and Serum Chemistry Effects ....
9.4.3.1 Animal Studies - Iri Vivo Exposures .
9.4.3.2 In Vitro Studies
9.4.3.3 Changes in Serum
9.4.3.4 Interspecies Variations
Reproductive and Teratogenic Effects
Chromosomal and Mutational Effects
9.4.5.1 Chromosomal Effects of Ozone
9.4.5.2 Mutational Effects of Ozone
Other Extrapulmonary Effects
9.4.6.1 Liver
9.4.6.2 The Endocrine System i.,
9.4.6.3 Other Effects
EFFECTS OF OTHER PHOTOCHEMICAL OXIDANTS ......
9.5.1 Peroxyacetyl Nitrate
9.5.2 Hydrogen Peroxide
9.5.3 Formic Acid
9.5.4 Complex Pollutant Mixtures
SUMMARY
9.6.1 Introduction
9.6.2 Regional Dosimetry in the Respiratory
9.6.3 Effects of Ozone on the Respiratory Tr
9.6.3.1 Morphological Effects ......
9.6.3.2 Pulmonary Function
9.6.3.3 Biochemical Effects
9.6.3.4 Host Defense Mechanisms ....
9. 6. 3. 5 To! erance
9.6.4 Extraoulmonarv Effects of Ozone
Tract
"act
9.6.4.1 Central Nervous System and Behavioral
Effects
9.6.4.2 Cardiovascular Effects
9.6.4.3 Hematological and Serum Chemistry
Effects
Page
9-110
9-113
9-119
9-120
9-125
9-132
9-141
9-144
9-153
9-153
9-158
9-159
9-159
9-166
9-170
9-172
9-173
9-176
9-176
9-189
9-190
9-190
9-197
9-204
9-205
9-205
9-206
9-209
9-209
9-217
9-217
9-218
9-221
9-221
9-224
9-228
9-235
9-243
9-244
9-245
9-245
9-246
VI
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TABLE OF CONTENTS (continued)
Page
9.6.4.4 Cytogenetic and Teratogenic Effects 9-247
9.6.4.5 Other Extrapulmonary Effects 9-248
9.6.5 Interaction of Ozone with Other Pollutants 9-249
9.6.6 Effects of Other Photochemical Oxidants 9-252
9.7 REFERENCES 9-257
APPENDIX A A-l
vn
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LIST OF TABLES
Table
Page
9-1 Morphological effects of ozone 9-17
9-2 Effects of ozone on pulmonary function: short-term exposures ... 9-53
9-3 Effects of ozone on pulmonary function: long-term exposures .... 9-58
9-4 Effects of ozone on pulmonary function: airway reactivity ...... 9-63
9-5 Changes in the lung antioxidant metabolism and oxygen
consumpti on by ozone 9-75
9-6 Monooxygenases 9-93
9-7 Lactate dehydrogenase and lysosomal enzymes , 9-96
9-8 Effects of ozone on lung protein synthesis 9-99
9-9 Effects of ozone exposure on lipid metabolism and content of
the lung . 9-109
9-10 Effects of ozone on lung permeability 9-111
9-11 Effects of ozone on host defense mechanisms: deposition and
clearance 9-122
9-12 Effects of ozone on host defense mechanisms: macrophage
alterations .' 9-126
9-13 Effects of ozone on host defense mechanisms: interactions
with infectious agents ., 9-135
9-14 Effects of ozone on host defense mechanisms: mixtures 9-138
9-15 Effects of ozone on host defense mechanisms: immunology 9-142
9-16 Tolerance to ozone 9-147
9-17 Central nervous system and behavioral effects of ozone 9-154
9-18 Hematology; animal—in vivo exposure 9-160
9-19 Hematology: animal—Tn vitro exposure 9-167
9-20 Hematology: human—in vitro exposure 9-168
9-21 Reproductive and teratogenic effects of ozone 9-175
9-22 Chromosomal effects from iji vitro exposure to high ozone
concentrations 9-177
9-23 Chromosomal effects from ozone concentrations at or below
1960 ug/m3 (1 ppm) 9-179
9-24 Hutational effects of ozone 9-184
9-25 Effects of ozone on the 1 i ver 9-191
9-26 Effects of ozone on the endocrine system, gastrointestinal
tract, and urine 9-198
9-27 Effects of complex pollutant mixtures 9-211
9-28 Summary Table: morphological effects of ozone in experimental
animal s 9-226
9-29 Summary Table: effects on pulmonary function of short-term
exposures to ozone in experimental animals 9-230
9-30 Summary Table: effects on pulmonary function of long-term
exposures to ozone in experimental animals 9-232
9-31 Summary Table: biochemical changes in experimental animals
exposed to ozone 9-237
9-32 Summary Table: effects of ozone on host defense mechanisms in
experimental animals ........ 9-242
9-33 Summary Table: extrapulmonary effects of ozone in experimental
animals ' 9-251
9-34 Summary Table: interaction of ozone with other pollutants in
experimental animal s 9-254
viii
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LIST OF FIGURES
Figure Page
9-1 Predicted tissue dose for several trachea! 03 concentrations
for rabbit and guinea pig 9-12
9-2 Tissue dose versus ozone for rabbit and guinea pig and tissue
dose versus airway generation for human. Trachea! 03 concen-
tration is 500 ug/ms (0.26 ppm) 9-13
9-3 Intracellular compounds active in antioxidant metabolism of
the 1 ung .- 9-73
9-4 Summary of morphological effects in experimental animals
exposed to ozone 9-225
9-5 Summary of effects of short-term ozone exposures on pulmonary
function in experimental animals _ 9-229
9-6 Summary of effects of long-term ozone exposures on pulmonary
function in experimental animals 9-231
9-7 Summary of biochemical changes in experimental animals
exposed to ozone 9-236
9-8 Summary of effects of ozone on host defense mechanisms in
experimental animals 9-241
9-9 Summary of extrapulmonary effects of ozone in experimental
animal s 9-250
9-10 Summary of effects in experimental animals exposed to ozone
combined with other pollutants 9-253
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LIST OF ABBREVIATIONS
A-V Atrioventrieular
ACh Acetylcholine
AChE Acetylcholinesterase
AM Alveo.lar macrophage
AMP Adenosine monophosphate
ATP Adenosine triphosphate
ATPS ATPS condition (ambient temperature and pressure,
saturated with water vapor)
BTPS BTPS conditions (body temperature, barometric
pressure, and saturated with water vapor)
CC Closing capacity
C. Dynamic lung compliance
CHEM Gas-phase chemiluminescence
C. Lung compliance
C. i Static lung compliance
CMP Cytidine monophosphate
CNS Central nervous system
CO Carbon monoxide
COHb Carboxyhemoglobin
COLD Chronic obstructive lung disease
COMT Catechol-£-methy1-transferase
COg Carbon dioxide
CPK Creatine phosphokinase
CV Closing volume
D. Diffusing capacity of the lungs
D.CO ' Carbon monoxide diffusing capacity of the lungs
DNA Deoxyribonucleic acid
E Elastance
EGG, EKG Electrocardiogram
EEG Electroencephalogram
ERV Expiratory reserve volume
m=,v The maximal forced expiratory flow achieved
max
during an FVC test
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LIST OF ABBREVIATIONS (continued)
FEF Forced expiratory flow
FEF2QO-12QQ Mean forced expiratory flow between 200 ml and
1200 ml of the FVC [formerly called the maximum
expiratory flow rate (MEFR)].
FEF25-75% Mean f°rced expiratory flow during the middle
half of the FVC [formerly called the maximum
mid-expiratory flow rate (MMFR)].
FEF75% Instanteous forced expiratory flow after 75% of
the FVC has been exhaled.
FEV Forced expiratory volume
FIVC Forced inspiratory vital capacity
fg Respiratory frequency.
FRC Functional residual capacity
FVC Forced vital capacity
G Conductance
G-6-PD Glucose-6-phosphate dehydrogenase
G,.. Airway conductance
dw
GMP Guanosine monophosphate
GS-CHEM Gas-solid chemiluminescence
GSH Glutathione
GSSG Glutathione disulfide
Hb Hemoglobin
Hct Hematocrit
HO* Hydroxy radical
H£0 Water
1C Inspiratory capacity
IRV Inspiratory reserve volume
IVC Inspiratory vital capacity
j/
o Average mucous production rate per unit area
LDH Lactate deyhydrogenase
LDgQ Lethal dose (50 percent)
LM Light microscopy
LPS Lipopolysaccharide
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LIST OF ABBREVIATIONS (continued)
MAO Monamine oxidase
MAST Kl-coulometric (Mast meter)
max ^g Maximum ventilation
max VOg Maximal aerobic capacity
MBC Maximum breathing capacity
MEFR Maximum expiratory flow rate
MEFV Maximum expiratory flow-volume curve
MetHb Methemoglobin
MMFR or MMEF Maximum mid-expiratory flow rate
MNNG N-methyl-N'-nitrosoguanidine
MPO Myeloperoxidase
MVV Maximum voluntary ventilation
NBKI Neutral buffered potassium iodide
(NH^gSO, Ammonium sulfate
NQg Nitrogen dioxide
NPSH Non-protein sulfhydryls
Og Oxygen •
Og- Oxygen radical
Og Ozone
P(A-a)Og Alveolar-arterial oxygen pressure difference
PABA Para-amiriobenzoic acid
P/\COg , Alveolar partial pressure of carbon dioxide
PaCQg Arterial partial pressure of carbon dioxide
PAN Peroxyacetyl nitrate
P*Qg Alveolar partial pressure of oxygen
PaOg Arterial partial pressure of oxygen
PEF • Peak expiratory flow
PEFV Partial expiratory flow-volume curve
PG Prostag!andin
pHa Arterial pH
PHA Phytohemagglutinin
P. Transpulmonary pressure
PMN Polymorphonuclear leukocyte
PPD Purified protein derivative
xii
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LIST OF ABBREVIATIONS (continued)
PS£ Static transpulmonary pressure
PUFA Polyunsaturated fatty acid
R Resistance to flow
Raw Airway resistance
RBCs Red blood cells
RC011 Collateral resistance
RL Total pulmonary resistance
RQ, R - Respiratory quotient
RJ..J Tissue resistance
RV Residual volume
Sa02 Arterial oxygen saturation
SCE Sister chromatid exchange
Se Selenium
SEM Scanning electron microscopy
SGaw Specific airway conductance
SH Sulfhydryls
SOD Superoxide dismutase
S02 Sulfur dioxide
SPF Specific pathogen-free
SRaw Specific airway resistance
STPD STPD conditions (standard temperature and
pressure, dry)
TEM transmission electron microscopy
TGV Thoracic gas volume
TIC Trypsin inhibitor capacity
TLC total lung capacity
TRH Thyrotropin-releasing hormone
TSH Thyroids-stimulating hormone
TV Tidal volume
UFA Unsaturated fatty acid
UMP Uridine monophosphate
UV Ultraviolet photometry
*
V^ Alveolar ventilation
V^/Q Ventilation/perfusion ratio
xi i i
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LIST OF ABBREVIATIONS (continued)
VC Vital capacity
*
VCOp Carbon dioxide production
Vp Physiological dead space
Vp Dead-space ventilation
^D anat Anatomical dead space
Ve Minute ventilation; expired volume per minute
Vj Inspired volume per minute
V^ Lung volume
V* Maximum expiratory flow
VOg Oxygen uptake
V0«j ^Og Oxygen consumption
125£ Radioactive iodine
5-HT 5-hydroxytryptamine
6-P-GD 6-phosphogluconate dehydrogenase
MEASUREMENT ABBREVIATIONS
g gram
hr/day hours per day
kg ' ki1ogram
kg-m/min kilogram-meter/min
L/min liters/min
ppm parts per million
mg/kg milligrams per kilogram
2
mg/m milligrams per cubic meter
min minute
ml milliliter
mm millimeter - .
}jg/m micrograms per cubic meter
urn micrometers
uM micromolar
sec second
xiv
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
Chapter 9: Toxicological Effects of Ozone and Other Photochemical Oxidants
Principal Authors
Dr. Donald E, Gardner
Northrop Services, Inc.
Environmental Sciences
P.O. Box 12313
Research Triangle Park, NC 27709
Dr. Judith A. Graham
Health Effects Reearch Laboratory
MD-82
U.S. Environmental Protection Aency
Research Triangle Park, NC 27711
Dr. Susan M. Loscutoff
16768 154th Ave., S.E.
Renton, WA 98055
Dr. Daniel B. Menzel
Laboratory of Environmental Toxicology
and Pharmacology
Duke University Medical Center
P.O. Box 3813
Durham, NC 27710
Dr. Daniel L. Morgan
Laboratory of Environmental Toxicology
and Pharmacology
Duke University Medical Center
P.O. Box 3813
Durham, NC 27710
Dr. John H. Overton, Jr.
Health Effects Research Laboratory
MD-82
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Mr. James A. Raub
Environmental Criteria and
Assessment Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Stephen C. Strom
Department of Radiology
Duke University Medical Center
P.O. Box 3808
Durham, NC 27710
Dr. Walter S. Tyler
Department of Anatomy
School of Veterinary Medicine
University of California,
Davis, CA 95616
xv
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Contributing Authors
Mr. James R. Kawecki
TRC Environmental Consultants, Inc.
701 W. Broad Street
Falls Church, VA 22046
Dr. Frederick J. Miller
Health Effects Research Laboratory
MD-82
U.S. Environmental Protection Agency
Research Triangle Park, NC. 27711
Ms. Elaine D. Smolko
Laboratory of Environmental Toxicology
and Pharmacology
Duke University Medical Center
P.O. Box 3813
Durham, NC 27710
Dr. Jeffrey L. Tepper
Northrop Services, Inc.
Environmental Sciences
P.O. Box 12313
Research Triangle Park, NC 27709
xvi
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Authors also reviewed individual sections of the chapter. The following addi-
tional persons reviewed this chapter at the request of the U.S. Environmental
Protection Agency. The evaluations and conclusions contained herein, however,
are not necessarily those of the reviewers.
Dr. Karim Ahmed
Natural Resources Defense Council
122 East 42nd Street
New York, NY 10168
Dr. Ann P. Autor
Department of Pathology
St. Paul's Hospital
University of British Columbia
Vancouver, British Columbia
Canada V6Z1Y6
Dr. David V. Bates
Department of Medicine
St. Paul's Hospital
University of British Columbia
Vancouver, British Columbia
Canada V6Z1Y6
Dr. Philip A. Bromberg
Department of Medicine
School of Medicine
University of North Carolina
Chapel Hill, NC 27514
Dr. George L. Carlo
Dow Chemical, U.S.A.
1803 Building, U.S. Medical
Midland, MI 48640
Dr. Larry D. Claxton
Health Effects Research Laboratory
MD-68
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Donald L. Dungworth
Department of Veterinary Pathology
School of Veterinary Medicine
University of California
Davis, CA 95616
Dr. Richard Ehrlich
Life Sciences Division
Illinois Institute of Technology
Research Institute
Chicago, IL 60616
Dr. Robert Frank
Department of Environmental
Health Sciences
Johns Hopkins School of Hygiene
arid Public Health
615 N. Wolfe Street
Baltimore, MD 21205
Dr. Milan J. Hazucha
School of Medicine
Center for Environmental Health
and Medical Sciences
University of North Carolina
Chapel Hill, NC 27514
Dr. Donald H. Horstman
Health Effects Research Laboratory
MD-58
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Steven M. Horvath
Institute of Environmental Stress
University of California
Santa Barbara, CA 93106
Dr. George Jf Jakab
Department of Envi ronmental
Health Sciences
Johns Hopkins School of Hygiene
and Publ-i-c-Health
615 N. Wolfe St.
Baltimore, MD 21205
Dr. Robert J.,Kavlock
Health Effects Research Laboratory
MD-67
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
xvn
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Reviewers (cont'd)
Dr. Thomas J, Kulle
Department of Medicine
School of Medicine
University of Maryland
Baltimore, MD 21201
Dr, Michael D. Lebowitz
Department of Internal Medicine
College of Medicine
University of Arizona
Tucson, AZ 85724
Dr. Robert C. MacPhail
Health Effects Research Laboratory
MD-74B
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
McDonnell
Research Laboratory
Dr. William F.
Health Effects
MD-58
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. Myron A. Mehlman
Environmental Affairs and
Toxicology Department
Mobil Oil Corporation
P.O. Box 1026
Princeton, NJ 08540
Dr. Harold A. Menkes
Department of Environmental
Health Sciences
Johns Hopkins School of Hygiene
and Public Health
615 N. Wolfe Street
Baltimore, MD 21205
Dr. Phyllis J. Mullenix
Forsyth Dental Center
140 The Fenway
Boston, MA 02115
Dr. Mohammad G. Mustafa
Division of Environmental and
Nutritional Sciences
School of Public Health
University of California
Los Angeles, CA 90024
Dr. Russell P. Sherwin
Department of Pathology
University of Southern California
Los Angeles, CA 90033
Dr. Robert J. Stephens
Division of Life Sciences
SRI International
333 Ravenwood Avenue
Menlo Park, CA 94025
Dr. David L. Swift
Department of Environmental
Health Sciences
Johns Hopkins School of Hygiene
and Public Health
615 N. Wolfe Street i
Baltimore, MD 21205
Ms. Beverly E. Tilton
Environmental Criteria and Assessment
Office
MD-52
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr.- Jaroslav J. Vostal
Executive Department
General Motors Research Laboratories
Warren, MI 48090
xvm
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SCIENCE ADVISORY BOARD
CLEAN AIR SCIENTIFIC ADVISORY COMMITTEE
The substance of this document was reviewed by the Clean Air Scientific
Advisory Committee of the Science Advisory Board in public sessions.
SUBCOMMITTEE ON OZONE
Chairman
Dr. Morton Lippmann
Professor
Department of Environmental Medicine
New York University Medical Center
Tuxedo, New York 10987
Members
Dr. Mary 0. Amdur
Senior Research Scientist
Energy Laboratory
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Dr. Eileen G. Brennan
Professor
Department of Plant Pathology
Martin Hall, Room 213, Lipman Drive
Cook College-NJAES
Rutgers University
New Brunswick, New Jersey 08903
Dr. Edward D. Crandall
Professor of Medicine
School of Medicine
Cornell University
New York, New York 10021
Dr. James D. Crapo
Associate Professor of Medicine
Chief, Division of Allergy, Critical
Care and Respiratory Medicine
Duke University Medical Center
Durham, North Carolina 27710
Dr. Robert Frank
Professor of Environmental Health
Sciences
Johns Hopkins School of Hygiene
and Public Health
615 N. Wolfe Street
Baltimore, Maryland 21205
Professor A. Myrick Freeman II
Department of Economics
Bowdoin College
Brunswick, Maine 04011
Dr. Ronald J. Hall
Senior Research Scientist and Leader
Aquatic and Terrestrial Ecosystems
Section
Ontario Ministry of the Environment
Dorset Research Center
Dorset, Ontario POA1EO
Dr. Jay S. Jacobson
Plant Physiologist
Boyce Thompson Institute
Tower Road
Ithaca, New York 14853
xix
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Dr. Warren B. Johnson
Director, Atmospheric Science Center
SRI International
333 Ravenswood Avenue
Menlo Park, California 94025
Dr. Jane Q. Koenig
Research Associate Professor
Department of Environmental Health
University of Washington
Seattle, Washington 98195
Dr. Paul Kotin
Adjunct Professor of Pathology
University of Colorado Medical School
4505 S. Yosemite, #339
Denver, Colorado, 80237
Dr. Timothy Larson
Associate Professor
Environmental Engineering and
Science Program
Department of Civil Engineering
University of Washington
Seattle, Washington 98195
Professor M. Granger Morgan
Head, Department of Engineering
and Public Policy
Carnegie-Mellon University
Pittsburgh, Pennsylvania 15253
Dr. D. Warner North
Principal
Decision Focus Inc., Los Altos
Office Center, Suite 200
4984 El Camino Real
Los Altos, California 94022
Dr. Robert D. Rowe
Vice President, Environmental and
Resource Economics
Energy and Resources Consultants, Inc.
207 Canyon Boulevard
Boulder, Colorado 80302
Dr. George Taylor
Environmental Sciences Division
P.O. Box X
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37831
Dr. Michael Treshow
,Professor
Department of Biology
University of Utah
Salt Lake City, Utah 84112
Dr. Mark J. Utell
Co-Director, Pulmonary Disease Unit
Associate Professor of Medicine and
Toxicology in Radiation Biology
and Biophysics
University of Rochester Medical
Center
Rochester, New York 14642
Dr. James H. Ware
Associate Professor
Harvard School of Public Health
Department of Biostatisties
677 Huntington Avenue
Boston Massachusetts 02115
Dr. Jerry Wesolowski
Air and Industrial Hygiene Laboratory
California Department of Health
2151 Berkeley Way
Berkeley, California 94704
Dr. James L. Whittenberger
Director, University of California
Southern Occupational Health Center
Professor and Chair, Department of
Community and Environmental Medicine
California College of Medicine
University of California - Irvine
19772 MacArthur Boulevard
Irvine, California 92717
Dr. George T. Wolff
Senior Staff Research Scientist
General Motors Research Labs
Environmental Science Department
Warren, Michigan 48090
xx
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PROJECT TEAM FOR DEVELOPMENT
OF
Air Quality Criteria for Ozone and Other Photochemical Oxldants
Ms. Beverly E. Tilton, Project Manager
and Coordinator for Chapters 1 through 5, Volumes I and II
Environmental Criteria and Assessment Office (MD-52)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Mr. Norman E. Chi Ids
Environmental Criteria and Assessment Office (MD-52)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. J.H.B. Garner
Coordinator for Chapters 7 and 8, Volume III
Environmental Criteria and Assessment Office (MD-52)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Mr. Thomas B. McMullen
Environmental Criteria and Assessment Office (MD-52)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Mr. James A. Raub
Coordinator for Chapters 10 through 13, Volumes IV and V
Environmental Criteria and Assessment Office (MD-52)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. David T. Tingey
Coordinator for Chapter 7, Volume III
Environmental Research Laboratory
U.S. Environmental Protection Agency
200 S.W. 35th Street
Corvallis, OR 97330
xxi
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9. TOXICOLOGICAL EFFECTS OF OZONE AND OTHER PHOTOCHEMICAL OXIDANTS
9,1 INTRODUCTION
This chapter discusses the effects of ozone on experimental animals.
Carefully controlled studies of the effects of ozone on animals are particu-
larly important in elucidating subtle effects not easily found in man through
epidemiological studies and in identifying chronic toxicity not apparent from
short-term controlled human exposures. Animal studies allow investigations
into the effects of ozone exposure over a lifetime, uncomplicated by the
presence of other pollutants. In the animal experiments presented here, a
broad range of ozone concentrations has been studied but emphasis has been
3
placed on recent studies at 1960 ug/m (1 ppm) of ozone or less. Higher
concentrations have been cited when the data add to an understanding of mechan-
isms. Concentrations of 1 ppm or greater cannot be studied ethically in man
because of the toxicity of even short-term exposures.
A majority of the literature describes the effects of ozone on the respira-
tory tract, but extrarespiratory system effects have now been noted and are
documented in this chapter. Most of the studies utilize invasive methods that
require sacrifice of the animals on completion of the experiment; thus, the
studies would be impossible to perform in human subjects. Noninvasive methods
of examining most of these endpoints are not readily available.
Emphasis has been placed on the more recent literature published after
the prior criteria document (U.S. Environmental Protection Agency, 1978);
however, older literature has been reviewed .again in this chapter. As more
information on the toxicity of ozone becomes available, a better understanding
of earlier studies is possible and a more detailed and comprehensive picture
of ozone toxicity is emerging. The literature used in developing this chapter
is, set out in a series of tables, Not all of..the. literature cited in the
\
tables appears in the detailed discussion of the text, but citations are
provided to give the reader more details on the background from which the text
is drawn.
In selecting studies for consideration, a detailed review of each paper
has been completed. This review included an evaluation of the exposure methods;
the analytical method used to determine the chamber ozone concentration; the
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calibration of the ozone monitoring equipment and the analytical methods used
(wherever possible); the species, strain, age and physical characteristics of
the animals; the technique used for obtaining samples; and the appropriateness
of the technique used to measure the effect. In interpreting the results, the
number of animals used, the appropriateness and results of the statistical
analysis, the degree to which the results conform with past studies, and the
appropriateness of the interpretation of the results are considered. No
additional statistical analysis beyond that reported by the author has been
undertaken. Unless otherwise stated, all statements of effects in the text
are statistically significant at p < 0.05. Many reports, especially in the
older literature, do not present sufficient information to permit the assessment
described above. However, should a particular study not meet all of these
criteria, but provide reasonable data for consideration, a disclaimer is
provided in the text and/or tables.
In this chapter, a discussion of the regional respiratory dosimetry of
ozone in common laboratory animal species is presented and compared to human
dosimetry. Morphological alterations of the lungs of animals exposed to 03
are described, followed by the effects of ozone on the pulmonary function of
animals. The biochemical alterations observed in the ozone-exposed animals
are then related to morphological changes and to potential mechanisms of toxi-
city and biochemical defense mechanisms. The influence of dietary factors,
such as vitamins E and C, in animals is discussed with consideration of poten-
tial roles in humans. It should be stressed, however, that no evidence for
complete protection against ozone toxicity has been found for any factor,
dietary or. therapeutic. The effects of ozone on the defense mechanisms of the
lung against respiratory infectious agents are discussed using the infectivity
model system and effects on alveolar macrophages as examples of experimental
evidence. This section is followed by a discussion of ozone tolerance in
animals. Last, the effects of ozone on a number of extrarespiratory organ
systems are discussed to provide insight into potential effects of ozone
inhalation in the respiratory system beyond those now well documented.
A brief discussion of the available literature on the effects of other
oxidants likely to occur in polluted air as a result of photochemical reactions
or other sources of pollution-is presented. Peroxyacetyl nitrate, hydrogen
peroxide, and automobile exhaust are the principal pollutants studied in these
experiments. This section is short because of the general lack of information
9-2
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in this area, but its brevity does not necessarily reflect a general lack of
importance.
A summary is provided for all of the sections of the chapter to set the
tone for a clearer understanding of the effects of ozone on animals. The
major emphasis of this chapter is to provide evidence for the toxicity of
ozone which can not, ethically or practically, be obtained from the study of
human subjects. The overall health effects of ozone can be judged from three
types of studies: animal exposures, controlled human exposures, and epidemio-
logical studies of adventitious human exposures. No single method alone is
adequate for an informed judgment, but together they provide a reasonable
estimate of the human health effects of ozone on man.
9.2 REGIONAL DOSIMETRY IN THE RESPIRATORY TRACT
A major goal of environmental toxicological studies on animals is the
eventual quantitative extrapolation of results to man. One type of information
necessary to obtain this goal is dosimetry, which is the specification of the
quantity of inhaled material, -in this instance ozone (0~), absorbed by specific
sites in animals or man. This information is needed because the local dose
(quantity of 03 absorbed per unit area), along with cellular sensitivity,
determines the type and extent of injury. At this time, only dosimetry is
sufficiently advanced for discussion here. Until both elements are advanced,
quantitative extrapolation cannot be conducted.
At present, there are two approaches to dosimetry, experimental and
deterministic mathematical modeling. Animal experiments have been carried out
to obtain direct measurements of 0- absorption; however, experimentally obtaining
local lower respiratory tract (tracheobronchial and pulmonary regions) uptake
data is currently extremely difficult. Nevertheless, experimentation is
important in assessing concepts and hypotheses, and in validating mathematical
models that can be used to predict local doses.
Because the factors affecting the transport and absorption of 03 are
general to all mammals, a model that uses appropriate species and/or disease-
specific anatomical and ventilatory parameters can be used to describe 0, ab-
sorption in the species and in different-si zed, aged, or diseased members of
the same species. Models may also be used to explore processes or factors
which cannot be studied experimentally, to identify areas needing additional
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research, and to test our understanding of 03 absorption in the respiratory
tract.
9.2.1 Absorption in Experimental Animals
There have been very few experiments in which measurements of the regional
uptake of 0, or other reactive gases have been determined. Of the several
results published, only one is concerned with the uptake of 03 in the lower
respiratory tract; the others deal with nasopharyngeal uptake.
9.2.1.1 Nasopharyngeal Absorption. Nasopharyngeal removal of 0~ lessens
the quantity of 03 delivered to the lung and must be accounted for when
estimating the 03 dose responsible for observed pulmonary effects. Vaughan
et al. (1969) exposed the isolated upper airways of beagle dogs to 03 at a
continuous flow of 3.0 L/min and collected the gas below the larynx in a
plastic (mylar) bag. One-hundred percent uptake by the nasopharynx was reported
for concentrations of 0.2 to 0.4 ppm. Using a different procedure, Yokoyama and
Frank (1972) observed 72 percent uptake at 0.26 to 0.34 ppm (3.5 L/min to
6.5 L/min flow rate). They also replicated the procedure of Vaughan et al. and
found that 03 was absorbed on the mylar bag wall. This may account for the dif-
ference between the observations of Yokoyama and Frank (1972) and of Vaughan et al.
(1969).
Yokoyama and Frank (1972) also observed a decrease in the percent uptake
due to increased flow rate, as well as to increased 0, concentration. For example,
with nose breathing and an 03 concentration of 0.26 to 0.34 ppm, the uptake
decreased from 72 percent to 37 percent for a flow rate increase from 3.5 to 6.5
to 35 to 45 L/min. An increase in concentration from 0.26 to 0.34 to 0,78 to
0.80 ppm decreased nose breathing uptake (3.5 to 6.5 L/min flow rate) from 72 per-
cent to 60 percent. Thejr data, however, indicate that the trachea! concentra-
tion increases with increased nose or mouth concentrations. They also demon-
strated that the concentration of 03 reaching the trachea depends heavily on the ....
route of breathing. Nasal uptake significantly exceeded oral uptake at flow
rates of both 3.5 to .6.5 and 35 to 45 L/min. For a given flow rate, nose
breathing removed 50 to 68 percent more 03 than did mouth breathing.
Moorman et al. (1973) compared the loss of 0, in the nasopharynx of acutely
e * *•*
and chronically exposed dogs. Beagles chronically exposed (18 months) to 1 to
3 ppm of 0~ under various daily exposure regimes had significantly higher tra-
chea! concentrations of 03 than animals tested after 1 day of exposure to cor-
responding regimes. Moorman et al. (1973) suggested that the differences were
9-4
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due to physiochemical alterations of the mucosal lining in the chronically ex-
posed beagles. When dogs were exposed for 18 months to 1 ppm for 8 hr a day,
they had significantly lower trachea! values than those continuously exposed.
The average trachea! concentration (0.01 ppm) for the acutely exposed group,
however, was not significantly different from that (0.023 ppm) of the 8 hr/day
chronic exposure group, when the relative insensitivity of the Mast 03 meter
(unmodified) used to measure the responses is taken into account. Thus, at
levels of 1.0 ppm or less, there is no significant evidence that chronic
exposure would result in trachea! 0- concentrations significantly greater than
those observed with acute exposure.
Nasopharyngea! removal of 0- in rabbits and guinea pigs was studied by
Miller et al. (1979) over a concentration range of 0.1 to 2.0 ppm. The trachea!
0., concentration in these two species was markedly similar at a given inhaled
concentration and was linearly related to the chamber concentration that was
drawn unidirectionally through the isolated upper airways. Ozone removal in
the nasopharyngeal region was approximately 50 percent in both species over
the concentration range of 0.1 to 2.0 ppm. The positive correlation between
the trachea! and chamber concentrations is in agreement with Yokoyama and
Frank (1972). Caution needs to be exercised in applying the above results to
relate ambient and trachea! concentrations of 0- since the effects.of naso-
pharyngeal volume and the cyclical nature of breathing are not taken into
account. For example, if the tidal volume was less than the nasopharyngeal
volume and convection was the only process of axial transport, then no 0~
would be delivered to the trachea! opening, regardless of the percent uptake
measured for unidirectional flow.
9.2.1.2 Lower Respiratory Tract Absorption. Morphological studies on animals
suggest that 03 is absorbed along the entire respiratory tract; it penetrates
further into the peripheral nonciliated airways as inhaled 0- concentrations
increase (Dungworth et al., 1975b). Lesions were found consistently in the
trachea and proximal bronchi and between the junction of the conducting airways
and the gaseous exchange area; in both regions, the severity of damage decreases
distally. In addition, several studies have reported the most severe or
prominent lesions to be in the centriacinar region (see section 9.3).
No experiments determining 0- tissue dose at the generational or regional
level have been reported; however, there is one experiment concerned with the
uptake of 0- by the lower respiratory tract. Removal of 0- from inspired air
9-5
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by the lower airways was measured by Yokoyama and Frank (1972) in dogs that
were mechanically ventilated through a tracheal cannula. In the two ranges of
Og concentrations studied, 0.7 to 0.85 ppm and 0.2 to 0.4 ppm, the rate of
uptake was found to vary between 80 and 87 percent when the tidal volume was
kept constant and the respiratory pump was operated at either 20 or 30 cycles/
min. This estimate of uptake applies to the lower respiratory tract as a
whole; it does not describe uptake of 03 by individual regions or generations.
9.2.2 Ozone Dosimetry Models
9.2.2.1 Modeling Nasal Uptake. LaBelle et al. (1955) considered the absorp-
tion of gases in the nasal passages to be similar to absorption on wetted
surfaces of distillation equipment and scrubbing towers and applied the theory
and models of these devices to the nasal passages of rats. By associating
biological parameters of rats with the chemical engineering device parameters
of the model, they calculated the percent of penetration of several gases to
the lung. They concluded that Henry's law constant is the major variable in
determining penetration. Based on these calculations, The National Academy of
Sciences (National Research Council, 1977) concluded that the model predicts
99 percent penetration for Og. This is much more than that measured by Yokoyama
and Frank (1972) or by Miller et al. (1979). Several possible reasons for the
differences were discussed (National Research Council, 1977), but the major
factor was considered to be that the model does not account for the reactions
of 03 in the mucus and epithelial tissue.
Aharonson et al. (1974) developed a model for use in analyzing data from
experiments on the uptake of vapors by the nose. The model was based on the
assumptions of quasi-steady-state flow, mass balance, and proportionality of
flux of a trace gas at the air-mucus interface to the gas-phase partial
pressure of the trace gas and a "local uptake coefficient" (Aharonson et al.,
1974). The model was applied to data from their own experiments on the removal
of acetone and ether in dog noses. They also applied the model to the 03
uptake data of Yokoyama and Frank (1972) and concluded that the uptake coef-
ficient (average mass transfer coefficient) for Og» as well as for the other
gases considered, increases with increasing air flow rate.
9.2.2.2 Lower Respiratory Tract Dosimetry Models. There are three models
for which published results are available. The model of McJilton et al.
(1972) has been discussed and simulation results for 0^ absorption in each
generation of the human lower respiratory tract are available (National Research
9-6
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Council 1977; Morgan and Frank, 1977). Two models have been developed by
Miller and co-workers. A detailed description of the formulation of the first
and earliest mathematical model of Miller and co-workers is found in Miller
(1977) and major features are given in Miller et al. (1978b) and Miller (1979).
Results, using this model, of simulations of the lower respiratory tract
absorption of Q, in humans, rabbits, and guinea pigs are in Miller (1977,
1979) and Miller et al. (1978b). The formulation of the second model of
Miller and co-workers, as well as results of simulating lower respiratory
tract absorption of 0- in humans, appears in Miller et al. (1985).
Because all of the above models were developed to simulate the local
absorption of 03, they have much in common. This is especially true with
respect to the following areas: formulation of 0^ transport in the airspaces
or lumen of the airways, use of morphometric models of the lower respiratory
tract, and inclusion of a liquid lining that coats the tissue walls of the
airspaces or lumen of the airways.
In each model the descriptions of 03 transport and absorption in the
lumen are based on a one-dimensional differential equation relating axial
convection, axial dispersion or diffusion, and the loss of 0- by absorption at
the gas-liquid interface. The use of a one-dimensional approximation has been
very common in modeling the transport of gases such as 0^, N^, etc., in the
lower respiratory tract (see Scherer et al., 1972; Paiva, 1973; Chang and
Farhi, 1973; Yu, 1975; Pack et al., 1977; Bowes et al., 1982). .The approxima-
tion is appropriate for 0^ as well.
The models of Miller and co-workers took into account effective axial
dispersion in the airways by using an effective dispersion coefficient based
on the results of Scherer et al. (1975). McJilton's model did not take this
factor into account (Morgan and Frank, 1977). However, this may not be an
important difference since Miller et al. (1985) report that results are little
affected by different values of the coefficient. Also, Pack et al. (1977) and
Engel and Macklem (1977) reported results that indicate an insensitivity of
airway concentrations to the effective dispersion coefficient.
Airway or morphometric zone models such as those of Weibel (1963) and
Kliment (1973) were used to define the lengths, radii, surface areas, cross-
sectional areas, and volumes of the airways and air spaces of each generation
or zone. The breathing pattern was assumed sinusoidal; however, dimensions
were held constant throughout the breathing cycle. The physical properties of
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the liquid lining were assumed to be those of water. The lining thickness
depended on generation or zone, being thicker in the upper airways than in the
lower.
The flux of Og from the lumen or airspaces to the liquid lining was
defined in each model in terms of a mass-transfer coefficient. McJilton et
al. (1972) made the assumption that radial mass transfer was controlled by the
absorbing medium and estimated the transfer coefficient from empirical data on
the physical properties (not chemical) of the medium and of 03 (National
Research Council, 1977). In the first model of Miller and co-workers
(Miller, 1977; Miller et al., 1978b) the radial dependence of the luminal 03
concentration was assumed to vary quadratically with the radius. From this
formulation, the gas-phase mass transfer was determined. For their later
model, the gas phase mass transfer coefficient was defined in terms of a
Sherwood number. In both models the gas phase coefficient was combined with
the mass transfer coefficient of the absorbing medium (which depended on the
chemical and physical properties of the absorbing medium and of 03) to obtain
the overall transfer coefficient. However, Miller et al. (1985) conclude,
based on the data available for the absorbing medium, that radial mass transfer
is controlled by the medium, making specification of the gas phase mass trans-
fer coefficient unnecessary.
The main differences in the models are the mechanisms of absorption and
their formulation. In the model of McJilton et al. (1972) and in the early
model of Miller and co-workers (Miller, 1977; Miller et al., 1978b) there is
only one compartment, the liquid lining, which can absorb unreacted Qg. In
the later model of Miller et al. (1985) there are three absorbing compartments,
liquid lining, tissue, and capillary or blood (in the pulmonary region where
the air-blood tissue barrier is very thin). Further, (k is known to react
chemically with constituents of the absorbing medium(s). This aspect, included
by Miller and co-workers, was not included in McJilton's model. The inclusion
resulted in significant differences between the tissue dose pattern curves in
the tracheobronchial region predicted by the models. In addition, McJilton's
model predicts a dose curve (equivalent to a tissue dose curve because of no
mucous reactions) in the tracheobronchial region that has its maximum at the
trachea and decreases distally to the thirteenth or fourteenth generation (see
Figure 7-5 in National Research Council, 1977). By contrast, the models of
Miller and co-workers predict the tissue dose to be a minimum at the trachea
and to increase distally to the pulmonary region.
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The concentration of ozone in tissue and at the liquid-tissue interface
was assumed to be zero by McJilton et al. (1972) (National Research Council,
1977; Morgan and Frank, 1977) and in the first model of Miller and co-workers
(Miller, 1977; Miller et al., 1978b). The interpretation was that this boundary
condition means that 03 reacts (chemically) instantaneously when it reaches
the tissue. Miller et al. (1978b) define the tissue dose as that quantity of
0~ per unit area reacting with or absorbed by the tissue at the liquid-tissue
interface.
The first model of Miller and co-workers took into account the reaction
of 03 with the unsaturated fatty acids (UFA) and ami no acids in the mucous-
serous lining. Reactions of 03 with other components (such as carbohydrates)
were not included in the model because of insufficient information (Miller,
1977; Miller et al., 1978b). The CL-UFA and Ov-amino acid reactions were
assumed fast enough so that an instantaneous reaction scheme based on that
outlined in Astarita (1967) could be used. The scheme required the specifica-
tion of the production rate of the UFA and ami no acids in each mucous-lined
generation. These rates were estimated by using trachea! mucous flow data,
the surface area of the tracheobronchial region, the concentrations of the
specific reactants known to react with Q,, and the assumption that the produc-
tion rate decreased distally (Miller, 1977; Miller et al., 1978b).
Although the instantaneous reaction scheme is a good preliminary approach
to treating 0, reactions in the mucous-serous lining, its use is not completely
justifiable. Second-order rate constants of 03 with some of the UFA present
in mucus indicate that although they are large (Razumovskii and Zaikov, 1972),
they are less than the diffusion-limited rates necessary for the instantaneous
reaction scheme. Experimental evidence (Mudd et al., 1969) suggests that the
reactions of 0- with amino acids are very rapid. Rate constants for these
»3 . ...
reactions and others are not known; thus, the information available is scanty,
which makes the specification of a reaction mechanism or reaction scheme
difficult and assumptions necessary.
The approach to chemical reactions used in the later model (Miller et
al., 1985) goes a long,way in addressing the above criticisms. The reaction
of 0~ with biochemical constituents is assumed to be bimolecular; however, the
concentration of the constituents is considered to be large enough so as not
to be depleted by the reactions. Hence, the model uses a pseudo first order
reaction scheme in which the pseudo first order rate constant is the product
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of the bimolecular rate constant and the concentration of the biochemical
constituents that react with Q~.
For modeling purposes, Miller et al. (1985) consider that only the reaction
of ozone with the UFA is important, using the 03~oleic acid rate constant of
Razumovskii and Zaikov (1972) for the 03-UFA reaction. They point out that
although 03 reacts with ami no acids and other constituents, rate constants are
not known and that Bailey (1978) estimates the reaction of 0~ with UFA to be
3
~10 times faster than the 03-amino acid reaction, justifying omitting amino
acids from consideration.
9.2.3 Predictions of Lower Respiratory Tract Ozone Dosimetry Modeling
The predictions of lower respiratory tract dosimetry models are reviewed
by illustrating the results of simulations, by comparing predictions to experi-
mental observations, and by describing uses for dosimetry models.
The following discussion of modeling results of lower respiratory tract
absorption is based mainly on simulations using the first model of Miller and
co-workers (Miller, 1977; Miller et al., 1978b). This is because the model
includes the important effects of 03 reactions in the mucous-serous lining and
because simulations of 03 absorption in laboratory animals are available.
Simulations of 03 absorption in different animals can be carried out by
modifying input parameters of the computer program that solves the mathematical
equations. These input parameters, which characterize an animal, include the
number and dimensions of the airways, tidal volume, length of time of one
breath, etc. The airway and alveolar dimensions of Weibel (1963) were used
for the simulation of 03 uptake in humans. For the rabbit and guinea pig,
Miller and co-workers used the morphometric zone models of Kliment (1973).
The zone model is a less detailed model than the generationally based airway
model of Weibel (1963) since more than one generation corresponds to a zone in
an animal; they were used because they were the only complete (tracheobronchial
and pulmonary regions) "airway" models available at the time. However, Schreider
and Hutchens (1980) criticize the guinea pig model of Kliment (1972, 1973) as
having a lung volume that is too low, suggesting the possibility of incomplete
casts. Since the same method also was used by Kliment (1972, 1973) for the
rabbit, this criticism may also apply to this model.
To illustrate simulation results, two aspects of the simulations by
Mi77er and co-workers are considered: (1) the effect of various trachea!
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concentrations on the tissue dose pattern (tissue dose as a function of zone
or generation) in guinea pigs and rabbits; and (2) the similarity between the
dose patterns of guinea pigs, rabbits, and humans.
9.2.3.1 I]1ustration of Dosimetry Simulations. Figure 9-1 is a set of
plots of the tissue dose- for one breath versus zone for various trachea! 0,,
concentrations for the rabbit and the guinea pig. All curves have the same
general characteristics. Independent of the inhaled concentration, the model
predicts that the first surfactant-lined zone (first non-mucous-lined or first
zone in the pulmonary region), zone 6, receives the maximal dose of Q~.
Although the model predicts uptake of 0Q by respiratory tissue (zones 6, 7,
3
and 8) for all trachea! concentrations studied (62.5 to 4000 ug/m ), the
penetration of 0^ to the tissue in the airways lined by mucus depended on the
trachea! concentration and the specific animal species. For example, as
2
illustrated in Figure 9-1 for the tracheal 03 concentration of 1000 jjg/m , no
0- reaches the airway tissue of the rabbit until zone 3, whereas 0- is predicted
to penetrate to the tissue in guinea pig airways in all zones. However, at
3
the two lowest tracheal concentrations plotted, 250 and 62.5 ug/m , no penetra-
tion occurs until zones 4 and 6, respectively, for both animals. The dependence
of penetration on tracheal 0, concentration is a result of the instantaneous
reaction scheme used to describe chemical reactions. Penetration does occur
in the simulation of uptake in humans using the newer model of Miller and
co-workers (1985), as depicted in Figure 9-2, and can be expected to occur in
future animal simulations.
The similarity of the predicted dose patterns in rabbits and guinea pigs
extends to the simulation of 0- uptake in humans. Figure 9-2 compares the
3
tissue dose for the three species for a tracheal concentration of 500 ug/m
(0.26 ppm) using results of the first model for the three species and the
newer model results for man. In Figure 9-2, the-guinea pig and rabbit tissue
doses are plotted in the form of a histogram to allow a comparison to human
dosimetry data that are expressed as a function of airway generation.
For the earlier model, the dose patterns of the three- species peak at the
first surfactant-lined zone (6) or in generation 17 (which is in zone 6).
Also, 03 penetrates to the tissue everywhere in the pulmonary region (zones >
5 and generations > 16); however, 03 is not predicted to penetrate to the
tissue before zone 3 for the rabbit and guinea pig or before zone 5 (generations
12-16) for man.
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104
i r~i i i i r~i r
105
m
Q)
O)
a.
O 106
u.
O
ui
CO
O
Q
Ul
CO
g 10-7
10-8
I I I I I I I
TRACHEAL
O3 CONG.
• 4000
A1000
• 250
• 62.5
ppm
2.041
0.510
0.128
0.032
0
TRACHEA
2 3
BRONCHI-
5678 MODEL ZONE
BR°N» A.D. A.S. MORPHOMETRIC ZOIME
' CHIOLES
Figure 9-1. Predicted tissue dose for several trachea! O3
concentrations for rabbit ( ) and guinea pig ( ).
See text for details. (A.D. = alveolar duct; A.S. = alveolar
sac).
Source: Adapted from Miller et al. (1978b).
9-12
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105
«J
l_
&
i
O
LL
O
LU
C/3
O
d
UJ
C/5
106
~ 107
1.0-8
TRACHEOBRONCHIAL
GENERATION
—ZONE
Figure 9-2. Tissue dose versus zone for rabbit (o) and guinea pig (•);
and tissue dose versus airway generation for human ( , earlier
model; , newer model). Tracheal O3 concentration is 500 ^g/m3
(0.26 ppm). See text for details.
Source: Guinea pig, rabbit and earlier human simulations adapted from
Miller et al. (1978b); newer model results for human adapted from
Miller etal. (1985).
9-13
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All of the earlier simulations presented by Miller and co-workers (Miller,
1977; Miller et al., 1978b; Miller, 1979) share the following characteristics:
(1) the maximal tissue dose occurs in the first surfactant-lined zone or
generation; (2) 0, penetrates to tissue everywhere in the pulmonary region
(zones >5), decreasing distally from the maximum; and (3) the onset of 0,
penetration to mucous-lined tissue, as well as dose in general, depends on
trachea! 0, concentration, animal species, and the breathing pattern. These
general characteristics of tissue dose pattern are independent of the two
airway models used.
Results using the newer model to simulate tissue dose in humans are also
illustrated in Figure 9-2. Comparison of dose values for the two human
simulations show.the most notable differences in the conducting airways (genera-
tions 0 to 16). The new and old results are similar in that both models predict
relatively low doses in the upper airways, a maximum in the first pulmonary
generation, and then a rapid decline in dose distally. There is no reason to
assume that these features would be missing in simulation results for laboratory
animals using the newer model.
9.2.3.2 Comparison of Simulations to Experimental Data. There are no quanti-
tative experimental observations with which to compare the results of modeling
the local uptake of 03 in the lower respiratory tract. Yokoyama and Frank
(1972) observed 80 to 87 percent uptake of 03 by the isolated lower respiratory
tract of dogs. With the earlier model, Miller et al. (1978b) predict a 47
percent uptake for humans; the newer model predicts 89 percent (Miller et al.,
1985). Because of differences between the two species, comparing the experi-
mental and simulated results is most likely inappropriate.
Morphological studies in animals report damage throughout the lower
respiratory tract. Major damage, and in some cases the most severe damage, is
observed to occur at the junction between the conducting airways and the gas
exchange region, and to decrease distally (see Section 9.3.1.1). For the
animals simulated by Miller and co-workers, the maximal tissue dose of 0, is
predicted to occur at this junction, with the dose curve decreasing rapidly
for more distal regions. Thus, in the pulmonary region, the model results are
in qualitative agreement with experimental observations.
Damage is also observed in the trachea and bronchi of animals (see Sections
9.3.1.1.1.2 and 9.3.1.1.1.3). In the animals modeled, the early model of
Miller and co-workers either predicts significantly less tissue dose in the
9-14
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upper airways compared to the dose in the first zone of the pulmonary region
or it predicts no penetration to the tissue in the upper portion of the conduc-
ting airways (see Figure 9-2). Based on simulation results using the newer
model for humans (Miller et al., 1985), one can infer that this model will
predict non-zero tissue doses in the upper airways of animals, but that these
doses also will be significantly less than predicted for the ceritriacinar
airways. The observations of damaged upper and lower respiratory tract airways
in the same animal and the predictions of significantly different tissue doses.
in the two regions appear inconsistent. However, much of the reported damage
in the trachea and bronchi is associated with the cilia of ciliated cells,
which in current model formulations are not part of the tissue. The cilia
extend into the hypophase (perciliary) portion of the mucous-serous layer, and
the dosimetry models do not distinguish the cilia of the ciliated cells as a
separate component of this layer. Thus, relatively low predicted tissue dose
should not be interpreted as predicting no damage to cilia. Likewise, the
frequent reporting of cilia being damaged following 03 exposure should not be
interpreted necessarily as an indication of 03 tissue dose since the model
definition of tissue does not currently include cilia. The inclusion of a
"cilia compartment" in future dosimetry models may be helpful. There are also
other factors that complicate our understanding of ozone toxicity, such as the
possibility of 0- reaction products being toxic or differences in cell sensiti-
vity that may prevent explanations of observed effects based on dosimetry
modeling alone.
9.2.3.3 Uses of Predicted Dose. Model-predicted doses can be used to estimate
comparable exposure levels that produce the same dose in different species or
different members of the same species for use in comparing toxicological data.
One can simulate tissue dose for several species for the same time for a range
of tracheal 0- concentrations. The doses for a specific zone or generation,
for each species, can be plotted versus tracheal or ambient Q~ concentration.
By using such plots and information on nasopharyngeal removal, the ambient
concentration necessary to produce the same dose in different species can be
estimated. Also, the relative quantity of 0~ delivered to a zone or generation
in a given species for the same time span and ambient concentration can be
predicted from the same graph. If the same biological parameters have not
been measured in these species at dose-equivalent exposure levels, the procedure
can be used to scale data and to design new studies to fill gaps in the current
data base.
9-15
-------
To illustrate the above procedure, Miller (1979) calculated exposure
levels of Og, giving the same respiratory bronchiolar dose in rabbits, guinea
pigs, and man. Considering the discussion on nasopharyngeal removal (Section
9.2.1.1) and the question concerning the guinea pig and rabbit anatomical
models (Section 9.2.3), these calculations are mainly useful for illustra-
tive purposes.
9.3 EFFECTS OF OZONE ON THE RESPIRATORY TRACT
9.3.1 Morphological Effects
The many similarities and differences in the structure of the lungs of
man and experimental animals were the subject of a recent workshop entitled
"Comparative Biology of the Lung: Morphology", which was sponsored by the Lung
Division of the National Institute of Blood, Heart, and Lung Diseases (National
Institutes of Health, 1983). These anatomical differences complicate but do
not necessarily prevent qualitative extrapolation of risk to man. Moreover,
because the lesions due to 0, exposure are similar in many of the species
studied (see Table 9-1), it appears likely that many of the postexposure
biological processes of animals could also occur in man.
9.3.1.1 Sites Affected. The pattern and distribution of morphological
lesions are similar in the species studied. Their precise characteristics
depend on the location (distribution) of sensitive cells and on the type of
junction between the conducting airways and the gaseous exchange area.
The upper or extrathoracic airways consist of the nasal cavity, pharynx,
larynx, and cervical trachea. Except for a few sites, the lining epithelium
is ciliated, pseudostratified columnar, with mucous (goblet) cells; it rests
on a lamina propria or s.ubmucosa that contains numerous mucous, serous, or
mixed glands and vascular plexi. Sites with differing structure include the
vestibule of the nasal cavity and portions of the pharynx and larynx, which
tend to have stratified squamous epithelium, and those portions of the nasal
cavity lined by olfactory epithelium, which contain special bipolar neurons
and glands associated with the sense of smell. Significant morphological dif-
ferences exist among the various animal species used for 0, exposures as well
as between most of them and man (Schreider and Raabe, 1981; Gross et al., 1982).
With the exception of the cervical trachea, these structures have received
little attention with respect to 0- sensitivity, but 0., removal through "scrub-
bing" has been studied (Yokoyama and Frank, 1972; and Miller et al., 1979).
9-16
-------
TABLE 9-1. MORPHOLOGICAL EFFECTS OF OZONE
f
Ozone
concentration
ug/m3
196
392
196
392
392
686
980
1568
392
686
392
980
1568
ppm
0.1
0.2
0.1
0.2
0.2
0.35
tf. 5
0.8
0.2
0.35
0.2
..0.5
0.8
Measurement, '
method
UV,
NBKI
UV,
NBKI
,
MAST,
NBKI
UV,
NBKI
j
UV,
NBKI
i
UV, :
NBKI
Exposure
duration
and
protocol
7 'days,
continuous
7 days,
continuous
30 days,
continuous
7 days,
8 hr/day
7 days.,
8 hr/day
7 days,
8 hr/day
or
24 hr/day
Observed effect(s) Species
Two of six fed with "basal" vitamin E diet had increased cen- Rat
triacinar AMs (SEM, LM).
Centriacinar accumulation of AMs, commonly in clumps of
3-5. Occasionally cilia were reduced in number, nonciliated
cells, some reduction in height.
Five of six fed E-deficient "basal" diet had centriacinar AMs Rat
and bronehiolar epithelial lesions (SEM). Four of six fed "basal"
diet +11 ppm E had lesser but similar lesions. One of six fed
"basal" diet +110 ppm E had lesser lesions.
Increased lung volume, mean chord length, and alveolar Rat
surface area. Lung weight and alveolar number did not
change. Decrease in lung tissue elasticity. Parenchyma
appeared "normal" by LM,
Respiratory bronchi olitis at all concentrations. Increased Monkey
AMs. Bronchiolar -epithelium both hyperplastic and hypertrophic. (Rhesus
Increased alveolar type 2 cells. Random foci of short, blunt and
cilia or absence of cilia (LM, SEM, TEM). Bonnet)
All exposed monkeys had LM & EM lesions. Trachea and bronchi Monkey
had areas of shortened or less dense cilia. RBs had AM (Bonnet)
accumulation and cuboidal cell hyperplasia. Alveoli off RBs
had AM accumulations and increased type 2 cells. RB walls of
the 0.35-ppra group were often thickened due to mild edema and
cellular infiltration.
Exposed groups gained less weight. -Focal areas of missing or Rat
damaged cilia in trachea and bronchi. TB nonciliated (Clara)
cells were shorter arid had increased surface granularity and less
smooth endoplasmic reticulum. Ciliated cells of TB had fewer
cilia and focal blebs.. Centriacinus had clusters of AMs and
PMNs. Type 1 cells swollen and fragmented and type 2 cells fre-
quently in pairs or clusters. Proximal IAS were minimally thick-
ened. Lesions in 0.2 rats were, mild (LM, SEM, TEM). Only slight
differences between rats exposed continuously 24 hr/day compared
to those exposed only 8 hr/day.
Reference
Plopper et al., 1979
Chow et al., 1981
Bartlett et al., 1974
Dungworth et al . , 1975b
Castleman et al.,-1977
Schwartz et al., 1976
-------
TABLE 9-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
figTi3 ppi
392
980
1568
W>
03 392
980
I960
392
1960
0,2
0.5
0.8
0,2
0.5
1.0
0,2
1.0
Exposure
k duration
Beasurement ' and
•ethod protocol
UV, 20, 50, or
NBKI 90 days;
8 hr/day
ND, ' 4 days,
NBKI 3 hr/day,
exercised in
a rotating
case alter-
nate 15 min
CHEM 7, 14, 30, 60,
90 days; con-
tinuous
10 days, con-
tinuous
Observed effect(s) Species Reference
Epithelial changes and PAH accumulations at 90 days were - Rat Boornan et al., 1980
similar to 7-day exposures, but less severe. 0,5- and 0.8-pptn
groups had increased centriacinar PAMs at all tines. 0.2 ppm
and controls could not be separated by "blind" LH examination,
nor were there distinguishing EH changes. 90-day 0,8-ppm group
had changed the terminal bronchiole/alveolar duct junction to
terminal bronchiole/respiratory bronchiole/alveolar duct junctions.
TBs had loss or shortened cilia. Nonciliated cells were flattened
lumenal surfaces that occasionally occurred in clusters. Proximal
alveoli of 20- and 90-day 0.8-ppn groups had thicker blood/air
barriers.
Exercised control mice have significantly smaller body weights. Mouse Fukase et al., 1978
Both unexercised and exercised mice exposed to 0.5 or 1.0 ppm (male,
had smaller body weights and larger lung weight. Exercised 5 weeks
mice exposed to 0,2 ppm also had larger lung weights. Other old,
pathology not studied. ICR-JCL)
Short-term exposures produced a slight degree of tonsil epithelial Rabbit Ikematsu, 1978
detachment. Cell infiltration below the epithelium was slight.
Long-term exposures caused slight edema of the lacunar epithelium
which was destroyed or detached in places. Lymphocyte infiltra-
tion also occurred.
Tonsil epithelium had a high degree of detachment. Cell satura-
tion occurred below the epithelium. Some protrusion of the tonsil
9800 5.0
3 hr
into the oral cavity.
Strong detachment of the tonsil epithelium. High degree of cell
saturation below the epithelium, including lymphocyte infiltra-
tration around the blood vessels and swelling of the endothelial
cells. Large amount of lymphocytes, viscous liquid, and detached
epithelial cells in the tonsilar cavity.
-------
TABLE 9-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
Jig/m"
490
510
980
1960
588
ppm
0.25
0.26
0.50
1.0
0.3
Exposure
• . duration
Measurement ' and
method protocol
CHEM ; 6 weeks,
| 12 hr/day
i
i
HAST, 4.7-6.6 hr,
NBKI ; endotracheal
tube
NBKI : '16 days,
3 hr/day
Observed effect(s)c Species Reference
Centriacinar or proximal alveoli had thicker interalveolar septa Rat Barry e£ al., 1983;
with significant increases in epithelium, cellular interstitiutn, Crapo et al., 1984
and endothelium. Type 1 and 2 alveolar epithelial cells and macro-
phages were increased in numbers. Type 1 cells had smaller volumes
and surface areas and were thicker.
Desquamation of ciliated epithelium. Focal swelling or Cat Boatman et al., 1974
sloughing of type 1 cells.
SEN, but not LM, showed swollen cilia with hemispheric Rat Sato et al., 1976a
extrusions and surface roughness. Some adhesion of
severely injured cilia otcurred. Small, round bodies were
frequently noted, mainly in the large airways and proximal
bronchioles. Luminal surfaces of the epithelium were often
covered with a pseudomembrane. The surfaces of Clara cells
showed swellings and round bodies. The surfaces of alveolar
ducts and walls showed scattered areas of cytoplasmic swelling
1
H
588
588
588
686
980
1372
1470
1960
686
980
0.3
0.3
0.3
0.35
0.50 •
0.70
0.75
1.00
0,35
0.50
NBKI ! 28 weeks,
: 5 days/week,
i 3 hr/day
i
UV ; 6 weeks,
1 5 days/week,
! 7 hr/day
ND 1, 5, 11, and
16 days,
r 3 hr/day
ND ; 1, 2, 4, 5, 6
or 8 days, con-
tinuous
i 4 days, contin-
'•. uous, followed
i by 0.50, 0.70,
; 0.75 or 1.00
: for 1-4 days
and attachment of round bodies. All responses were pronounced •
in vitamin E-deficient rats. Some rats had chronic respiratory
disease.
No morphological differences noted between vitamin E-defi- • Rat
cient and vitamin E-supplemented groups with the use of SEM
and TEM. Exposed and control rats had chronic respiratory
disease.
Increased LDH positive cells stated to be type 2 cells. Mouse
Rats were fed a basal diet with or without vitamin E supplement. Rat
Volume density of lamellar bodies in type 2 alveolar epithelial
cells were increased. Giant lamellar bodies were seen after 11
days exposure.
Dividing cells were labeled with tritiated thymidine and Rat
studied with autoradiographic techniques by using LM. All
labeled cells increased and then decreased to near control
levels within 4 days. Type 2 cells showed largest change
in labeling index.
Type 2 cells from groups showing adaptation to 0, were
exposed to higher concentrations. Groups exposed to low
initial concentration of Oa (0.35 ppn) did not maintain
tolerance. Groups exposed to higher initial concentration
(0.50 ppm) demonstrated tolerance.
Sato et al . , 1980
Sherwin et al. , 1983
Shimura et al. , 1984
Evans et al. , 1976b
-------
TABLE 3-1. MORPHOLOGICAL EFFECTS OF OZOME (continued)
to
o
Ozone
concentration Mtasureaent
pg/a3 ppi method
784 0.4 HAST
784 0.4 NBKI
980 0.5 NO
980 0.5 UV,
or or NBKI
1568 0.8
980 0.5 UV,
NBKI
Exposure
k duration
>b and
protocol
10 months,
5 days/week,
6 hr/day
7 hr/day,
5 days/week,
6 weeks
2 to 6 hr
7 continuous
days;
2, 4, 6, 8,
or E4 hr/
day
7, 21, and
35 days,
continuous
Observed effect(s)c
All (exposed and control) lungs showed some degree of inflam-
matory infiltrate possibly due to Intel-current disease.
A "moderate" degree of "emphysema" was present in 5 of the 6
exposed rabbits. Lungs of the 6th were so congested that
visualization of the mural frwework of the alveoli was
difficult. Small pulmonary arteries had thickened tunica iiedias,
sometimes due to edema, other times to muscular hyperplasia.
Lung growth which follows pneumonectomy also occurred
following both pneumonectomy and 03 exposure.
Centriacinar type 1 cells were swollen then sloughed.
Type 2 cells were not damaged and spread over the denuded
basement membrane. In some areas of severe type 1 cell
damage, endothelial swelling occurred. Damaged decreased
rapidly with distance from TB. Damage was most severe only
in the most central 2-3 alveoli. Interstitial edema occasionally
observed.
Centriacinar inflammatory cells (mostly AMs) were counted in
SEMs. Dose- related increase in inflammatory cell numbers except
in the continuously (24-hr/day) 0,8-ppm exposed rats. Rats
exposed 0.5 and 0.8 ppm 24 hr/day had the same intensity of
effect.
Most severe damage at terminal bronchiole/alveolar duct
junction. TB had focal hyperplastic nodules of non-
ciliated cells. Proximal alveoli had accumulations of
macrophages and thickening of IAS by mononuclear cells
at 7 days. At 35 days, changes much less evident, but
increased type 2 cells.
Species
Rabbit
(New
Zealand)
Rabbit
Rat
(young
males)
Rat
House
(Swiss-
Webster;
60 days
old; 35-40
Reference
P'an et al., 1972
Boatman et al., 1983
Stephens et al . , 1974b
Brutnmer et al., 1977
Zitnik et al., 1978
9)
980 0.5
NO
2 days
Tolerance was induced by exposure to 0.5 ppm 03 for 2 days.
Challenge was by exposure to 6.0 ppm Q3 for 24 hours. Toler-
ance was present at 3 days and declined at 7 and 15 days after
the initial exposure. When the animals were tolerant, the type 1
alveolar epithelium was thicker, had a smaller surface area and a
smaller surface-to-volume ratio.
Rat
Evans et al., 1985
980
1568
0.5
0.8
UV, 7, 28, or Principal lesion was a "low-grade respiratory bronchiolitis"
NBKI 90 days, characterized by "intraluminal accumulations of macrophages
continuous, and hypertrophy and hyperplasia of cuboidal bronchiolar
8 hr/day epithelial cells." Conducting airway lesions not apparent
by LM, but parallel linear arrays of uniform shortening and
reduction of density of cilia by SEN. Kulschitzky-type cells
appeared more numerous in exposed.
Monkey Eustis et al., 1981
(Bonnet)
-------
TABLE 9-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
fig/in3
980
1568
980
1960
f 980
& to
H 3920
ppm
0.5
0.8
0,5
1.0
0.5
to
2.0
Measurement3 '
method
UV,
NBKI
MAST
NBKI
UV,
NBKI
Exposure
duration
and
protocol
7 days ,
8 hr/day
60 days
6 hr/day
30 days
3 hr/day
7 days,
24 hr/day
Observed effect(s) , Species Reference
All exposed, monkeys had lesions. Lesions similar in 0.5- Monkey - Mellick et al., 1975,
and 0.8-, less severe in 0.5-ppm exposure groups. Patchy (Rhesus, 1977
areas of epithelium devoid of cilia in trachea and bronchi. adult)
Luminal surfaces of RB and proximal alveoli coated with
macrophages, a few neutrophils and eosinophils and debris.
Nonciliated cuboidal bronchiolar cells were larger, more
numerous, and sometimes stratified. Proximal alveolar
epitheliuui thickened by increased numbers of type 2 cells.
Progressive decrease in intensity of lesions from proximal to
distal orders of RBs..
Both immersion and infusion fixed lungs were studied by LH. Rats Yokoyama et al., 1984
Immersion fixed large and middle size bronchi had deeper than
normal infolding of the mucosa with increased secretions. The
low concentration rats had less severe mucosal infolding, but
a greater accumulation of secretions.
Elevated collagen synthesis rates and histologically Rat • Last et al., 1979
discernible fibrosis was present at all levels of 03.
O.i
to
1.5
14 days
and
21 days,
24 hr/day
0.5 ppm Minimal or no thickening of walls or evidence of
fibrosis. Increased number of cuboidal cells and
•acrophages present.
0.8-2.0 ppm: Moderate-thickening of AD walls and associated
IAS by fibroblasts, reticulin and collagen with
narrowing of the ducts and alveoli. Thickening
decreased with increased length of exposure.
0.5 ppm Sometimes minimal thickening of alveolar duct
walls with mildly increased reticulin and
collagen.
980 0.5
0.5 03
10 mg/m3
CHEM, 6 months, Only 03 caused pulmonary lesions. Only LM histopathology,
NBKI 5 days/week, no SEM nor TEM. Rats did not have exposure-related pulmonary
6 hr/day lesions, except 2 of 70 rats in the 03 group, which had
type 2 hyperplasia and focal alveolitis; 2 of 70 rats from the
H2S04 03 + H2S04 group, had slight hypertrophy and hyperplasia of
bronchiolar epithelium. Guinea pigs exposed to 03 or 03 +
H2S04 had lesions "near" the TB. Epithelium was hypertrophied
and hyperplastic. Macrophages were in centriacinar alveoli.
Occasionally proliferation of type 2 cells. Trachea and bronchi
had slight loss of cilia, reduction of goblet cells, and mild
basal cell hyperplasia. Ozone alone had no effect on body weight
gain; lung/body weight ratio; RBCs, hemoglobin, or hematocrit.
Rat
and
Guinea
pig
Cavender et al., 1978
-------
TABLE 9-1. HORPHOLOGICAL EFFECTS OF OZONE (continued)
f
to
to
Ozone
concentration
ug/raa pp"5
980
1058
1725
0.5
0.5 03
1 nig/13
0.54
0.88
Exposure
k duration
Measurement ' and
method protocol
UV, 3, 50, 90,
NBKI or 180 days;
continuous,
plus 62 days
. H2S04 postexposure,
24 hr/day
NO 2, 4, 8, 12,
or 48 hr
Observed effect(s)c Species * Reference
H2S04 did not potentiate effects of Og alone. Fixed lung Rat Moore and Schwartz, 1981
wolmes were increased at 180 days, but decreased at 62 days
postexposure/ After SO, 90, 94 180 days all 03 exposure
rats had "bronchiolization of alveoli" or formation of an RB
between the TB and ADs. Centriacinar inflammatory cells were
significantly increased at all exposure times and after 62 days
postexposure. TB lesions were qualitatively similar at 3, 50,
90, and 180 days. Cilia were irregular in number and length.
Nonciliated secretory (Clara) cells had flattened apical pro-
trusions and a blebbed granular surface. At 90, but not 180 days,
small clusters of nonciliated cells were present in the TB. At
180 days, 2 of 12 rats had larger nodular aggregates of noncili-
ated cells which bulged into the lumen. Most rats had a very nild
interstitial thickening of alveolar septa in the centriacinar
region (LH, SEM, TEH),
Severe loss of cilia from T8 after 2 hr. TB surface more Rat Stephens et al., 1974a
uniform in height than controls, Necrotic ciliate cells in
1058 0.54
6 months,
24 hr/day
TB epithelium and free in lumen after 6-12 hr of a 0.88-ppm
exposure. Ciliated cell necrosis continued until 24 hr, when
little evidence of further cell damage or loss was seen. Only
mi nilgai loss of ciliated cells in 0.5-ppm rat group. Non-
ciliated cells were "resistant" to injury from 03 and hyper-
trophic at 72 hr. Damage to the first 2 or 3 alveoli after
0.54-ppm for 2 hr. Type 1 cell "fraying" and vesiculation.
Damage was greater after 0.88 for 2 hr. "Basement lamina"
denuded. Type 2 and 3 cells resistant. Hacrophages
accumulated in proximal alveoli. Endothelium appeared
relatively normal.
Repair started at 20 hr. Type 2 cells divide, cuboidal
epithelium lines proximal alveoli where type 1 cells were
destroyed. Continued exposure resulted in thickened alveolar
walls and tissue surrounding TBs, Exposure for 8-10 hr followed
by clean air until 48 hr resulted in a proliferative response
(at 48 hr) about equal to that observed after continuous exposure
(LM, SEH, TEH).
No mention in either the results or discussion of the 6 months
at 0.54-ppra group.
-------
TABLE 9-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
ug/m3 ppm
1058 0.54
1725 0.88
1764 0.9 Oa
0.9 N02 -
490 0.25 03
2.5 N02
1176 0.6
vo 2548 1.3
1
NJ
US
1254 0.64
1254 0.64'
1882 0.96
5000 (NH4)2S04
1254 0.64
Exposure
, duration
Measurement ' and,
method protocol
ND 4 hr
to
3 weeks
60 days
6 months
NO 1 or 2 days,
6 hr/day or
7 hr/day
UV 1 year
8 hr/day
UV 3, 7 or 14 days
UV 23 hr/day
3 or 7 days
Observed effect(s)c
Ozone-exposed lungs heavier and larger than controls. Increased
centriacinar macrophages. Hyperplasia of distal airway epithelium
Increased connective tissue elements. Collagen-like strands
formed bridges across alveolar openings. Fibrosis more pronounced
in 0.88-ppm group.
Respiratory distress during first month. Several rats died.
Gross and microscopic appearance of advanced experimental
emphysema as produced by NOZ earlier (Freeman et al., 1972).
Ozone potentiated effect of nitrogen dioxide. .
"At 6 months the pulmonary tissue seemed quite normal." Proximal
orders of ADs minimally involved.
Endothelial cells showed the most disruption. The lining mem-
branes were fragmented. Cell debris was often present in the
alveoli as well as the capillaries. Some disorganization of
the cytoplasm of the large alveolar corner or wall cells was
evident.
LM and TEM morphometry revealed increased volume density and
volumes of RBs which had thicker walls and narrower lumens.
PeHbronchiolar and perivascular connective tissue was increased
by increased inflammatory cells and amorphous extracellular
matrix rather than stainable fibers. In RBs cuboidal bronchiolar
cells were increased and type 1 cells decreased. The media and
intima of small pulmonary arteries were thicker.
Ammonium sulfate aerosol enhanced the effects of 03 alone and
accelerated the occurrence of these effects. The same numbers of
lesions were seen in lungs from rats exposed to either 03 alone or
to 03 with the aerosol, but the lesions were larger in the latter
group. Lesions in the 03 plus aerosol rat lungs had more inflam-
matory cells, fibroblasts and stainable collagen fibers.
SEM and TEH, including TEM morphoraetry, revealed necrosis of
ciliated cells, decreased numbers of ciliated cells and loss of
Species
Rat
(month
old)
Rat
(month
old)
Rat
(month
old)
Mouse
(young^
Bonnet
monkey
Rat
Bonnet
monkey
Reference
Freeman et al . , 1974
Bils, 1970
Fujinaka et al. , 1985
Last et al . , 1984a
Wilson et al. , 1984
cilia. Extracellular space was increased and focal areas of epi-
thelial stratification were seen. Small mucous granule cells
were increased and an intermediate cell was described. Regular
mucous cells had decreased density and smaller irregularly sized
secretory granules which contained only filamentous or granular
material. TEH morphometry indicated the most severe lesions
occurred at 3 days of exposure and that the epithelium had returned
towards normal after 7 days of exposure.
-------
TABLE 9-1. HQRPHOLOGICflL EFFECTS OF OZOHE (continued)
Ozone
concentration
jjgTi3"
1372
1568
1372
ppffl
0.7
to
0,8
0,7
Exposure
b duration
Measurement ' and
method
UV,
NBKI
NO
protocol
7 days,
continuous
24 hr,
continuous
Observed effect(s)c Species
|n situ cytochenrical studies of lungs from 03 exposed and Rat
control rats. Ozone-exposed rats had increased acid phosphatase,
both in lysosoties and in the cytoplasm, in nonciliated bronchiolar
(Clara) cells, alveolar macrophages , type 1 and Z cells, and
fibroblasts.
Exposure end: General depletion of cilia from TB surface. Rat
Nonciliated cells were shorter and contained
fewer dense granules, less SER, and lore free
ribosomes.
Reference
Castleiaan et al,, 1973a
Evans et al., 1976a
Post exposure: TB returned towards normal.
(0-4 days)
1568 0.8 UV, 6, 10, 20
NBKI days
exposure,
24 hr/day
or
20 days
exposure +
10 days
postexposure
1568 0.8 ' UV, 7 days,
NBKI continuous
with samples
at 6, 24,
72 and
168 hr.
1568 0.8 UV, 4, 8, 12,
NBKI 18, 26, 36,
50, and post
48 and
188 hr,
continuous
SEH of distal trachea and primary bronchi: Mouse Ibrahim et al,, 1980
6 days: Cilia of variable length. (Swiss
10 days: Marked loss of cilia. Very few cells had Webster)
normal cilia. Some nonciliated cells were
in clusters and had wrinkled corrugated
surfaces.
20 days: Similar to 10 days.
10 days Cilia nearly normal.
postexposure: Clusters of nonciliated cells were present and
elevated above the surface.
Clusters of nonciliated cells were interpreted as proliferative
changes.
Exposure-related epithelial changes. TB cell populations Rat Lura et al., 1978
changed after Os exposure; fewer ciliated and more non-
ciliated secretory cells.
Degeneration and necrosis of RB type 1 cells predominates Monkey Castlentan et al., 1980
from 4-12 hr. Type 1 cell most sensitive of RB epithelial (Rhesus)
cells. Labeling index highest at 50 hr. Mostly cuboidal
bronchiolar cells but some type 2 cells. Bronchiolar
epithelium hyperplastic after 50 hr exposure, which
persisted following 7 days postexposure. Intraluminal
•aerophages increased during exposure, but marked clusters
of K cells at 26-36 hr.
-------
TABLE 9-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
(ig/nia ppm
1568 0.8
Exposure
. duration
Measurement ' and
method protocol
UV, 3 days,
NBKI continuous
0, 2, 6, 9,
16, and 30
days post-
" exposure;
2nd 3 days
continuous
after 6, 13,
and 27 days
postexposure
Observed effect(s)c
1st Exposure; TB epithelium flattened and covered with debris.
Ciliated cells either unrecognizable or had
.shortened cilia. The type of most epithelial
cells could not be determined. Proximal
alveoli had clumps of macrophages and cell
debris. Type 2 cells lined surfaces of many
proximal alveoli. Occasionally, denuded basal
lamina or type 1 cell swelling.
Postexposure:
6 days: Most obvious lesions were not present. TB epithelium
Species Reference
Rat Plopper et al., 1978
(Sprague-
Dawley;
70 days
old)
had usual pattern. Clumps of macrophages had cleared
from the lumen. Host proximal alveoli lined by normal
type 1 and 2 cells.
NJ
UT
1666 0.8S ND
Similar
exposure
regimen
for 14 ppm
N02) but not
mixtures.
1960 1.0 NBKI
|
' i 1, 2, 3 .
I days ,
. continuous
"50 weeks,
~5 days/week,
6 hr/day
(268 expo-
posnres)
30 days: Lungs indistinguishable from controls.
2nd 3-Day 6 or 27 days after the end of the 1st. Lesions
Exposure: same as 1st exposure.
Birth to weaning at 20 days: "Very little indication of
response" or "tissue nodules" with dissecting microscope.
12 days: N02 lesions but no 03 lesions.
22 days old: 03, loss of cilia, hypertrophy of TB cells,
tendency towards flattening of luminal
epithelial surface..
32 days old: 03, loss of cilia, and significant hypertrophy
of TB epithelial cells.
21 days old Alveolar injury, including sloughing to type 1
and older: cells resulting in bare basal lamina.
Response plateau is reached at 35 days of age.
Chronic injury occurred in the lungs of each species of small
animal. The principal site of injury was In the terminal air-
way, as manifested by chronic bronchiolitis and bronchiolar
wall fibrosis resulting in tortuosity and stenosis of the
passages.
Rat Stephens et al . , 1978
(Sprague-
Dawl ey ;
1, 5, 10,
15, 20, 25,
30, 35, and
40 days old)
Mouse, Stokinger et al., 1957
Hamster,
Rat
-------
TABLE 9-1. MORPHOLOGICAL EFFECTS OF OZONf (continued)
Ozone
concentration
ug/«3 ppi
1960
to
5880
1960
1960
I960
3920
5880
1.0
to
3.0
1.0
1.0
1.0
2.0
3.0
Exposure
, duration
Measurement ' and
method protocol
NO 18 months,
8-24 hr/
day
A = 8 hr/day
8 = 16 hr/day
C = 24 hr/day
0 = 8 hr/day
; E = 8 hr/day
Observed effect(s)c Species Reference
Result: Dog Freeman et al., 1973
A 1 ppm, 8 hr/day: Minimal fibrosis occasionally
and randomly in the periphery of an alveolar duct.
A few "extra" macrophages in central alveoli.
B 1 ppm, 16 hr/day: Occasional fibrous strands
in some alveolar openings of RBs and ADs. A few more
"extra" macrophages.
1 ppm, 24 hr/day: More extensive fibrosis of
centriacinus. Thickened AD walls. More "extra"
macrophages. Sporadic hyperplasia of epithelium of
RB and AD.
NJ
1960 1.0
and and
3920 2.0
Also
mixtures
with
H2S04
' D & More fibrosis. Epithelial hyperplasia and squamous
: E metaplasia.
CHEH, ! 2 or 7 days, Results: Rat Cavender et al., 1977
NBKI 6 hr/day 03 alone: Lesions limited to centriacinus. and
; 1 ppm: Hypertrophy and hyperplasia of TB epithelium. Guinea
[ Centriacinar alveoli had increased type 2 pig
i cells, increased macrophages, and thickened
i walls. Some edema in all animals.
; Lesions less severe at 7 days than at 2 days.
i This adaptation was more rapid in rats than
; guinea pigs.
2 ppm: Same plus loss of cilia in bronchi.
} 03 plus No additive or synergistic morphological
i H2S04 changes.
Measurement method: MAST = KI-coulonetric (Mast meter); CHEM = gas phase chemiluminescence); NBKI = neutral buffered potassium iodide;
UV = UV photometry; NO = not described.
Calibration method: NBKI = neutral buffered potassium iodide.
Abbreviations used: LM = light (nicroscope; EM = electron microscope; SEN = scanning electron microscope; TEM = transmission electron microscope; RAM = pulmonary
alveolar macrophage; RB = respiratory bronchiole; TB = terminal bronchiole; AD = alveolar duct; IAS = interalveolar septa; LDH = lactic dehydrogenase;
SER = smooth endoplasnric reticulum; RER = rough endoplasmic reticulum.
-------
The lower, or intrathoracic conducting airways include the thoracic tra-
chea, bronchi, and bronchioles. Species variation of lower airway structure
is large, as recorded at the NIH workshop on comparative biology of the lung
(National Institutes of Health, 1983). The thoracic trachea and bronchi have
epithelial and subepithelial tissues similar to those of the upper conducting
airways. In bronchioles, the epithelium does not contain mucous (goblet)
cells, but in their place are specialized nonciliated bronchiolar epithelial
cells, which in some species can appropriately be called "Clara" cells.
Subepithelial tissues are sparse and do not contain glands.
The ciliated cell is the cell in the upper and lower conducting airways
in which morphological evidence of damage is most readily seen. This cell is
primarily responsible for physical clearance or removal of inhaled foreign
material from conducting airways of the respiratory system (see Section 9.3.4).
The effects of ozone on this cell type, which is distributed throughout the
length of conducting airways, are detected through various physiological tests
and several types' of morphological examination (Kenoyer et al., 1981; Oomichi
and Kita, 1974; Phalen et al., 1980; Frager et al., 1979; Abraham et al., ||
1980).
The other portion of the respiratory system directly damaged by inhala-
tion of 0, is the junction of the conducting airways with the gaseous exchange
area. The structure and cell makeup of this junction varies with the species.
In man, the most distal conducting airways, the terminal bronchioles, are
followed by several generations of transitional airways, the respiratory
bronchioles, which have gas exchange areas as a part of their walls. In most of
the species used for experimental exposures to 0,, (i.e., mouse, rat, guinea
pig, and rabbit), the terminal bronchioles are followed by alveolar ducts
rather than respiratory bronchioles. The only common experimental animals
with respiratory bronchioles are the dog and monkey, and they have fewer
generations of nonrespiratory bronchioles than does man as well as differences
in the cells of the respiratory bronchioles (National Institutes of Health,
1983).
9.3.1.1.1 Airways
9.3.1.1.1.1 Upper airways (nasal cavity, pharynx, and larynx). The
effects of 03 on the upper extrathoracic airways have received little attention.
The effect of upper airway scrubbing on the level of 03 reaching the more
distal conducting airways has been studied in rabbits and guinea pigs (Miller
et al., 1979). They demonstrated removal of approximately 50 percent of
9-27
-------
3
ambient concentrations between 196 and 3920 (jg/m (0.1 and 2.0 ppm). Earlier,
Vokoyama and Frank (1972) studied nasal uptake in dogs. They found uptake to
3
vary with flow rate as well as with 03 concentration. At 510 to 666 ug/m
(0.26 to 0.34 ppm) of 03, the uptake at low flows of 3.5 to 6.5 L/min was 71.7
±1.7 percent, and at high flow rates of 35 to 45 L/min the uptake was 36.9 ±
q
2.7 percent. At 1529 to 1568 pg/m (.0.78 to 0.8 ppm), the uptakes at low and
high flows were 59.2 ±1.3 percent and 26.7 ±2.1 percent, respectively. The
scrubbing effect of the oral cavity was significantly less at all concentrations
and flow rates studied. Species variations in uptake by the nasal cavity
probably relate to species differences in the complexity and surface areas of
the nasal conchae and meatuses (Schreider and Raabe, 1981).
No studies of the effects of 0~ on the nasal cavity were found, but two
references to articles in the Japanese literature were cited by Ikematsu
(1978). At least one study of the morphological effects of ambient levels of
0~ on the nasal cavity of nonhuman primates is in progress, but not published.
o
The effects of 392, 1960, and 9800 pg/m (0.2, 1.0, and 5.0 ppm) of Q3 on
the tonsils, the primary lymphoreticular structures of the upper airways, were
o
studied. In palatine tonsils from rabbits exposed to 392 ug/m (0.2 ppm) of
0~ continuously for 1 and 2 weeks, Ikematsu (1978) reported epithelial detach-
ment and disarrangement and a slight cellular infiltration. The significance
of these observations to the function of immune mechanisms in host defense is
unknown.
9.3.1.1.1.2 Trachea. Trachea! epithelial lesions have been described
O
in several species following exposure to less than 1960 ug/m (1 ppm) of Og.
Boatman et al. (1974) exposed anesthetized, paralyzed cats to 510, 980, or
1960 ug/m3 (0.26, 0.50, or 1 ppm) of 03 via an endotracheal tube for 4.7 to
6.6 hr. This exposure technique bypassed the nasal cavity, resulting in
higher trachea! concentrations than in usual exposures. They reported desquama-
3
tion of ciliated epithelium-at 1960 ug/m (1 ppm) of 03, but none,at 510 or
980 ug/m3 (0.26 or 0.5 ppm).
In rats exposed to 960 or 1568 ug/m (0.5 or 0.8 ppm) of 03, 8 or 24
hr/day for 7 days, Schwartz et al. (1976) described focal areas of the trachea
in which the cilia were reduced in density and were of variable diameter and
length. Mucous cells appeared to have been fixed in the process of discharging
mucigen droplets. These changes were more easily seen with the scanning
2
electron microscope (SEM) and were not obvious in rats exposed to 392 ug/m
(0.2 ppm) of 0- for the same times. Cavender et al. (1977), when using only
O *
9-28
-------
light microscopy (LM), studied tracheas from rats and guinea pigs exposed for
7 days to 1960 or 3920 ug/m3 (1 or 2 ppm) of 03 or sulfuric add (HgSO^) or
both. The article does not state the hours of exposure per day. Trachea!
lesions, which consisted of reduced numbers of cilia and goblet cells, were
reported only for guinea pigs exposed to 03. Animals exposed to both pollu-
tants had lesions similar to those exposed to 0, alone.
By using SEM and transmission electron microscopy (TEM), Castleman et al.
(1977) described shortened and less dense cilia in tracheas from bonnet mon-
keys exposed to 392 or 686 pg/m (0.2 or 0.35 ppm), 8 hr/day for 7 days.
Lesions occurred as random patches or longitudinal tracts. In these areas,
the nonciliated cells appeared to be more numerous. The TEM study revealed
that cells with long cilia had the. most severe cytoplasmic changes, which
included dilated endoplasmic reticulum, swollen mitochondria, and condensed
nuclei, some of which were pyknotic. In lesion areas, evidence of ciliogene-
sis was seen in noncilated cells with a microvillar surface. Mucous cells did
not appear to be significantly altered, but some had roughened apical surfaces.
o
The changes were more variable and less severe in the 392 ug/m (0.2 ppm)
group. More extensive and severe lesions of similar nature were seen in
3
tracheas from rhesus monkeys exposed to 980 or 1568 pg/m (0.5 or 0.8 ppm) of
03 in the same' exposure regimen (Dungworth et al., 1975b; Mellick et al.s
1977).
Wilson et al. (1984) evaluated the response of the tracheal epithelium
3
from bonnet monkeys exposed continuously for 3 or 7 days to 1254 ug/m (0.64
ppm) Og using SEM, quantitative TEM, and autoradiography. Extracellular space
was increased and foci of stratified epithelium were reported. Changes in
ciliated cells were generally similar, but more severe, than those reported by
Castleman et al. (1977). These changes were more severe at 3 days than at 7
days when both the volume percentage and population density had returned to
control values. They also reported fewer and smaller granules in regular, as
opposed to small-mucous-granule (SMG), mucous cells. These granules also
lacked the biphasic appearance of those seen in control monkeys. At both time
periods SMG cells in exposed monkeys were more numerous and had greater numbers
of granules than controls.
3
After mice were exposed to 1568 yg/m (0.8 ppm) of 0, 24 hr/day for 6,
10, or 20 days and for 20 days followed by a 10-day postexposure period during
which the animals breathed filtered air, the suffice of .the tracheas was
examined by SEM by Ibrahim et al. (1980). Short and normal-length cilia were
9-29
-------
seen at 6 days, but at 10 and 20 days, a marked loss of cilia and few normal
cilia were seen. Some of the nonciliated cells occurred as clusters and had
wrinkled or corrugated surfaces. After the mice breathed filtered air for
10 days, the surface morphology of the cilia returned to near normal, but the
clusters of nonciliated cells were still present. Earlier, Penha and Wer-
thamer (1974) observed metaplasia of the trachea! epithelium from mice exposed
o
to high concentrations of Q3 (4900 jjg/m , 2.5 ppm) for 120 days. After the
mice breathed clean air for 120 days, the metaplasia disappeared, and the
epithelium had a nearly normal frequency of ciliated and nonciliated cells.
9.3.1.1.1.3 Bronchi. Bronchial lesions were studied in many of the
same animals as those whose tracheal lesions are described above and were
reported as gene-rally similar to the tracheal lesions. -At low concentrations
(Castleman et a!., 1977), lesions tended to be more severe in the trachea and
proximal bronchi than in distal bronchi or in the next segment of the conduc-
tion airways, the nonalveolarized bronchioles. Eustis et al. (1981) reported
lesions in lobar, segmental, and subsegmental bronchi from bonnet monkeys
exposed to 980 or 1568 |jg/m3 (0.5 or 0.8 ppm) of 03 8 hr/day for 7, 28, or 90
days. Lesions at 7 days were similar to those previously described by Mellick
et al. (1977) and Castleman et al. (1977), as summarized above. At 28 and 90
days, lesions were not readily apparent by LM, but extensive damage to ciliated
cells was seen using SEM. Uniform shortening and reduced density of cilia
were seen in linear, parallel arrays. In these areas, the numbers of cells
with a flat surface covered by microvilli increased. Wilson et al. (1984)
reported similar changes in primary bronchi from bonnet monkeys exposed to
q
1254 pg/m (0.64 ppm) 03 continuously for 3 or 7 days.
Sato et al. (1976a,b) studied bronchi from vitamin E-deficient and control
q
rats exposed to 588 jjg/m (0.3 ppm) of 03 3 hr/day for up to 16 days. Using
LM, they did not see bronchial lesions with asymmetrical swelling and surface
roughness, which were obvious with SEM. The observations of Sato et al.
(1976a) support the concept that lesions in conducting airways are best seen
with SEM and that LM tends to underestimate damage to these ciliated airways.
In these short-term studies, lesions were more prominent in vitamin E-deficient
rats. This is in contrast to later studies in which Sato et al. (1978, 1980)
2
exposed vitamin E-deficient and supplemented rats to 588 |jg/m (0.3 ppm) of 03
3 hr/day, 5 days/week for 7 months following which they did not see clear
differences due to vitamin E with SEM or TEM.
9-30
-------
Yokoyama et al. (1984) studied effects of 0~ on "middle-sized bronchi" of
rats fixed by immersion rather than infusion via the airways. They compared
the effects of 3 hr/day exposures to 1960 jjg/m (1.0 ppm) 0, for 30 days with
3
those following 980 ug/m (0.5 ppm) 03 6 hr/day for 60 days, They reported
increased mucus and irregular loss of cilia, especially on the projections of
mucosal folds typical of this type of fixation. Changes were less severe in
the 0.5 ppm group.
9.3.1.1.1.4 Bronchioles. There are two types of bronchioles with
similar basic structure: those without alveoli in their walls (i.e., nonalveo-
larized) and those with alveoli opening directly into their lumen (respiratory
bronchioles). Man and the larger experimental animals (e.g., nonhuman primates
and dogs) have both nonrespiratory and respiratory bronchioles, whereas most
of the smaller experimental animals (e.g., mice, rats, and guinea pigs) have
only nonrespiratory bronchioles. Because the types, functions, and lesions of
epithelial cells are different, these two types of bronchioles will be discussed
separately.
Nonrespiratory bronchioles are conducting airways lined by two principal
types of epithelial cells: the ciliated and nonciliated bronchiolar cells.
The latter cell is frequently called the Clara cell. Although man and most
animals have several generations of nonrespiratory bronchioles, some nonhuman
primates have only one (Cast!eman et al., 1975). The last-generation con-
ducting airway before the gas exchange area of the lung is the terminal bron-
chiole. Terminal bronchioles may end by forming respiratory bronchioles, as
in man, monkeys, and dogs, or by forming alveolar ducts, as in mice, rats, and
guinea pigs. The acinus, the functional unit of the lung, extends distally
from the terminal bronchiole and includes the gas exchange area supplied by
the terminal bronchiole and the vessels and nerves that service the terminal
bronchiole and its exchange area.
A major lesion due to 0~ exposure occurs in the central portion of the
acinus, the centriacinar region, which includes the end of the terminal bronchi-
ole and the first'few generations of either respiratory bronchioles or alveolar
ducts, depending on the species. The centriacinar region is the junction of
the conducting airways with the gas exchange tissues. The 0,, lesion involves
both the distal portion of the airway and the immediately adjacent alveoli,
the proximal alveoli. In animals with respiratory bronchioles, the lesion is
a respiratory bronchiolitis. Regardless of species differences in structure,
the lesion occurs at the junction of the conducting airways with the gas
exchange area.
9-31
-------
o
Terminal bronchiolar lesions in rats due to inhalation of < i960 ug/m
(< 1.0 ppm) of 0~ for 2 hr to 1 week have been described by Stephens et al.
(1974a), Evans et al. (1976a,c), Schwartz et al. (1976), and others (Table 9-1}.
These changes were recently reviewed by Evans (1984). Ciliated cells are the
most damaged of the airway cells, and fewer of them are found in the bronchiolar
epithelium of exposed animals. Those ciliated cells present tend to have
cilia with focal blebs and blunt ends. Damaged ciliated cells are replaced by
nonciliated bronchiolar (Clara) cells (Evans et al., 1976a; Lum et al., 1978),
which become hyperplastic. The typical projection of the nonciliated or Clara
cell into the lumen is reduced, and the luminal surface has increased granular-
ity. The reduction in projection height appears to be due to a reduction in
agranular (smooth) endoplasmic reticulum (Schwartz et al., 1976). Many ciliated
cells contain basal bodies and precursors of basal bodies indicative of cilio-
genesis (Schwartz et al., 1976). The few brush cells present in nonrespiratory
bronchioles appeared normal (Schwartz et al., 1976). The lesions were more
severe in higher generation, more distal nonrespiratory bronchioles than in
the lower generation, more proximal nonrespiratory bronchioles.
In an earlier study, Freeman et al. (1974) exposed month-old rats con-
tinuously to 1058 or 1725 ug/m3 (0.54 or 0.88 ppm) of Q3 for 4 hr to 3 weeks.
In addition to the centriacinar accumulations of macrophages and the hyper-
plasia of the distal airway epithelium, they reported an increase in connective
tissue elements and collagen-like strands which formed bridges across alveolar
o
openings. In the 1725-ug/m (0.88-ppm) 0~ group, the fibrosis was pronounced
and sometimes extended into terminal bronchioles. In the same study, Freeman
3
et al. (1974) exposed month-old rats to a mixture of 1764 ug/m (0.9 ppm) 0,
3 3
and 1690 ug/m (0.9 ppm) nitrogen dioxide, or to a mixture of 490 ug/m (0.25
q
ppm) 03 and 4700 ug/m (2.5 ppm) nitrogen dioxide. After 60 days of exposure
to the 0.9/0.9 mixture, Freeman et al. (1974) reported that "both grossly and
microscopically, the appearance of the lungs was characteristic of advanced
experimental emphysema," referring to earlier nitrogen dioxide exposures
(Freeman et al., 1972). Freeman et al. (1974) did not report emphysema in
rats exposed to 0^ alone, only in those rats exposed to the 0.9/0.9 mixture.
The topic of emphysema is discussed later (Section 9.3,1.4.2).
Results differ in four studies of long-term (3- to 6-month) exposures of
o
rats to < 1960 ug/m (<1.0 ppm) for 6 or 8 hr/day. The differences appear to
be due at least in part to the methods used to evaluate the bronchioles. When
9-32
-------
3
using only LM to study effects in rats exposed for 6 months to 980 ug/m (0.5
ppm) for 6 hr/day, Cavender et al. (1978) did not find exposure-related lesions. .
Barr (1984) exposed rats to 1862 pg/m (0.95 ppm) 03 8 hr/day for 90 days.; : He '
examined the bronchioles with LM, TEM, and SEM. Using SEM he reported loss of
apical projections of nonciliated cells and loss of both density and height of
cilia. Boorman et al. (1980) and Moore and Schwartz (1981) reported signi-
3
ficant bronchiolar lesions following exposure to 980 or 1568 ug/m (0.5 or 0.8
ppm) of 03 8 hr/day for 90 or 180 days. In both studies, loss or shortening of
cilia and flattening of the luminal projections of nonciliated bronchiolar
cells were observed in terminal bronchioles at each time period, including the
end of exposure at 90 or 180 days. Clusters of four to six nonciliated bronchio-
lar cells, in contrast to dispersed individual cells in controls, were seen at
90 days in both studies, but not at 180 days. However, in 2 of the 12 rats
exposed 180 days, larger nodular aggregates of hyperplastic cells projected
into the bronchial lumen. After 50, 90, and 180 days of exposure, the nature
of the junction between the terminal bronchiole and the alveolar ducts changed
from the sharp demarcation seen in controls to a gradual transition with the
appearance of a respiratory bronchiole. The presence of this change in distal
airway morphology was confirmed by Barr (1984). Both ciliated and nonciliated
bronchiolar cells were seen on thickened tissue ridges between alveoli. This
change could result from either alveolarization of the terminal bronchiole or
bronchiolization of alveolar ducts. Although this change in the airway mor-
phology persisted, the changed segment reduced in length after the 180-day-
exposed rats had breathed filtered air for 62 postexposure days. The addition
of 1 to 10 mg/m HgSQ* to these concentrations of 0., for the same exposure
times did not potentiate the lesions seen in the CU-alone rats (Moore and
Schwartz, 1981; Cavender et al., 1978; Juhos et al., 1978).
Ozone-induced bronchiolar lesions in mice are similar to those seen in
rats, But the hyperplasia of the nonciliated cells is more severe (Zitnik et
al., 1978); Ibrahim et al., 1980). Following high concentrations (4900 ug/m ,
2.5 ppm), Penha and Werthamer (1974) noted persistence (unchanged in frequency
or appearance) or micronodular hyperplasia of noncilated bronchiolar cells for
120 postexposure days following 120 days of exposure. At lower 0~ levels (980
3
or 1568 ug/m , 0.5 or 0.8 ppm), the hyperplasia was pronounced (Zitnik et al.,
1978; Ibrahim et al., 1980). Ibrahim et al. (1980) noted hyperplastic clusters
of nonciliated cells 10 days after exposure but did not make observations
after longer postexposure periods.
9-33
-------
Guinea pigs were exposed by Cavender et al. (1977, 1978) continuously to
•3
1 or 2 ppm of Og for 2 or 7 days in acute studies and to 980 pg/m (0.5 ppm) 6
hr/day, 5 days/week for 6 months. Morphological effects were studied only by
LM. The acute, higher concentration distal-airway lesions were similar to
those seen in rats and included loss of cilia and hyperplasia of nonciliated
cells. The authors reported that the long-term, lower concentration lesions
were more severe in guinea pigs than those in rats exposed to a similar regimen.
The lesions were no more severe in guinea pigs exposed to a combination of
H2SCL aerosol and Oj.
9.3.1.1.2 Parenchyma. Ozone does not affect the parenchyma in a uniform
manner. The centriacinar region (i.e., the junction of the conducting airways
with the gas exchange area) is the focus of damage, and no changes have been
reported in the peripheral portions of the acinus.
9.3.1.1.2.1 Respiratory bronchioles. Respiratory bronchioles are the
focus of effects, because they are the junction of the conducting airways with
the gas exchange area. However, not all animals have respiratory bronchioles.
They are well developed in man but are absent or poorly developed in the
common laboratory animals frequently used for On study, with the exception of
dogs (Freeman et al., 1973) and macaque monkeys (Mellick et al., 1975, 1977;
Dungworth et al., 1975b; Castleman et al., 1977, 1980; Eustis et al., 1981).
3
Short-term exposures of monkeys to 392, 686, 980, or 1568 yg/m (0.2, 0.35,
0.5, or 0.8 ppm) of 0, 8 hr/day for 7 days resulted in damage to type 1 cells
and hyperplasia of nonciliated bronchiolar cells, which were visible by either
light or electron microscopy. At lower concentrations, these lesions were
limited to the proximal, lower generation respiratory bronchioles. At higher
concentrations, the lesions extended deeper into the acinus. The lesions were
focused at the junction of the conducting airways with the gas exchange area
and extended from that Junction with increasing 0, concentration.
3
The pathogenesis of the lesions due to 1568 ug/m (0.8 ppm) of Og for
periods up to 50 hr of exposure was studied quantitatively by Castleman et al.
(1980). Damage to type 1 cells was very severe following 4, 8j and 12 hr of
exposure. Type 1 cell necrosis, which resulted in bare basal larninas reached
a maximum at 12 hr. The absolute and relative numbers of these cells decreased
throughout the exposure. Only a few type 2 cells had mild degenerative changes
* and only at 4 or 12 hr of exposure. Cuboidal bronchiolar cells had mild
degenerative changes, swollen mitochondria, and endoplasmic reticulum at all
times except 18 hr. Both cuboidal bronchiolar and type 2 cells functioned as
9-34
-------
stem cells in renewal epithelium, and both contributed to the hyperplasia seen
at the latter exposure times. The inflammatory exudate included both fibrin
and a variety of leukocytes in the early phases. In the latter phase's, the
inflammatory cells were almost entirely macrophages. Inflammatory cells were
also seen in the walls of respiratory bronchioles and alveoli opening into
them. These lesions were not completely resolved after 7 days of filtered air
breathing.
3
Monkeys exposed to 960 or 1568 ug/m (0.5 or 0.8 ppm) of 0^ 8 hr/day for
90 days had a low-grade respiratory bronchiolitis characterized by hypertrophy
and hyperplasia of cuboidal bronchiolar cells and intraluminal accumulation of
macrophages (Eustis et a!., 1981). After the 90-day exposure, the percentage
of cuboidal respiratory bronchiolar epithelial cells was 90 percent rather
than the 60 percent found in controls. Intraluminal cells, mostly macrophages,
reached a maximum of a thirty-seven-fold increase after 7 days of exposure.
Their numbers decreased with continued exposure, but at 90 days of exposure,
they were still sevenfold higher than those of controls. This study did not
include a postexposure period.
3
Fujinaka et al. (1985) exposed adult male bonnet monkeys to 1254 jjg/m
(0,64 ppm) 03 8 hr/day for one year. They reported significantly increased
volume of respiratory bronchioles which had smaller internal diameters.
Respiratory bronchiolar walls were thickened by epithelial hyperplasia and
increased peribronchiolar connective tissue. Several small nodules of hyper-
plastic and hypertrophied cuboidal bronchiolar cells were reported near the
openings of alveoli into the respiratory bronchiole. The authors interpret
the morphometry of respiratory bronchioles as indicating an extension of
bronchiolar epithelium into airways Which were formerly alveolar ducts similar
to the formation of respiratory bronchioles reported in 03-exposed rats by
Boorman et al. (1980) and Moore and Schwartz (1981).
In an earlier study, Freeman et al. (1973) exposed female beagle dogs to
3
1960 |jg/m (1 ppm) of 0, 8, 16, or 24 hr/day for 18 months. Dogs exposed to
3
1960 ug/m (1 PPm) °f 03 f°r 8 hr/day had the mildest lesions, which were
obvious only in the terminal airway and immediately adjacent alveoli, where
minimal fibrosis and a few extra macrophages were seen. More fibrous strands
and macrophages were seen in centriacinar areas of lungs from dogs exposed 16
hr/day. Lungs from dogs exposed 24 hr/day had terminal airways distorted by
fibrosis and thickened by both fibrous tissue and a mononuclear cell infiltrate.
Relatively broad bands of connective tissue were reported in distal airways
9-35
-------
and proximal alveoli. Epithelial hyperplasia was seen sporadically in the
bronchiolar-ductal zone. Other dogs in that study exposed to 3920 or 5880
fjg/m (2 or 3 ppm) of 03 8 hr/day for the same period had more severe fibrosis,
greater accumulations of intraluminal macrophages, and areas of both squamous
and mucous metaplasia of bronchiolar epithelia.
9.3.1.1.2.2 Alveolar ducts and alveoli. Alveoli in the centriacinar
region, but not those at the periphery of the acinus, are damaged by ambient
levels of 03 (Stephens et a!., 1974b; Schwartz et a!., 1976; Mellick et a!.,
1977; Crapo et a!., 1984). The lesion is characterized by the destruction of
type 1 alveolar epithelial cells exposing the basal lamina; an accumulation of
inflammatory cells, especially macrophages; hyperplasia of type 2 alveolar
epithelial cells that recover the denuded basal lamina; and thickening of the
interalveolar septa. In animals with respiratory bronchioles (e.g., dog,
monkey) the alveoli involved at low concentrations are those opening directly
into the respiratory bronchiolar lumen of low-generation respiratory bronchioles
(Dungworth et al,, 1975b). As the concentration is increased, the lesions
include alveoli attached to but not, seemingly, opening into the respiratory
bronchioles and extending distally to higher-generation respiratory bronchioles
(Mellick et al., 1977). In monkeys exposed to 1568 ug/m (0.8 ppm) of 03,
alveoli opening into alveolar ducts are minimally involved (Mellick et al.,
1977), In animals that lack respiratory bronchioles (e.g., rat, mouse, guinea
pig) the alveoli involved open into or are immediately adjacent to the alveolar
ducts formed by the termination of the terminal bronchiole.
In the centriacinar region of animals which lack respiratory bronchioles,
damage to type 1 cells has been reported as early as 2 hours following exposure
o
to 980 ug/m (0.5 ppm) 03 using LM (Stephens et al., 1974a). While not an 0-
concentration studied comprehensively or illustrated in that article, the same
authors comment in the results section of their report that TEM evaluation of
rats exposed to 392 ug/m (0.2 ppm) 0,, for 2 hours revealed "...considerable
damage and loss of type 1 cells from proximal alveoli..." Recovering of
denuded basal lamina by type 2 cells has been reported to start as early as 4
hr (Stephens et al,, 1974b). The type 2 cell labeling index following tritiated
thymidine reached a maximum at 2 days of continuous exposure to either 686 or
980 ug/m3 (0.35 or 0.5 ppm) of 0,, (Evans et al., 1976b). Although the labeling
«5
index decreases as the exposure continues (Evans et al., 1976b), clusters of
type 2 cells and cells intermediate between types 2 and 1 were reported following
90 days of exposure to 1568 ug/m (0.8 ppm) by Boorman et al. (1980). They
9-36
-------
interpreted these intermediate cells as due to delay or arrest of the transfor-
mation from type 2 to type 1 epithelial cells. Type 1 cell damage and occasional
sloughing were observed by Barry et al. (1983) in newborn rats exposed to 490
3
ug/m (0.25 ppm) of 0- 12 hr/day for 6 weeks. By using LM and TEM morphometric
techniques, these authors also found that centriacinar alveoli also had more
type 1 and 2 epithelial cells and macrophages. The type 1 cells were smaller
in volume, covered less surface, and were thicker. Evans et al. (1985) studied
this effect and suggested that 03 tolerance exists when the surface area of a
cell is small enough so that antioxidant mechanisms contained in that volume
can protect it from damage. Sherwin et al. (1983) found increased numbers of
lactate dehydrogenase (LDH)-positive cells, presumed to be type 2 alveolar
epithelial cells, by automated LM morphometry of lungs from mice exposed to
3
588 ug/m (0.3 ppm) of 0- for 6 weeks. Moore and Schwartz (1981) reported
nonciliated bronchiolar cells lined some alveoli opening into the transformed
airways located between terminal bronchioles and alveolar ducts of rats exposed
3
to 1568 ug/m (0.8 ppm) of 0- 8 hr/day for 180 days. Sulfuric acid aerosol
did not potentiate this lesion.
The inflammatory cell response appears to occur immediately following or
concurrent with the type 1 cell damage and has been reported in monkeys as
early as after 4 hr of 1568 |jg/m (0.8 ppm) of 03 exposure (Castleman et al.s
1980). In rats, the numbers of inflammatory cells per centriacinar alveolus
3
appear to be related to 0- concentration, at levels between 392 and 1568 ug/m
(0.2 and 0.8 ppm), during 7-day (Brummer et al., 1977) and 90-day exposures
(Boorman et al. , 1980). Using the same technique, Moore and Schwartz (1981)
found statistically significant increases after 3, 50, 90, and 180 days of
3
exposure 8 hr/day to 980 ug/m (0.5 ppm) of 0~ and after 62 days of filtered
3
air following 180 days of exposure. The addition of 1 mg/m H^SO* aerosol did
not result in larger increases. In monkeys, the intensity of the response was
3
greater than in rats exposed to the same 0~ concentration (1568 ug/m , 0.8
ppm) in the same regimen (8 hr/day) for 7, 28, or 90 days (Eustis et al. ,
1981). Although in both species the numbers of inflammatory cells per alveolus
decreased with increasing length of exposure, the decrease was not as rapid in
the monkey as in the rat (Eustis et al., 1981).
Several investigators (Boorman et al., 1980; Moore and Schwartz, 1981;
Fujinaka et al., 1985; Barr, 1984) have presented evidence of bronchiolization
of airways which were previously alveolar ducts; i.e., bronchial epithelium
replaces the type 1 and 2 alveolar epithelium typical of alveolar ducts.
9-37
-------
This phenomenon is most easily seen and quantitated in species which normally
do not have respiratory bronchioles or have one very short generation of them.
In these species, airways with the characteristics of respiratory bronchioles
are seen between the terminal bronchioles and the alveolar ducts of exposed
but not control animals (Boorman et a!., 1980; Moore and Schwartz, 1981; Barr,
1984). In species which normally have several generations of respiratory
bronchioles, bronchiolization is detected morphometrically by increases in the
volume fraction or total volume of respiratory bronchioles. Using morphometric
techniques, bronchiolization was reported in nonhuman primates exposed to 1254
3
ug/m (0.64 ppm) CU 8 hr/day for one year (Fujinaka et a!., 1985).
The interalveolar septa of centriacinar alveoli are thickened following
exposure to ambient concentrations of 03. After 7 days of continuous expo-
sure, the thickening was attributed to eosinophilic hyaline material and
mononuclear cells (Schwartz et al., 1976). Loose arrangements of cells and
extracellular materials suggested separation by edema fluid. Castleman et al.
(1980) also reported edema of interalveolar septa of monkeys exposed to 1568
3
ug/m (0.8 ppm) of 0, for 4 to 50 hr. Boorman et al. (1980) used morphometric
techniques on electron micrographs to quantitatively evaluate the thickness of
centriacinar interaveolar septa. The arithmetic mean thickness was increased
3
in rats exposed to 1568 ug/m (0.8 ppm) of 03 8 hr/day for 20 or 90 days. The
increased total thickness was due to thicker interstitium. Although several
components could contribute to this increased thickness, the subjective im-
pression was one of a mild interstitial fibrosis. Crapo et al. (1984) made a
more comprehensive morphometric study of centriacinar interalveolar septa from
o
young adult rats exposed to 490 yg/m (0.25 ppm) 03 12 hr/day for 6 weeks.
They reported significant increases in tissue thickness and suggested that the
increased thickness was due to significant increases in all cell types except
type 2 cells, and to increased interstitium.
2
Moore and Schwartz (1981), after exposing rats to 980 ug/m (0.5 ppm) of
0- 24 hr/day for 180 days, reported very mild interstitial thickening of
centriacinar interalveolar septa, which they concluded was due to collagen.
Earlier, Freeman et al. (1973) morphologically demonstrated fibrosis in beagle
3
dogs exposed to 1960 to 5880 \sg/m (1 to 3 ppm) of 03 8 to 24 hr/day for 18
months.
In several biochemical studies (see Section 9.3.3.6), Last and colleagues
have shown that 03 is collagenic. In one of these (Last et al., 1979), the
9-38
-------
biochemical observations were correlated with histological observations of
slides stained for collagen and reticulin. Elevated collagen synthesis rates
were found at all concentrations and times studied. Mildly increased amounts
of collagen were seen morphologically in centriacinar alveolar duct septa from
3
most rats exposed to 980 ug/m (0.5 ppm) of 0~ 24 hr/day for 14 or 21 days.
3
More severe lesions were seen in rats exposed to 1568 to 3920 ug/m (0.8 to
2.0 ppm) of 03 24 hr/day for 7, 14, or 21 days. Last et al. (1983, 1984a)
have reported synergistic increases in morphometrically determined volume of
centriacinar lesions, inflammatory cells, and "stainable" collagen in rats
exposed to a mixture of 0,, and ammonium sulfate aerosols. The 1984 report
3
concerns this synergism in rats exposed to 1254 or 1882 ug/m (0.64 or 0.96 ppm)
3
03 and 5 mg/m ammonium sulfate for 3, 7, or 14 days. The results correlate
well with biochemically determined apparent collagen synthesis rates (see
Section 9.3.3.6).
Two studies address the biologically important question of the morphological
effects that follow multiple sequential exposures to 0~ with several days of
clean air interspersed between 0~ exposures (i.e., a multiple episode exposure
3
regime). Plopper et al. (1978) exposed rats to 1568 ug/m (0.8 ppm) 03 continu-
ously for 3 days, held them in the chambers breathing filtered air until
o
postexposure day 6, 13, or 27, when they were again exposed to 1568 |jg/m
(0.8 ppm) of 03 continuously for 3 days. Rats were also examined 2, 6, 9, 16,
and 30 days after the first 0« exposure. Lungs from rats breathing filtered
air for 9 days after one 3-day exposure had only minimal lesions and after
30 days of filtered air were indistinguishable from controls. When the second
3-day 03 exposure started 6 or 27 days after the end of the first exposure,
the lesions appeared identical to each other and to those seen at the end of
3
the first exposure. Barr (1984) compared lesions in rats exposed to 1862 |jg/m
(0.95 ppm) 03 8 hours every day for 90 days with both control rats and with
rats exposed to the same concentration 8 hours per day in 5-day episodes
followed by 9 days of filtered air repeated 7 times during an 89-day period.
The lesions were similar but less severe in the episodically exposed rats.
9.3.1.1.3 Vasculature, blood, and lymphatics. Although edema is the apparent
cause of death due to inhalation of high concentrations of Oa, there is very
3
little morphological evidence of pulmonary vascular damage due to £ 1960 ug/m
(^ 1.0 ppm) of 0, exposure. Bils (1970) reported capillary endothelial damage
3
in mice less than I month of age exposed for 7 hr to 1960 jjg/m (1 ppm) of 03,
but this experiment has not been confirmed by others. Boatman et al. (1974)
9-39
-------
did demonstrate endothelial damage in cats exposed via an endotracheal tube to
510, 980, or 1960 ug/m3 (0.26, 0.5, or 1.0 ppm) of Og for 4 to 6 hr, but it is
not clear whether all exposure levels resulted in endothelial damage. In
later studies that used pneumonectomized and control rabbits, Boatman et al.
(1983) reported occasional swelling of capillary endotheliurn in both groups
3
exposed to 784 ug/m (0.4 ppm) of 03 7 hr/day, 5 days/week for 6 weeks.
Stephens et al. (1974b) reported occasional areas of endothelial swelling but
concluded "the endothelium remains intact and rarely shows signs of significant
injury." Stephens et al. (1974a) reported that "endothelium retained a rela-
3
tively normal appearance" in rats exposed to 980 or 1764 ug/m (0.5 or 0.9
3
ppm) of Oo for 2 to 12 hr or 980 ug/m (0.5 ppm) for up to 6 months. In rats
3
exposed by the usual methods to 980 or 1568 ug/m (0.5 or 0.8 ppm) of 03 8 or
24 hr/day, centriacinar interalveolar septa had a loose arrangement of cells
and extracellular material, indicating separation by edema fluid (Schwartz et
al.» 1976). These investigators did not find morphological evidence of damage
to endothelial cells. Evidence of intramural edema in centriacinar areas was
3
found by Castleman et al. (1980) in monkeys exposed to 1568 ug/m (0.8 ppm)
for 4 to 50 hr, but they did not report morphological evidence of vascular
endothelial damage.
Arterial lesions have been only rarely reported. P'an et al. (1972)
reported increased thickness of the media and intima in pulmonary arteries
3
from rabbits exposed to 784 ug/m (0.4 ppm) 03 6 hr/day, 5 days/week for
10 months. These rabbits had evidence of intercurrent disease which was more
severe in exposed animals. The LM description indicates "some degree of
inflammatory infiltrate" in all lungs, and in one exposed rabbit^the lesions
were so severe that "visualization of the mural framework of the alveoli was
difficult." The pulmonary artery media and intima were also significantly
3
thickened in bonnet monkeys exposed to 1254 yg/m (0.64 ppm) 03 8 hr/day for
one year (Fujinaka et al., 1985).
No references to morphological damage of lymphatic vessels were found.
3
This is not surprising, because following nasal inhalation of £1960 ug/m
(<1 ppm) of 03, blood capillary endothelial damage has not been reported, and
edema has been reported only in centriacinar structures. In the more genera-
lized edema that follows exposure to higher concentrations, Scheel et al.
(1959) reported perivascular lymphatics were greatly distended.
9-40
-------
9.3.1.2 Sequence In which Sites are Affected as a Function of Concentration
and Duration of Exposure. The sequence in which anatomic sites are affected
appears to be a function of concentration rather than of exposure duration.
At sites that are involved by a specific concentration, however, the stages in
pathogenesis of the lesion relate to the duration of exposure. Multiple
anatomical sites in the conducting and exchange areas of the respiratory
system have been studied at sequential, time periods in only a few studies.
Stephens et al. (1974a), in a study of the short-term effects of several concen-
trations of 0~, reported finding by LM significant damage to centriacinar
3
type 1 alveolar epithelial cells in rats exposed to 980 ug/m (0.5 ppm) 03 for
2 hours. Using TEM, they also reported, but did not document by figures,
3
minimal damage to centriacinar type 1 cells in rats exposed to 392 ug/m
(0.2 ppm) 0- for 2 hours. Boatman et al. (1974) found lesions in both the
3
conducting airways and parenchyma of cats exposed to 510, 980, or 1960 |jg/m
(0.26, 0.5, or 1.0 ppm) via an endotracheal tube for times as short as 4.7 and
6.6 hr. Thus, if there is a time sequence in effect at various sites, it is a
short time.
Increasing concentration not only results in more severe lesions, but
also appears to extend the lesions to higher generations of the same type of
respiratory structure (i.e., deeper into the lung) (Dungworth et al., 1975b).
Several investigators who have described gradients of lesions have related
them to assumed decreases in concentration of 0- as it progresses through
increasing generations of airways and to differences in protection and sensi-
tivity of cells at various anatomic sites. For the conducting airways, Mellick
et al. (1977) reported more severe and extensive lesions in the trachea and
major bronchi than in small bronchi or terminal bronchioles of rhesus monkeys
3
exposed to 980 or 1568 ug/m (0.5 or 0.8 ppm) of 03 8 hr/day for 7 days. For
the acinus, they noted the most severe damage in proximal respiratory bronchioles
and their alveoli rather than in more distal, higher generation ones. Proximal
portions of alveolar ducts were only minimally involved. The predominant
lesion was at the junction of the conducting airways with the exchange area.
In monkeys, as in man, the proximal respiratory bronchioles, not alveolar
*
ducts, are in the central portion of the acinus. Similar gradients of effects
in the conducting airways and the centriacinar region were reported by Castleman
et al. (1977), who studied bonnet monkeys exposed to 392 and 686 ug/m (0.2
and 0.35 ppm) of 03 8 hr/day for 7 days. In the centriacinar region, this
9-41
-------
gradient is most easily demonstrated by the hyperplasia of cuboidal cells in
respiratory bronchioles that extended further distally in monkeys exposed to
686 rather than 392 ug/m (0.35 than 0.2 ppm) of Og.
The effects of duration of exposure are more complex. In the time frame
of a few hours, an early damage phase has been observed at 2 or 4 hr of expo-
sure (Stephens et a!., 19,74a,b; Castleman et al., 1980). Repair of the damage,
as indicated by DNA synthesis by repair cells, occurs as early as 18 hr (Castle-
man et al., 1980) or 24 hr (Evans et al., 1976b; Lum et al., 1978). Stephens
et al. (1974a) reported little change in the extent of damage after 8 to 10 hr
of exposure. Full morphological development of the lesion occurs at about 3
days of continuous exposure (Castleman et al., 1980). Damage continues while
repair is in progress, but at a lower rate. This phenomenon has been termed
adaptation (Dungworth et al., 1975b). When the time frame is shifted from
hours to days, severity of the lesion at 7 days differs little between exposures
of 8 hr/day and 24 hr/day (Schwartz et al., 1976). When the time frame is
again shifted from days to months of daily exposures, the centriacinar lesions
diminish in magnitude, but a significant lesion remains (Boorman et al., 1980;
Moore and Schwartz, 1981; Eustis et al., 1981).
9.3.1.3. Structural Elements Affected
9.3.1.3.1 Extent ofinjurytoindividualcell types. The extent of injury
to an individual cell is related to the product of the sensitivity of that
cell type and the dose of 03 delivered to the specific site occupied by that
cell. Other factors, e.g. maturity of the individual cell, may also be in-
volved. The dose to an individual cell is determined by the concentration of
0« at that specific site in the respiratory system and the surface area of the
cell exposed to that concentration of 03. Thus, sensitivity and extent of
morphologically detected injury are not the same. While 0- concentrations at
any specific site in the respiratory system can not be determined using the
usual analytical methods., the.,, concentration can _be estimated us ing., mode ling
techniques (See Section 9.2). The literature does contain extensive informa-
tion on the extent of morphologically detected injury to individual cells at
specific sites in the respiratory system. That information is reviewed in the
following paragraphs. Ciliated cells of the trachea and proximal, lower
generation bronchi are subject to more damage than those located in distal,
higher generation bronchi or in lower generation bronchioles proximal to the
terminal bronchiole (Schwartz et al., 1976; Mellick et al., 1977). Ciliated
9-42
-------
cells in terminal bronchioles of animals without respiratory bronchioles
(i.e., rats) are severely damaged by even low concentrations of 0, (Stephens
et a!., 1974a; Schwartz et a!., 1976), whereas those in terminal bronchioles
of animals with respiratory bronchioles (i.e., monkeys) are much less subject
to damage (Castleman et al., 1977; Mellick et a!., 1977).
In a similar manner, type 1 alveolar epithelial cells located in the
centriacinar region are subject to damage by low concentrations of Qg, but
those in the peripheral portions of the acinus appear undamaged by the same or
higher concentrations (Stephens et al., 1974a,b; Schwartz et al., 1976; Castleman
et al., 1980; Crapo et al., 1984).
Although type 2 alveolar epithelial cells appear to be damaged less by
Oo, some type 2 cells in centriacinar locations develop mild lesions detectable
with the TEM (Castleman et al., 1980). There is one report of larger than
normal lamellar bodies in type 2 cells from rats fed a special basal diet with
3
or without vitamin E supplementation and exposed to 588 ug/m (0.3 ppm) 03 3
hr/day for 11 or'16 days (Shimura et al., 1984). Type 2 cells are progenitor
cells that recover basal lamina denuded by necrosis or sloughing of type 1
cells and transform (differentiate) into type 1 cells when repairing the
centriacinar Oq lesion (Evans et al., 1976b). Increased DMA synthesis by
type 2 cells, as evaluated by autoradiography, may be a very sensitive indica-
tor of 03 damage.
Although nonciliated cuboidal bronchiolar cells appear less damaged
morphologically by 0~ than ciliated cells and are the progenitor cells for
replacement of damaged ciliated cells (Evans et al., 1976a,c; Lum et al.,
1978), they are a sensitive indicator of 0~ damage (Schwartz et al,, 1976).
3
Following exposure to 1960 ug/m (< 1 ppm) of 0~, their height is reduced and
their luminal surface is more granular (Schwartz et al., 1976). The reduction
in height appears to be due to a loss of smooth endoplasmic reticulum (Schwartz
et a-1., 1976). - -.-.•. _
Several investigators report that type 3 alveolar epithelial cells, the ,
brush cells, are not damaged by less than 1 ppm of 0, (Stephens et al., 1974a;
Schwartz et al., 1976). No reports are available of damage to type 3 cells by
higher concentrations.
Vascular endothelial cells in capillaries of the interalveolar septa may
be damaged much less than earlier reports indicated, because lesions are not
described in detailed studies using TEM (Stephens et al., 1974a,b; Schwartz et
al., 1976; Mellick et al., 1977; Crapo et al., 1984). In the earlier reports,
9-43
-------
damaged endothelial cells were those located immediately deep to denuded basal
lamina and resulted from sloughing of type 1 epithelial cells in the eentri-
acinar region (Bils, 1970; Boatman et al., 1974). Stephens et al. (1974b)
reported occasional areas of endothelial swelling but the endothelium in these
areas appeared relatively normal and the capillary bed was intact (Stephens
et al,, 1974a»b).
Morphological damage to the various types of interstitial cells in the
interalveolar septa has not been reported. During Q3 exposure, inflammatory
cells migrate into the centriacinar interalveolar septa (Schwartz et al.,
1976). Later, more collagen and connective tissue ground substance is found in
the interalveolar septa (Moore and Schwartz, 1981). Boorman et al. (1980)
2
reported centriacinar interalveolar septa from rats exposed to 1568 pg/m
(0.8 ppm) Og 8 hr/day for 20 or 90 days had thicker .blood-air barriers which
contained more interstitium. Crapo et al. (1984) made a more comprehensive
morphometric study of centriacinar interalveolar septa from young adult rats
2
exposed to 490 pg/m (0.25 ppm) Q3 12 hr/day for 6 weeks. They reported
significant increases in tissue thickness and suggested that the increased
thickness was due to significant increases in all cell types except type 2
cells, and to increased interstitium.
Mucous-secreting cells in conducting airways appear relatively resistant
3
to 0~. Boatman et al. (1974), in studies of cats exposed to £ 1960 pg/m (S
1,0 ppm) of 03 via an endotracheal tube for short periods, did find limited
desquamation of these cells, but the authors also observed that most appeared
intact and increased in size. Castleman et al. (1977) noted roughened apical
surfaces of mucous cells, which appeared to be associated with mucigen drop-
lets near the cell surface, but did not find other alterations in pulmonary
2
mucous cells from monkeys exposed to 392 or 686 |jg/m (0.2 or 0.35 ppm) of 0,
8 hr/day for 7 days. Mellick et al. (1977) mention that mitochondrial swell-
ing and residual bodies seen in ciliated cells were not seen in mucous cells
3
in conducting airways of monkeys exposed to 980 or 1568 pg/m (0.5 or 0.8 ppm)
of 03 8 hr/day for 7 days. Schwartz et al. (1976), who reported mucigen
droplets being released from the apical surfaces t)f mucous cells and mucous
droplets trapped among cilia, did not find changes suggesting damage to organ-
ell es in rats exposed to 392, 980, or 1568 ug/m3 (0.2, 0.5, or 0.8 ppm) of 03
8 or 24 hr/day for 7 days. Wilson et al. (1984) reported only minor changes
in trachea! mucous cells from bonnet monkeys continuously exposed for 3 or 7
3
days to 1254 ng/m (0.64 ppm) 03. Using TEM they reported more prominent
9-44
-------
small-mucous-granule (SMG) cells which had more abundant cytoplasm and more
specific granules. They speculated that SMG cells may relate to the repair
process. In regular mucous cells they reported fewer mucous granules dispersed
in more cytoplasm. The mucous granules appeared smaller and differed from
controls in that they lacked the typical biphasic appearance and had only
filamentous or granular secretory material. No reports of damage to conducting
airways other than to ciliated and mucous cells were found.
9.3.1.3.2 Extracellular elements (structural proteins). Although physiologic
and biochemical changes following 03 exposure suggest changes in the extracel-
lular structural elements of the lung, no direct morphological evidence has
been given of changes in the extracellular structural elements themselves, in
contrast to changes in their location or quantity. These physiologic and
biochemical studies are discussed elsewhere, in this document (See Sections
9.3.2.2 and 9.3.3.6, respectively). Three studies provide morphological
evidence of mild fibrosis (i.e., local increase of collagen) in centriacinar
interalveolar septa following exposure to < 1960 pg/m (< I ppm) of 03 (Last
et al., 1979; Boorman et a!., 1980; Moore and Schwartz, 1981). Changes in
collagen location or amounts, or both, which occur with the remodeling of
the distal airways, were reported in two of those studies (Boorman et al.,
1980; Moore and Schwartz, 1981). One study (Fujinaka et al., 1985) reported
increased connective tissue surrounding respiratory bronchioles from bonnet
2
monkeys exposed to 1254 pg/m (0.64 ppm) 03 8 hr/day for one year. This
increase was due to Increased amorphous extracellular matrix rather than
stainab!e connective tissue fibers. Evidence of more collagen or changes in
3
collagen location is in the report of dogs exposed to 1960 or 5880 pg/m (1 or
3 ppm) of 03 for 18 months (Freeman et al., 1973).
9.3.1.3.3 Edema. Morphologically demonstrable alveolar edema, or alveolar
flooding—an effect of higher-than-ambient levels of 0, (Scheel et al., 1959;
3
Cavender et al., 1977)—is not reported after exposures to <_ 1960 ug/m (S 1.0
ppm) of 0-j for short or long exposure periods (Schwartz et al., 1976; Cavender
et al.s 1978; Mellick et al., 1977; Eustis et al., 1981; Boorman et al., 1980;
Moore and Schwartz, 1981). Mild interstitial edema of conducting airways
(Mellick et al., 1977) and centriacinar parenchymal structures (Schwartz et
al., 1976; Castleman et al., 1980; Mellick et al., 1977) is seen following
3
exposure of monkeys or rats to < 1960 ug/m (< 1 ppm) of 03 for several hours
to 1 week. Interstitial edema is not reported following longer-term (i.e.,
o
weeks to months) exposure to < 1960 ug/m (< 1 ppm) or less (Cavender et al. ,
9-45
-------
1977; Eustis et a!., 1981; Boorman et al., 1980; Moore and Schwartz, 1981;
Zitnik et al., 1978). Biochemical indicators of edema are described in Sec-
tion 9.3.3.
9.3.1.4 Considerations of DegreeofSusceptibility to Morphological Changes
9.3.1.4.1 Compromised experimental animals. Compromised experimental animals
(e.g., those with a special nutritional or immunological condition) in a
disease state or of young or old age may respond to 0- exposure with greater,
lesser, or a different type of response than the normal, healthy, young adult
animals usually studied. Some of these may represent "at risk" human popula-
tions.
9.3.1.4.1.1 Vitamin E deficiency. Rats maintained on vitamin E-deficient
diets tended to -develop more morphological lesions follbwing exposure to low
levels of 03 than did rats on the usual rations (Plopper et al., 1979; Chow et
al., 1981). Rats maintained on a basal vitamin E diet equivalent to the
average U. S. human adult intake were exposed to 196 or 392 ug/m (0.1 or
0.2 ppm) of 03 24 hr/day for 7 days. According to LM studies, two of the six
rats on the basal vitamin E had increased numbers of macrophages in their
centriacinar alveoli, a typical response to higher levels of 0., (Schwartz et
3
al., 1976). Of five rats on the usual rat chow diet exposed to 196 ug/m (0.1
ppm) 0- for the same period, LM revealed no increased centriacinar macrophages.
LM analysis showed neither dietary group had lesions in the ciliated terminal
bronchiolar epithelium. Rats in which lesions were observed by LM had increased
macrophages, according to SEM analysis. Analysis by TEM showed that all rats
3
exposed to 196 ug/m (0.1 ppm) of 03 differed from controls in that some of
the centriacinar type 1 alveolar epithelial cells contained inclusions and
were thicker.
Chow et al. (1981) fed month-old rats a basal vitamin E~deficient diet or
that diet supplemented with 11 or 110 ppm vitamin E for 38 days, after which
3
they were exposed either to filtered air or to 196 ug/m (0.1 ppm) of 03
continuously for 7 days. The morphology of six rats from each diet and exposure
group was studied using SEM. None of the filtered-air control animals had
lesions. Of the rats exposed to 0-, five of the six on the vitamin E-deficient
diet, four of six on the deficient diet supplemented by 11 ppm vitamin E, and
one of the six on the deficient diet supplemented by 110 ppm vitamin E developed
the typical 03 lesion as seen with SEM (Schwartz et al., 1976).
Sato et al. (1976a,b, 1978, 1980) exposed vitamin E-deficient and supple-
3
mented rats to 588 ug/m (0.3 ppm) of 03 3 hr daily for 16 consecutive days or
9-46
-------
5 days a week for 7 months. The short-term experiments (Sato et al., 1976a,b)
were marred by the presence of chronic respiratory disease in the rats, which
may explain the investigators' finding of large amounts of debris and numerous
small bodies "so thick that the original surface could not be seen" and their
failure to find the typical centriacinar 0, lesions reported by others (Stephens
et al., 1974a; Schwartz et al., 1976). In the latter experiments, Sato et al.
(1978, 1980) did not find morphological differences between the vitamin
E-depleted and supplemented, filtered-air control rats or between vitamin
E-depleted and supplemented, (k-exposed rats. They did find mild centriacinar
Og lesions in exposed rats from both vitamin E-deficient and supplemented
groups.
Stephens et al. (1983) reported results of exposure of vitamin E-depleted
3
and control young and old rats to 1764 ug/m (0.9 ppm) of 03 for I, 3, 6, 12,
34, 48, and 72 hr. Vitamin E depletion was evaluated by determination of lung
tissue levels. Lung response to ozone was based on characteristic tissue
nodules previously reported by these authors when using a dissecting microscope
rather than on conventional LM, SEM, or TEM. They concluded that response to
injury and repair of the lung was independent of the level of vitamin E in
lung tissue. Most of these studies included concurrent biochemical evaluations
of oxidant metabolism and are discussed in Section 9.3.3.
9.3.1.4.1.2 Age at start of exposure. Although most exposures use
young adult experimental animals, there are a few reports of exposures of very
young animals (i.e., either before weaning or very soon thereafter).
Bartlett et al. (1974) exposed 3- to 4-week-old rats to 392 ug/m3 (0.2
ppm) of 0- for 30 days. Lung volumes, but not body weights, were significantly
greater in the exposed rats. Light microscopy of paraffin sections of conven-
tionally fixed lungs did not reveal differences between exposed and control
rats in the lung parenchyma or terminal bronchioles, with the exception of two
control animals which had lesions of "typical murine pneumonia." Morphometry
was done on thick sections cut by hand with a razor blade from the dorsal and
lateral surfaces of air-dried lungs rather than on the paraffin sections of
conventionally fixed lungs. Morphometry using these nonrandom samples revealed
significantly increased mean alveolar chord lengths and alveolar surface area,
but no difference in alveolar numbers.
Freeman et al. (1974) exposed month-old rats to 1058 or 1725 ug/m (0.54
or 0.88 ppm) of 0- for periods of 4 hr to 3 weeks. In addition to the centri-
acinar accumulations of macrophages and hyperplasia of distal airway epithelium
9-47
-------
seen by others following exposures of young adult animals, they reported an
increase in connective tissue elements and collagen-like strands that formed
o
bridges across alveolar openings. Fibrosis was pronounced in the 1725
(0.88 ppm) group and sometimes extended into terminal bronchioles. Although
3
fixed lung volumes were not determined, the 1725-pg/m (0.88 ppm) group required
greater volumes of fixing fluid, evidence of larger lung volumes. In the same
research report, Freeman et al. (1974) studied month-old rats exposed to 1764
3 3
pg/w (0.9 ppm) of Q3 and 1690 pg/m (0.9 ppm) nitrogen dioxide combined.
After 60 days of exposure, they observed the gross and microscopic appearance
of advanced experimental emphysema of the type they earlier described following
nitrogen dioxide exposure (Freeman et al., 1972). Although others have reported
larger fixed lung volumes in exposed young adult rats (Moore and Schwartz,
1981}, reports of emphysema following 0- exposures are uncommon and are dis-
cussed in the next subsection of this document.
Stephens et al. (1978) exposed rats ranging in age from 1 to 40 days old
o
to 1666 jjg/m (0.85 ppm) 03 continuously for 24, 48, or 72 hr. Rats exposed
to Og before weaning at 20 days of age developed little or no evidence of
injury, as evaluated by light and electron microscopy. When exposure was
initiated after weaning at 20 days of age, centriacinar lesions increased
progressively, plateaued at 35 days of age, and continued until approximately
1 year of age.
Barry et al. (1983) exposed 1-day-old male rats and their mother to 490
3
(jg/m (0.25 ppm) of 03 12 hr/day for 6 weeks. They observed persistence of
the centriacinar damage to type 1 epithelial cells and increased centriacinar
tnacrophages. By using LM and TEM morphometry of centriacinar regions, they
reported an increase in both type 1 and 2 alveolar epithelial cells. The
type 1 cells were smaller in volume, covered less surface, and were thicker.
The authors were aware of the above study by Stephens et al. (1978) and dis-
cussed the possibility that much of the damage they observed may have occurred
in the last 3 weeks of exposure (i.e., after weaning). Changes in lung function
evaluated by Raub et al. (1983a) are discussed in Section 9.3.2.
Bils (1970) studied the effects of 1176 to 2548 M9/m3 (0.6 to 1.3 ppm) of
On on mice 4 days old and 1 and 2 months old. From his study, Bils concluded
that the endothelium appeared to be the main target of the 03, a conclusion
not supported by more recent studies, which deal mostly with other species.
81 Is did note the lesions were more severe in the 4-day-old mice than in the
1- or 2-month-old mice.
9-48
-------
9.3.1.4.1.3 Effect of pneumbnectomy. Two to four weeks following
pneumonectomy of rabbits, the contra!ateral lung increases in volume, weight,
collagen, and protein content to approximate that of both lungs from controls,
but alveolar multiplication appears dependent on age at surgery. Boatman
3
et al. (1983) exposed pneumonectomized and control rabbits to 784 [jg/m (0.4 ppm)
of 03 7 hr/day, 5 days/week for 6 weeks. They examined the lungs with standard
LM and TEM morphometric techniques, but not methods for alveolar numbers.
Boatman and co-workers concluded that the lung growth that follows pneumonectomy
occurred after 03 exposure and that no difference existed between males and
females in this response.
9.3.1.4.2 Emphysema following ozone exposure. The previous criteria document
for 03 (U.S. Environmental Protection Agency, 1978) cites three published
research reports in which emphysema was observed in experimental animals
3
following exposure to < 1960 ug/m (< 1 ppm) of 03 for prolonged periods (P'an
et al., 1972; Freeman et al. , 1974; Stephens et al., 1976). Since then, no
similar exposures (i.e., same species, 03 concentrations, and times) have been
documented to confirm these observations-. An additional consideration is the
similarity of the centriacinar lesion following 0, exposure to that seen in
young cigarette smokers (Niewoehner et al., 1974; Schwartz et al., 1976; Cosio
et al., 1980; Wright et al., 1983; Fujinaka et al., 1985) and the relationship
between cigarette smoking and emphysema in humans (U.S. Department of Health,
Education, and Welfare, 1967, 1969). Further, animals exposed to 1960 pg/m
(1 ppm) of 03 reportedly have more voluminous lungs than controls (Bartlett
et al., 1974; Moore and Schwartz, 1981). Morphometry was used to demonstrate
enlarged subpleural alveoli in one of these reports (Bartlett et al., 1974).
However, these authors indicate that these subpleural alveoli may not be
representative of the whole lung and do not conclude that emphysema was present.
Thus, a restudy of these three reports in the 1978 document appears appropriate.
The precise definition of emphysema is critical to reevaluation of these
reports. Several professional groups have presented definitions of emphysema
(Fletcher et al. , 1959; World Health Organization, 1961; American Thoracic
Society, 1962). The most recent is the report of a National Heart, Lung and
Blood Institute, Division of Lung Diseases Workshop (National Institutes of
Health, 1985). 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" (National Institutes of Health, 1985). Destruction is
9-49
-------
further defined: "Destruction in emphysema is defined as non-uniformity 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
further indicates "Destruction . . . may be recognized by subgross examination
of an inflation-fixed lung slice . . .," In order to stimulate additional
research, the definition of emphysema in animal models was less restrictive.
The document states: "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 qualita-
tively in appropriate specimens and quantitatively by stereologic methods."
Thus in animal models airspace wall destruction need not be present. However,
where information from air pollution exposures of animals is to be extrapolated
to hazards for humans, the definition of human emphysema must be considered and
the presence of airspace wall destruction documented.
Stokinger et al. (1957) reported emphysematous changes in lungs from
guinea pigs, rats and hamsters, but not mice or dogs, exposed 6 hr/day, 5 days/
3
week for 14.5 months to a mean concentration of slightly more than 1960 pg/m
(1 ppm) of Og. With the exception of the dogs, mortality rates were high in
both control and exposed animals, ranging in the controls from 25 to 78 percent
and in exposed from 11 to 71 percent. The published report indicates that
emphysema was present but does not further characterize it as to the presence
of only enlarged air spaces (Fletcher et al., 1959) or enlarged air spaces
accompanied by destructive changes i,n alveolar walls (World Health Organiza-
tion, 1961; American Thoracic Society, 1962). The lungs were fixed via the
trachea, making.them suitable for studies of experimentally induced emphysema
(American Thoracic Society, 1962; National Institutes of Health, 1985).
Stokinger et al. (1957) attributed the emphysema in the guinea pigs to the
observed bronchial stenosis. Also in the guinea pigs were foci of "extensive
linear fibrosis . . . considered to be caused by organization of pneumonic areas."
In exposed rats, the mild degree of emphysema "did not exceed the emphysema
found in the unexposed control rats." In exposed hamsters, "mild to moderate"
emphysema was present, but not in controls. Emphysema is not mentioned in the
figure legends, but three of them mention "alveoli are overdistended . . .
alveolar spaces are dilated . . . dilation of alveolar ducts and air sacs."
Evidence of destruction of alveolar walls is not mentioned. Later, however,
Gross et al. (1965), in an unrefereed publication abstracted from a presenta-
tion at the seventh Aspen Conference on Research in Emphysema, reviewed the
9-50
-------
lesions in the hamsters from this exposure and described a "destructive process"
that resulted in contraction of interalveolar septa not associated with enlarge-
ment of air spaces.
Because the interpretation in this EPA Criteria Document differs from
that in the previous document, the details need to be presented. The signifi-
cance will be discussed in a paragraph at the end of this section. The earlier
0, criteria document (U.S. Environmental Protection Agency, 1978) cites Stephens
et al. (1976), a "long abstract" that appears not to be refereed. This brief
article states "rats exposed continuously for long periods (3-5 months) to
28,200 M9/m3 (15.0 ppm) N02 or 1568 pg/m3 (0.8 ppm) 03 develop a disease that
closely resembles emphysema", but does not provide additional evidence other
than citing five earlier studies by the Stanford group of investigators. Each
of those five references was checked for studies of animals exposed to 0.,.
Three articles describe only N09-exposed animals. The fourth reference (Freeman
3
et al., 1973) is to an exposure of dogs to 1960 to 5880 |jg/m (1 to 3 ppm) of •
03 8 to 24 hr daily for 18 months and was cited earlier in this document.
Emphysema is not mentioned in that article. Neither is emphysema mentioned in
the fifth reference (Stephens ,et al., 1974b), which was also cited earlier in
this document. These investigators did describe (Freeman et al., 1974) a
3
group of month-old rats exposed continuously for 3 weeks to 1725 jjg/m (0.88 ppm)
of 0-j, half of which died and had "grossly inflated, dry lungs." In this same
3
study, they also exposed month-old rats to a mixture of 1690 pg/m (0.9 ppm)
of NOp and 0, continuously for 60 days, at which time the lungs were grossly
enlarged, and "both grossly and microscopically, the appearance of the lungs
was characteristic of advanced experimental emphysema" of the type they earlier
reported following N0? alon6 at much higher concentrations (Freeman et al.,
1972).
The third citation in the 0, criteria- document (U.S. Environmental Protection
Agency, 1978). is to~-P'an et al. (1972). These investigators exposed-rabbits
3
to 784 M9/m (0-4 ppm) of 03 6 hr/day, 5 days/week for 10 months. Tissues
were fixed apparently by immersion rather than infusion via the trachea, which
is not in accord with the American Thoracic Society's diagnostic standard for
emphysema, making emphysema lesions much more difficult to evaluate accu-
rately. The lesions related to emphysema are only very briefly described and
illustrated in only one figure. The authors also report that "all lungs
showed some degree of inflammatory infiltrate" and "lungs of the sixth were so
9-51
-------
congested that visualization of the mural framework of the alveoli was diffi-
cult." This is more reaction than reported in other species exposed to this
comparatively low CL concentration. The rabbits were not specified pathogen-
free, nor was the possibility considered that some lesions could be due to
infectious agents. Neither did these investigators consider the possibility
of spontaneous "emphysema and associated inflammatory changes" which Strawbridge
(1960) described in lungs from 155 rabbits of various ages and breeds.
In the studies reviewed in this section, enlargement of air spaces distal
to the terminal bronchiole have been described following 03 exposure of several
species of experimental animals. In one study, the enlargement was quantitated
using morphometry of air-dried lungs (Bartlett et al., 1974). Destruction of
alveolar walls was only briefly described in two reports (P'an et al., 1972;
Gross et al., 1965). In one of these studies (P'an et al., 1972) the lungs
were apparently fixed by immersion rather than infusion, making a diagnosis of
emphysema less reliable (American Thoracic Society, 1962). The other study
(Gross et al., 1965) appears to be an unrefereed long abstract rather than a
full research report article. Neither of these reports describes lesions
which unequivocally meet the criteria for human emphysema as defined by either
the American Thoracic Society (1962) the or the National Institutes of Health
(1985).
9.3.2 Pulmonary Function Effects
9.3.2.1 Short-Term Exposure. Results of short-term 03 exposures of experi-
mental animals are shown in Table 9-2. These studies were designed to evalu-
ate the acute changes in lung function associated with 03 exposure in a variety
of species (mice, rats, guinea pigs, sheep, rabbits, cats, monkeys, and dogs)
when compared to filtered-air exposure.
The effects of short-term local Q~ exposure of the lung periphery have
been examined in dogs by Gertner et al. (1983a,b). A fiber-optic bronchoscope ....
with an outside diameter of 5.5 mm was wedged into a segmental airway and a
3
continuous flow of 196 or 1960 jjg/m (0.1 or 1.0 ppm) of 0- was flushed through
this airway and allowed to escape through the system of collateral channels
normally present in the lung periphery. During exposure to either 196 or
o
1960 ug/m (0.1 or 1.0 ppm) of 03, airflow resistance through the collateral
channels increased during the first 2 min of exposure. Resistance of the
3
collateral channels continued to increase throughout exposure to 1960 ug/m
9-52
-------
TABLE 9-2. EFFECTS OF OZONE ON PULMONARY FUNCTION: SHORT-TERM EXPOSURES
Ozone
concentration
Ma/n1*
196
1960
431
804
1568
470 to
2156
ppm
0.1
1.0
0.22
0.41
0.80
0.24 to
1.1
Measurement3
method
MAST
CHEM,
NBKI
NBKI
, Exposure
' duration
& protocol Observed effects
30 orin Collateral system resistance increased
rapidly during exposure, falling to
control levels at 0.1 ppm but con-
tinuing to increase at 1.0 ppm of Oa.
2 hr Concentration-dependent increase in fn
for all exposure levels. No change in
R., TV, or MV. Decreased C.dyn during
exposure to 0.4 and 0.8 pp> of 08.
12 hr Premature airway closure at 6 hr, and
1 and 3 days following exposure, ref lee-
Species
Dog
Guinea pig
(200-300 g)
Rabbit
Reference
Gertner et al.
1983a,b
Aindur et al . ,
Inoue et al . ,
,
1978
1979
ted by increased RV, CC, and CV (6 hr
and 1 day only). Maximum effect 1 day
following exposure, all values returned
to normal by 7 days. Distribution of
ventilation less uniform 6 hr following
exposure. Increased lung distensibility
in the mid-range of lung volumes (25-75%
TLC) 7 days following exposure.
510
980
1960
666
1333
2117
2646
980
0.26 MAST
0.5
1.0
0.34 NBKI
0.68
1.08
1.35
0.5 NBKI
2.0 to
6.1 hr
2 hr
2 hr
Concentration-dependent increase in R,
during exposure. Decreased C, and D,CO
but less frequent and less marked than
changes in R, . No change in VC or
deflation pressure- volume curves.
Increased f« and decreased TV during
exposure to all 03 concentrations.
Increased R during exposure to 1.08
and 1.35 ppm of 03.
Slight increase in fg and R (to 113%
of control values) during exposure.
Cat
Guinea pig
(300-400 g)
Guinea pig
(280-540 g)
Watanabe et al . ,
1973
Murphy et al . , 1964
Yokoyama, 1969
-------
TABLE 9-2. EFFECTS OF OZONE ON PULMONARY FUNCTION: SHORT-TERM EXPOSURES (continued)
(Jl
Ozone L Exposure
concentration Measurement ' duration
yg/H85 ppM netted & protocol
1470 0.75 CHEM
1960 1.0
3920 2.0
1960 1 NBKI
1960 1 NBKI
Continuous
1, 3, 7 or
14 days
3 hr
6 hr/day,
7 to 8 days
Observed effects'" Species Reference
The validity of this study is ques- Rat Pepelko et al.,
tioned because of low airflow through 1980
the exposure chambers and high mortality
of exposed animals (66% mortality in rats
exposed to 1 pp« of Q3).
Reduced TLC at air inflation pressure Rabbit Yokoyaaa, 1972, 1973
of 30 cii H20, 1 to 3 days postexposure
but not at 7 days. No difference in
lung pressure- volume characteristics
during lung inflation with saline.
Increased R, and decreased C.dyn Rabbit Yokoyama, 1974
1 day following exposure. No change
in HEFV curves.
Measurement method: HAST = Kl-coulometric (Hast meter); CHEM = gas phase chemiluminescence; NBKI = neutral buffered potassium iodide.
Calibration Method: NBKI = neutral buffered potassium iodide.
See Glossary for the definition of pulmonary symbols.
-------
(1.0 ppm) of 0~, but decreased again to control levels during continued expo-
3
sure to 196 ug/m (0.1 ppm) of 03- Based on these observations, the authors
reported that tolerance appears to develop in the collateral airways to locally
3
delivered 0- at concentrations of 196 but not 1960 jjg/m (0.1 but not 1.0 ppm)
of 03.
Amdur et al. (1978) measured bresrthing pattern (tidal volume, respiration
rate, and minute volume), pulmonary resistance, and dynamic pulmonary compli-
3
ance in guinea pigs during 2-hr exposures to 431, 804, or 1568 ug/m (0.22,
0.41, or 0.8 ppm) of 0~, Accelerated respiration rates with no significant
changes in tidal volume were measured during exposures to all 03 concentra-
tions,. The onset and magnitude of these changes in respiration rate were
concentration-dependent, and values of respiration rate remained elevated
during a 30-min recovery period following exposure. Pulmonary compliance was
significantly lower than pre-exposure values following 1 and 2 hr of exposure
3
to 804 or 1568 ug/m (0.41 or 0.8 ppm) of 0-, and values of compliance remained
low during the 30-min recovery period. Changes in dynamic compliance were
3
essentially the same during exposure to either 804 or 1568 ug/m (0.41 or
0.8 ppm) of Q3. These investigators observed no significant change in pulmo-
nary resistance during exposure to 0,. If anything, resistance tended to
decrease throughout the exposure and recovery period.
The lack of a significant increase in pulmonary resistance in the Amdur
et al. (1978) study is in contrast to the 113 percent increase over pre-exposure
values in total respiratory flow resistance measured in guinea pigs exposed to
980 ug/m3 (0.5 ppm) of 03 for 2 hr by Yokoyama (1969). Watanabe et al. (1973)
also found increased pulmonary flow resistance in cats artificially ventilated
through an endotracheal tube with 510, 980, or 1960 ug/m (0.26, 0.50, or
1.00 ppm) of 03 for 2 to 6.5 hr. Pulmonary resistance had increased to at
least 110 percent of control values in all animals after 105 min of exposure
to 510 ug/m3 (0.26 ppm) of 0,, after 63 min of exposure to 980 ug/m (0.50 ppm)
3
of 03, and after 49 min of exposure to 1960 ug/m (1 ppm) of 03- Dynamic lung
compliance was decreased during 03 exposure in the, Watanabe et al. (1973)
study, as it was in the Amdur et al. (1978) study. However, changes in pul-
monary compliance measured by Watanabe et al. (1973) occurred less frequently
and were less severe (based on percentage changes from pre-exposure control
values) than changes in pulmonary resistance.
Like Amdur et al. (1978) and Yokoyama (1969), Murphy et al. (1964) also
measured breathing pattern and respiratory flow resistance in guinea pigs
9-55
-------
during 2-hr 0~ exposures. These investigators found concentration-related
3
increases in respiration rate during exposure to 666, 1333, 2117 or 2646 pg/rn
(0.34, 0,68, 1,08, or 1,35 ppm) of 0-. Respiratory flow resistance was increased
(to 148 and 170 percent of pre-exposure values) in guinea pigs exposed to 2117
o
and 2646 ug/m (1.08 and 1.35 ppm) of 0~ respectively. Pulmonary compliance
was not measured.
The variability in measurements of pulmonary resistance following 0-
exposure can be attributed to a number of factors including the following:
frequency characteristics of the monitoring equipment and measurement tech-
niques utilized, the influence of anesthetics, and the intraspecies differ-
ences in airway reactivity of guinea pigs. The latter point was the subject
of critical review in the assessment of toxicological effects from particulate
matter and sulfur oxides (U.S. Environmental Protection Agency, 1982).
Recovery of guinea pigs from short-term Og exposure was substantially
different in the above three studies. Animals exposed by Amdur et al. (1978)
showed little or no return toward pre-exposure values for any of the measured
parameters during a 30-min recovery period following exposure. In guinea pigs
exposed by Murphy et al. (1964) and Yokoyama (1969), respiration rates had
returned almost to pre-exposure values by 30 min following exposure. The
development of more persistent lung-function changes following 0~ exposure in
the Amdur et al. study (1978) may be attributed to the small size and associ-
ated immaturity of these guinea pigs (200 to 300 g) compared with those in the
studies by Murphy et al. (1964) (300 to 400 g) and Yokoyama (1969) (280 to
540 g). In an earlier study, Amdur et al. (1952) showed that young guinea
pigs 1 to 2 months old were significantly more sensitive to inhaled sulfuric
acid aerosols than 12- to 18-month-old animals. The use of ether anesthesia
and placement of an intrapleural catheter by Amdur et al. (1978) but not by
Murphy et al. (1964) or Yokoyama (1969) may also have sensitized the animals
exposed by Amdur et al. (1978) to effects of 0~.
O .3
Inoue et al. (1979) exposed rabbits to 470 to 2156 M9/m (0.24 to 1.1 ppm)
of 03 for 12 hr and performed lung function tests 6 hr, and 1, 3, and 7 days
following exposure. These rabbits showed functional evidence of premature
airway closure with increased trapped gas at low lung volumes 6 hr, I day, and
3 days following exposure. Functional changes indicating premature airway
closure included increased values of closing capacity, residual volume, and
closing volume. Lung quasistatic pressure-volume measurements showed higher
lung volumes at lung distending pressures from 0 to -10 cm of H~0. These lung
9-56
-------
function changes were greatest 1 day following exposure and had disappeared by
7 days following exposure. Distribution of ventilation in the lung was less
uniform in 03-exposed animals only at 6 hr following exposure. By 7 days
following the initial 12<-hr 0- exposure, the only significant functional
change was an increased lung distensibility in the midrange of lung volumes
(from 25 to 75 percent total lung capacity).
Earlier studies by Yokoyama (1972), in which rabbits were exposed to
1 ppm of 0- for 3 hr, showed a timing of lung function changes similar to that
observed by Inoue et al. (1979). For both studies, maximum changes in 0,,-
exposed animals were observed 1 day following exposure and had disappeared by
7 to 14 days following exposure. However, in some aspects, the Yokoyama (1972)
study was substantially different from that of Inoue et al. (1979). Yokoyama
(1972) found reduced maximum lung volume at an air inflation pressure of 30 cm
of FLO, whereas Inoue et al. (1979) found no difference in maximum lung volume.
Yokoyama (1972) does not show lung pressure-volume curves at pressures less
than atmospheric pressure, so premature airway closure and gas trapping cannot
be evaluated in this study. One factor that may contribute to differences
between these two studies is the use of an excised lung preparation by Yokoyama
(1972) compared with evaluation of intact lungs in anesthetized rabbits by
Inoue et al. (1979).
Yokoyama (1974) also evaluated lung function in rabbits following exposure
to 1960 ug/m (1 ppm) of 03, 6 hr/day, for 7 to 8 days. He found increased
pulmonary resistance and decreased dynamic compliance in 0~-exposed animals
«J
compared to air-exposed control animals. Static pressure-volume curves and
maximum expiratory flow-volume curves were not significantly different between
the two groups.
9.3.2.2 Long-Term Exposure. Table 9-3 summarizes results of long-term 0^
exposures. Raub et al. (1983a) exposed neonatal and young adult (6-week-old)
rats to 157, 235, or 490 ug/m3 (0.08, 0.12, or 0.25 ppm) of 03 12 hr/day,
7 days/week for 6 weeks. Lung function changes were observed primarily in
neonatal rats following 6 weeks of O- exposure. Peak inspiratory flow measured
in these animals during,spontaneous respiration was significantly lower follow-
3
ing exposure to 235 or 490 ug/m (0.12 or 0.25 ppm) of 03- Lung volumes
measured at high distending pressures were significantly higher in neonatal
o
animals exposed to 490 ug/m (0.25 ppm) of 03 for 6 weeks than in control
animals. These results are consistent with increased lung volumes measured
during lung inflation with either air or saline by Bartlett et al. (1974)
9-57
-------
TABLE 9-3. EFFECTS OF OZONE ON PULMONARY FUNCTION: LONG-TERM EXPOSURES
vo
ui
oo
Ozone
concentration
(jg/«d ppi
157 0.08
235 0.12
490 0.25
392 0. 2
392 0.2
1568 0.8
784 0.4
882 0.45
980 0. 5
1568 0.8
Measurement3 '
method
CHEH
MAST,
NBKI
UV or CHEM
NBKI
NBKI
i
MAST
|w.
NBKI
i
Exposure
duration
& protocol
6 weeks,
12 hr/day,
7 days/week
28 to 32 days,
continuous
62 exposures,
6 hr/day,
5 days/week
6 weeks,
7 hr/day,
5 days/week
6 to 7 weeks,
6 hr/day,
6 days/week
7, 28, or
90 days;
8 hr/day
Observed effects0
Increased end expiratory lung volume
in adult rats and increased lung
volumes at high distending pres-
sures in neonatal rats exposed to
0.25 ppm of 03. Reduced peak inspira-
tory flow in neonatal rats exposed
to 0.12 or 0.25 ppm of 03.
Increased lung distensibility in
03-exposed rats at high lung
volumes (95-100% TLC) during in-
flation with air or saline.
Increased R. (not related to con-
centration) in rats exposed to
0.2 or 0.8 ppm of 03. Lung volumes at
high distending pressures (VC and TLC)
were increased at 0.8 ppm and FEF2s and
FEF10 were decreased at 0.2 and 0.8 ppm
of 03.
Increased alveolar wall extensibility
at yield and break points, increased
hysteresis ratio, and decreased stress
at moderate extensions. Fixed lung
volume increased 15%. Lung growth'
following pneumonectomy prevented
these changes to 03 exposure.
No effect of exposure on lung pressure-
volume curves.
Decreased quasistatic compliance (not
related to concentration).
Species
Rat
(neonate or
6-week-old
young adult)
Rat
(3 to 4 weeks)
Rat
(10 weeks)
Rabbit
Rat
Monkey
(Bonnet)
Reference
Raub et al . , 1983a
Bartlett et al. ,
1974
Costa et al . , 1983
Martin et al.,
1983
Yokoyama and
Ichikawa, 1974
Eustis et al., 1981
-------
TABLE 9-3. EFFECTS OF OZONE ON PULMONARY FUNCTION: LONG-TERM EXPOSURES (continued)
vo
Ozone
concentration Meas
|jg/raa ppm • me
980 0.5 MA
1960 1.0 NB
. Exposure
urement * duration
thod & protocol Observed effects0 Species Reference
ST, 3 and 6 hr/day Increased resistance of central airways Rat Yokoyama et al.,
O for up to 60 after 3-hr daily exposures to 1.0 ppm 1984
days for 30 days; increased resistance of
peripheral airways after 6-hr daily
exposures to 0.5 ppm 03 for SO days.
1254 0.64 UV', 7 or 20 days Increased peripheral resistance in Rat Kotlikoff et al.,
NBKI rats exposed for 7 days but not 1984
. 20 days; decreased lung reactance
! at high frequencies in both groups.
1254 0.64 UV
NB
, 1 year, Following 6 months of exposure, venti- Monkey Wegner, 1982
O 8 hr/day, lation was less homogeneous and R. (Bonnet)
7 days/week was increased. Following 12 months
of exposure, R, remained elevated
and forced expiratory maneuvers showed
small airway dysfunction (decreased
FEVj and FEFja-s). During the 3-nonth
recovery period following exposure,
C.st decreased.
Measurement method: MAJST = Kl-coulometric (Mast meter); CHEM = gas phase chemiluminescence; UV = UV photometry.
b-
Calibration method: NBKI = neutral buffered potassium iodide.
See Glossary for the definition of pulmonary symbols.
-------
o
following exposure of young rats (3- to 4-week-old) to 392 ug/m (0.2 ppm) of
Og continuously for 28 to 32 days. Moore and Schwartz (1981) also found an
increased fixed lung volume (following lung perfusion at 30 cm inflation
pressure with Karnovsky's fixative) after 180 days of continuous exposure to
2
980 ug/m (0.5 ppm) of 0-. Yokoyama and Ichikawa (1974) found no change in
3
lung static pressure-volume curves' in mature rats exposed to 882 ug/m
(0.45 ppm) of Og 6 hr/day, 6 days/ week for 6 to 7 weeks.
Martin et al. (1983) studied the mechanical properties of the alveolar
3
wall from rabbits exposed to 784 ug/m (0.4 ppm) of 0~, 7 hr/day, 5 days/week
for 6 weeks. A marked increase in the maximum extensibility of the alveolar
wall and a greater energy loss with length-tension cycling (hysteresis) were
found following exposure. A 15-percent increase in fixed lung volume following
perfusion at 20 cm of H90 was also reported following 0, exposure, which is
-------
former exposure while R, increased only at lower pressures after the latter
exposure. The authors interpreted these changes as an indication of increased
3
central airway resistance in rats exposed to 1960 |jg/m (1 ppm) of 0, for
3
30 days and increased peripheral airway resistance in rats exposed to 980 ug/m
(0.5 ppm) of 0,, for 60 days. These changes were also consistent with morpholog-
ical findings of greater mucous secretions in large bronchi of the animals
2
exposed to 1960 jjg/m (1 ppm) of 0, and in the peripheral airways of animals
3
exposed to 980 (jg/m (0.5 ppm) of 0~. No changes in static deflation volume-
pressure curves of the lungs were found after either exposure nor were there
any differences in effects that could be attributed to the age of rats at the
start of exposure.
Eustis et al. (1981) evaluated.lung function in bonnet monkeys (Macaca
radiata) exposed to 980 or 1568 jjg/m3 (0.5 or 0.8 ppm) of 0,,, 8 hr/day for 7,
28, or 90 days. This study appeared to be preliminary (range-finding) for the
long-term study reported by Wegner (1982). Only a limited number of animals
were evaluated at each time point (1 per exposure group at 7 days, 2 at 28 days,
and 3 at 90 days). With so few animals tested and tests made following three
different exposure periods, little significant lung-function data related to
0- exposure were generated. When pooling results from all exposure times and
0- concentrations, quasi-static lung compliance was significantly different in
Og-exposed animals than in control animals. Compliance tended to decrease
from pre-exposure values in control animals and increase in 0,-exposed animals.
Wegner (1982) evaluated lung function in 32 bonnet monkeys, 16 of which
o
were exposed to 1254 ug/m (0.64 ppm) of 03, 8 hr/day, 7 days/week for 1 year.
Lung function tests were performed pre-exposure, following 6 and 12 months of
exposure, and following a 3-month postexposure recovery period. In addition
to measurements of carbon monoxide diffusion capacity of the lungs (D,co),
lung volumes, quasi-static pulmonary compliance (C .,.O and partial and
maximum expiratory flow-volume curves by standard techniques, frequency depen-
dence of compliance and resistance and pulmonary impedance from 2-32Hz were
measured by a forced oscillation technique. The addition of these latter
measurements may elucidate more clearly than ever before the site and nature
of lung impairment caused by exposure to toxic compounds.
Following six months of 03 exposure, pulmonary resistance and frequency
dependence of pulmonary compliance were significantly increased. After 12
months, the 0- exposure had significantly increased pulmonary resistance and
inertance (related to the pressure required to accelerate air and lung tissue),
9-61
-------
and forced expiratory maneuvers showed decreased flows at low lung volumes
(12.5 percent VC) and decreased volume expired in 1 sec (FEV-,). Wegner (1982)
suggested that because lung volumes and pulmonary compliance were not affected
in Og~exposed animals, changes in forced expiratory function-were more likely
caused by narrowing of the peripheral airways than by decreased small airway
stiffness. Rigid analysis of the pulmonary impedance data by linear-lumped-
parameter modeling suggested that the increase in pulmonary resistance was due
to central as well as peripheral airway narrowing.
During the 3-month recovery period following exposure, static lung compli-
ance tended to decrease in both 03-exposed and control animals. However, the
decrease in compliance was significantly greater in 0~-exposed animals than in
control animals.. No other significant differences were measured following the
3-month recovery period, although values for 03-exposed animals remained
substantially different from those for control animals, suggesting that full
recovery was not complete.
The forced oscillation technique has also been utilized in rats exposed
q
to 1254 (jg/m (0.64 ppm) of 0- for either 7 or 20 days (Kotlikoff et a!.,
1984). In an attempt to further characterize 0~-induced changes in central
and peripheral distribution of mechanical properties of the respiratory system,
impedance spectra of Og-exposed rats were compared to the spectra of normal
rats. The effective resistance was higher at all frequencies in the 7-day
exposed rats but no consistent differences were observed by 20 days. The
effective reactance, however, was significantly lower than control in both the
7- and 20-day exposed rats. These changes in respiratory system impedance
demonstrate evidence of mechanical alterations in the peripheral airways of
rats for as long as 20 days of 0, exposure.
9.3.2.3 Airway Reactivity. Ozone potentiates the effects of drugs that con-
strict airway smooth muscle in mice, guinea pigs, dogs, sheep, and humans
(Table 9-4). Early experimental evidence for hyperreactivity to broncho-
constrictive drugs following 03 exposure was provided by Easton and Murphy
(1967). Although much of their work was done with very high 0, concentrations
o
(9800 to 11760 (jg/m , 5 to 6 ppm), they did show that mortality from a single
subcutaneous injection of histamine was higher in guinea pigs exposed to 980
or 1960 ng/rn3 (0.5 or 1 ppm) of 03 for 2 hr (33 and 50 percent mortality,
respectively) compared with the mortality of air-exposed control animals. The
animals appeared to die from massive bronchoconstriction, with the lungs
remaining fully inflated instead of collapsing when the chest was opened.
9-62
-------
TABLE 9-4. EFFECTS OF OZONE ON PULMONARY FUNCTION: AIRWAY REACTIVITY
Ozone
concentration
ug/m3
196
1568
196
1568
196
1960
vo
S 980
980
1568
980
2156
980
1960
3920
ppm
to 0.1 to
0.8
0.1
0.8
0.1
1.0
0.5
0.5
0.8
0.5
1.1
0.5
1
2
Exposure
Measurement duration
method & protocol
CHEM 1 hr
CHEM 1 hr
MAST 10-30 min
CHEM 2 hr
MAST Continuous,
13 to 16 days
of 03 ex-
posure in
four periods -»
(3 to 5 days
each) separ-
ated by 3 to
8 days of
breathing
air
NBKI 2 hr
CHEM 2 hr
Observed effects Species
Subcutaneous injection of histamine 2 hr following 03 Guinea pig
exposure caused a greater increase in R. following expo- (200-300 g)
sure to 0.8 ppm of 03 and a greater decrease in C.dyn
following exposure to all 03 concentrations (magni-
tude of C, changes not related to 03 concentration).
Decreased diaphragm and lung cholinesterase activity; Guinea pig
parathi on-treated animals had increased peak airway
resistance compared to controls, but the difference was
not statistically significant.
Bilateral vagotomy: completely blocked increased peri- Dog
pheral lung resistance from 0.1 ppm of 03 but not histamine;
only partially blocked response from 1.0 ppm of 03.
Histamine- induced airway reactivity increased during
1.0 ppm but not 0.1 ppm of 03 exposure and was not blocked
by atropine or vagotomy.
Increased number of mast cells and lymphocytes in tracheal Sheep
lavage 24 hr after exposure.
Repeated exposures to 0.5 or 0.8 ppm of 03 plus aerosolized Mouse
ovalbumin resulted in greater mortality from anaphy lactic
shock produced by intravenous injection of ovalbumin
compared with effects of ovalbumin injection in mice
repeatedly exposed to ovalbumin aerosols but no 03.
Increased histamine- induced mortality immediately Guinea pig
following exposure to 0.5 or 1.1 ppm of 03.
Increased airway reactivity to aerosolized carbachol Sheep
24 hr but not immediately following exposure to
980 ug/m3 (0.5 ppm) of 03 with no change in R,,
FRC, C.st, or tracheal mucous velocity. Increased
R. 24 hr following exposure and airway reactivity
immediately and 24 hr following exposure (1 ppm).
Reference
Gordon and Amdur,
1980
Gordon et al . , 1981
Gertner et al . ,
1983a,b,c, 1984
Kaplan et al. ,
1981
Sielczak et al., 1983
Osebold et al. ,
1980
Easton and Murphy,
1967
Abraham et al . , 1980
-------
TABLE 9-4. EFFECTS OF OZONE OH PULMONARY FUNCTION: AIRWAY REACTIVITY (continued)
Ozone
concentration
pg/M^ ppm
980 0.5
980 0.5
1960 1.0
Exposure
Measurement duration .
nethod & protocol Observed effects
CHEH 2 hr/day No effect on airway responsss to inhaled carbachol
for 2 days 1 day after Oa exposure; airway reactivity increased
34X and airway sensitivity increased 31% with intra-
venous carbachol challenge.
CHEH 2 hr Airway responsiveness and airway permeability to hista-
mine increased 1 day after exposure to 0.5 ppm 03
(n=6) and in 4/7 exposed to 1.0 ppm 03; directional
changes in airway responsiveness paralleled direc-
tional changes in airway permeability.
Species Reference
Sheep Abraham et al., 1984a
Sheep Abraham et al., 1984b
1100 to 0.56 to CHEM
1666 0.85
I
O\
2 hr
Abnormal, rapid, shallow breathing in conscious dogs
while walking on a treadmill following 03 exposures.
Maximal 1- to 3-hr postexposure, normal 24-hour post- •
exposure. Abnormal breathing not affected by drug-
induced bronchodilatation (inhaled isoproteronol) but
abolished by vagal cooling. Increased respiration rate
caused by inhalation of aerosolized histamine after 03
exposure also blocked by vagal cooling but not by
isoproteronol.
Dog Lee et al., 1979
1313
0.67
CHEH
2 hr
Abnormal, rapid, shallow breathing during exposure to
air containing low 02 or high C02 immediately following
Q3 exposure. Abnormal breathing not affected by
inhaled atropine aerosols or inhaled isoproteronol
aerosols but abolished by vagal cooling.
Dog Lee et al., 1980
1372 to
2352
0.7 to
1.2
CHEH
2 hr
Greater increase in R, caused by histamine aerosol
inhalation 24 hr following 03 exposure. No hyper-
reactivity to histamine 1 hr following 03 exposure.
Drug-induced bronchodilatation (inhaled isoproteronol)
blocked any increase in R, before or after 03 exposure.
Inhalation of atropine or vagal cooling (to block reflex
bronchoconstriction) prevented 03-induced reactivity
to hi stamina.
Dog Lee et al., 1977
-------
TABLE 9-4. EFFECTS OF OZONE ON PULMONARY FUNCTION: AIRWAY REACTIVITY (continued)
Ozone Exposure
concentration Measurement duration
W>
CTi
01
ug/m3
I960
2352
1960
4312
5880
5880
ppm method & protoco
1.0 CHEM 1 hr
1.2
1.0 UV 2 hr
2,2
3.0
3.0 UV 2 hr
1 . Observed effects
R. increased and C. decreased with subcutaneous
hTstamine 2 hr after exposure; responsiveness was not
blocked by atropine or vagotomy. No change in static
compliance after subcutaneous histamine injection.
Marked increase in airway responsiveness to inhaled ACh
and histamine 1 hr after exposure; increased to a lesser
degree 1 day later, and returned to control levels by
1 week. Effects possibly linked to acute inflamma-
tory response.
Airway responsiveness to inhaled ACh was prevented
by indomethacin pretreattnen not the airway infiltra-
tion by neutrophils. Both responsiveness and neutrophil
infiltration were prevented by hydroxyurea pretreatment.
Species Reference
Guinea pig Gordon et al.,
Dog Holtzman et al
1983a,b
Fabbri et al . ,
Dog 0' Byrne et al.
1984a,b
1984
" >
1984
1
5880 3.0
UV,
2 hr
SR measured with intravenous ACh and/or inhaled ACh or
metnacholine increased similarly 14 hr after after expo-
sure; airway reactivity to inhaled bronchoconstrictors
returned to baseline levels 2 days after exposure while
responses to Intravenous ACh persisted.
Guinea pig Roun and Hurl as, 1984
5880 3.0
UV
2 hr
SR increased with intravenous ACh; maximal response
2 nr after exposure; remission by the 4th day. Airway
infiltration of neutrophils occurred later and lasted
longer than airway reactivity.
Guinea pig Murlas and Roum (1985)
Measurement method: MAST - Kl-coulometric (Mast meter); CHEM = gas phase cherailuminescence; NBKI = neutral buffered potassium iodide.
See Glossary for the definition of pulmonary symbols.
-------
Abraham et al. (1980) evaluated airway reactivity in sheep from measure-
ments of pulmonary resistance following inhaled carbachol aerosols. Carbachol
causes bronchoconstriction by stimulating airway smooth muscle at receptor
sites that are normally stimulated by release of acetylcholine from terminals
of the vagus nerve. Pulmonary resistance during inhalation of carbachol
aerosols was significantly higher than pre-exposure values at 24 hr postexposure
o
but not immediately following a 2-hr exposure to 980 jjg/m (0.5 ppm) of Og.
This 07 exposure did not affect resting end-expiratory lung volume (functional
3
residual capacity) or static lung compliance. In sheep exposed to 1960 ug/m
(1 ppm) of 0~ for 2 hr, baseline resistance (before carbachol aerosol inhala-
tion) was elevated 24 hr following exposure, and airway reactivity to carbachol
was increased immediately and 24 hr following 0~ exposure.
To determine if 0,,-induced airway secretions limit the penetration of an
inhaled bronchoconstrictor, Abraham et al. (1984a) compared airway respon-
siveness to inhaled and intravenous carbachol before and 24 hours after exposure
3
to Og. Adult female sheep were exposed to 980 pg/m (0.5 ppm) of 0~, 2 hr/day
for 2 consecutive days. Airway sensitivity was defined as the largest increase
in specific lung resistance after carbachol challenge and airway reactivity as
the slope of the dose-response curve. There were no significant differences
between pre- and postexposure responses to inhalation challenge. However, Og
exposure increased mean airway reactivity and sensitivity by 34 and 31 percent,
respectively, using intravenous challenge. Since carbachol causes direct
stimulation of airway smooth muscle, the authors .suggested that 0- may have
decreased penetration of the inhaled carbachol to the airway smooth muscle as
the result of increased airway secretion. This hypothesis is supported by a
previous study showing that changes in airway responsiveness to inhaled his-
tamine following exposure to 03 may have been related to changes in airway
permeability to histamine (Abraham et al., 1984b).
A study in awake guinea pigs comparing changes'in airway reactivity with ~
intravenous and inhaled bronchoconstrictors after exposure to a high concentra-
tion of CL has suggested that mechanisms other than increased airway permeabi-
lity may be involved. Roum and Murlas (1984) measured changes in specific
airway resistance with intravenous and/or inhaled acetylcholine or methacholine
3
from 4 hr to 2 days after exposure to 5880 M9/m (3.0 ppm) of 03 for 2 hr.
Airway hyperreactivity by either route was similar within 14 hr of exposure.
Two days after exposure airway reactivity to bronchoconstrictor inhalation
9-66
-------
returned to baseline levels while responses 'to intravenous acetylcholine
persisted. The consistent early changes in airway reactivity after 0- exposure
with either intravenous or inhaled bronchoconstrictors indicated that this
response may be independent of the route of delivery. However, it is also
possible that there may be more than one mechanism responsible for 0,-induced
airway hyperreactivity depending on the concentration of 0, reaching the
airway tissue and on interspecies differences in cellular responsiveness to
°3- .
Gordon and Amdur (1980) evaluated airway reactivity to subcutaneously
injected histamine in awake guinea pigs following a 1-hr exposure to 196 to
3
1568 |jg/m (0.1 to 0.8 ppm) of 0,. Airway reactivity to histamine was maximal
2 to 6 hr following 03 exposure and returned to control levels by 24 hr follow-
ing exposure. The histamine-induced increase in pulmonary resistance was
3
greater in guinea pigs exposed to 1568 ug/m (0.8 ppm) of 0, than in air-exposed
control animals. Pulmonary compliance decreased more following histamine
injection in all O^-exposed groups than in air-exposed controls, but there
were no differences in the histamine-induced decreases in pulmonary compliance
3
between any of the 03 concentrations (from 196 to 1568 ug/m ; 0.1 to 0.8 ppm).
Gordon et al. (1981) studied the effect of 03 on tissue cholinesterases
to see if they were responsible for the bronchial reactivity observed following
challenges with bronchoconstrictor analogs of acetylcholine. Guinea pigs were
3
exposed to clean air or 196 or 1568 ug/m (0.1 or 0.8 ppm) of 0~ for 1 hr.
After 2 hr, brain, lung, and diaphragm samples were analyzed for cholines-
terase activity. Brain cholinesterase activity was not affected, but lung
3
cholinesterase underwent a 17 percent decrease in activity at 196 ug/m
3
(0.1 ppm) and a 16 percent decrease at 1568 ug/m (0.8 ppm). Ozone at
3
1568 |jg/m (0.8 ppm) also decreased the diaphragm cholinesterase activity by
14 percent. To provide long-term inhibition of cholinesterase, guinea pigs
were treated with parathion, an irreversible cholinesterase inhibitor. Airway
resistancei tended to increase following histamine challenge in the parathion-
treated guinea pigs, but the difference was not statistically significant
because of large variations in response. The authors suggested that cholines-
terase inhibition by 0^ may contribute to the Q,,~induced bronchial reactivity,
as already reported. Presumably, the decreased cholinesterase activity could
result in higher acetylcholine concentrations in the bronchial muscle. A
cholinergic-related stimulus, such as occurs with 0- exposure, should then
9-67
-------
increase the contraction of the bronchus. The persistence of this activity is
not known.
Gordon et al. (1984) extended these studies to determine the site of the
airway hyperresponsiveness to histamine-induced airway constriction after Og
exposure. Anesthetized guinea pigs were evaluated for response to subcutaneous
o
histamine 2 hr after exposure to 1960 or 2352 ug/m (1.0 or 1.2 ppm) of 0, for
1 hr. Respiratory resistance increased and dynamic compliance decreased in
Qg-exposed animals, as previously reported (Gordon and Amdur, 1980). However,
static compliance changes after histamine were similar in the air- and Q~-
exposed animals, suggesting that the site of hyperresponsiveness was in the
conducting airways rather than the parenchyma. In addition, enhanced airway
responsiveness to histamine was not blocked by atropine or vagotomy, indicating
a minimal level of vagal involvement. The significantly greater increase in
respiratory resistance caused by efferent electrical stimulation of the vagus
in 0_-exposed animals suggested that other mechanisms, such as changes in the
airway smooth muscle, may be responsible for the hyperexcitability following
DO exposure. However, i_n -vitro studies on isolated parenchyma! strips removed
from the lungs .of air- and Qg-exposed animals failed to show any differences
in the contractile responses to histamine or carbachol.
Lee et al. (1977) evaluated airway reactivity in Og~exposed dogs from
changes in pulmonary resistance induced by histamine aerosol inhalation. Dogs,
were exposed to 1372 to 2352 ug/m3 (0.7 to 1.2 ppm) of 03 for 2 hr. Airway
reactivity to inhaled histamine aerosols was significantly greater 24 hr but
not 1 hr after 0, exposure. Bronchodilatation induced by inhalation of isopro-
teronol aerosols prevented any change in resistance following histamine expo-
sure. This experiment showed that the increased resistance normally observed
following histamine exposure was caused by constriction of airway smooth
muscle and not by edema or increased mucous production, which would not be
prevented by isoproterenol bronchodilatation. Administration of atropine
(which blocks bronchoconstrictor activity coming from the"vagus nerve) or
vagal cooling (which blocks both sensory receptor activity traveling from the
lung to the brain and bronchoconstrictor activity going from the brain to the
lung) decreased the response to histamine both before and following 03 exposure
and abolished the hyperreactive airway response. These experiments showed
"that the increased sensitivity to histamine following 03 exposure was caused
by heightened activity of vagal bronchoconstrictor reflexes.
9-68
-------
Although the work of Lee et al. (1977) provides evidence that stimulation
of vagal reflexes by histamine is in part responsible for the increased airway
reactivity found in dogs following 03 exposure, Kaplan et al. (1981) found
that local responses to histamine in the lung periphery may not be mediated by
a significant vagal component. When monodispersed histamine aerosols were
delivered to separate sublobar bronchi in dogs through a 5.5 mm diameter
fiber-optic bronchoscope, collateral airflow resistance increased both before
and after bilateral cervical vagotomy. In follow-up studies that used similar
techniques, Gertner et al. (1983a,b,c; 1984) described the role of vagal
reflexes in the response of the lung periphery to locally administered histamine
and DO- Collateral resistance increased during separate 30-min exposures to
3 ~6 3
either 196 (jg/m (0.1 ppm) of 0, or 1.5 xlO mg/m of histamine. However,
although parasympathetic blockade (atropine or bilateral cervical vagotomy)
prevented the responses to 03, it did not prevent the responses to histamine
(Gertner et al. , 1983b). To determine if 03~induced increases in collateral
resistance in the lung periphery were dependent on vagal reflexes, aerosolized
neostigmine was administered locally to maintain parasympathetic tone. Re-
3
sponses to 196 (jg/m (0.1 ppm) of 03 in the lung periphery were enhanced only
if the vagi were intact and were limited to the challenged region (Gertner
et al. , 1984). When larger areas were exposed, vagally mediated responses
occurred in both lungs. In addition, the characteristics of responses to high
concentrations of 0- differ markedly from responses to the lower concentrations.
3
A 30-min exposure to 196 (jg/m (0.1 ppm) of 0, did not affect the airway
responsiveness to histamine, but when the 0- exposure was increased to 1960
3
|jg/m (1.0 ppm) for 10 min, histamine produced greater increases in collateral
resistance that were not abolished by parasympathetic blockade (Gertner et al.,
1983c). Exposure to 1960 (jg/m (1.0 ppm) of 03 for 30 min produced an increase
in collateral resistance that was mediated by the parasympathetic system in
the early phase of the response and related in part to histamine release in
the late phase of the response (Gertner et al., 1983a). Results from this
series of studies by Kaplan et al. (1981) and Gertner et al. (1983a,b,c; 1984)
are difficult to interpret because of the small numbers of animals in each
test group and large variations in response. In addition, because peripheral
resistance contributes only a small part to total pulmonary resistance, the
findings of these authors do not necessarily contradict the work of Lee et al.
(1977). Rather, all the studies taken together suggest that the periphery of
9-69
-------
the lung may respond differently from the larger conducting airways during
exposure to Qg and that factors in addition to vagal bronchoconstrictor reflexes
can produce an increased airway reactivity to histamine.
Holtzman et al. (1983a) reported the time course of Q~-induced airway
~ o
hyperreactivity in dogs exposed to 1960 and 4312 ug/m (1.0 and 2.2 ppm) of 0-
for 2 hrs. Airway responsiveness to acetylcholine in 7 dogs increased markedly
3
1 hr, and to a lesser extent, 24 hr after exposure to 4312 jjg/m (2.2 ppm) of
0~, returning to control levels by 1 week after exposure. Ozone- induced in-
creases in airway responsiveness to histamine were similar following exposure
3
to 1960 |jg/iii (1.0 ppm) of 03, but data were reported for only 2 dogs. The
authors suggested that the time course of the 03 effect may be linked to acute
airway inflammation. In a coincident publication, Holtzman et al. (1983b)
found a strong association between airway hyperreactivity and trachea! inflam-
2
mation in dogs 1 hr following a 2-hr exposure to 4116 jjg/m (2.1 ppm) of 0,.
Airway reactivity was assessed from the increase in pulmonary resistance
following inhalation of acetylcholine aerosols, and airway reactivity was
increased in 6 of 10 0,,-exposed dogs. The number of neutrophils present in a
trachea! biopsy, a measure of inflammation, was increased only in the 6 dogs
that were hyperreactive to acetylcholine. These observations have recently
been extended to show an association of 0,-induced increases in airway respon-
siveness with inflammation in more distal airways (Fabbri et al., 1984). The
number of neutrophils as well as ciliated epithelial cells in fluid recovered
from bronchoalveolar lavage was increased in 5 dogs that were hyperreactive to
q
acetylcholine following a 2-hr exposure to 5880 jjg/m (3.0 ppm) of 03 without
significant changes in the numbers of macrophages , lymphocytes, or eosinophils.
In dogs depleted of neutrophils by treatment with hydroxyurea, 0™ exposure to
3 '
5880 ug/m (3.0 ppm) of 0- for 2 hr caused the desquamation of epithelial
cells but airway responsiveness to inhaled acetylcholine was prevented (O'Byrne
et al., 1984a). This observation suggests that Ov"induced hyperresponsiveness
may depend on the mobilization of neutrophils into the airways.
The authors have expanded their work to speculate that the neutrophils
produce mediators that are responsible for the increased responsiveness of
airways (O'Byrne et al., 1984b). In dogs pretreated with indomethacin, a
3
prostaglandin synthetase inhibitor, exposure to 5880 jjQ/m (3.0 ppm) of 03 for
2 hr had no effect on airway responsiveness to inhaled acetylcholine but there
was a significant increase in the number of neutrophils in the airway epithe-
lium. While these results suggest that oxidation products of arachidonic
9-70
-------
acid, possibly prostaglandins or thromboxane (O'Byrne et al., 1984c), may be
released by the neutrophils, they are not conclusive. Therefore, the identity
of the specific inflammatory cells or of the responsible mediators is still
uncertain.
3
The results of trachea! lavage in sheep exposed to 980 ug/m (0.5 ppm) of
Og for 2 hr suggest that the migration of mast cells into the airways may also
have important implications for reactive airways and allergic airway disease
(Sielczak et al., 1983). Nasotracheal-tube exposure to 0., in 7 sheep resulted
in an increased number of mast cells and lymphocytes 24 hr after exposure, sug-
gesting an association between an enhanced inflammatory response and 03~induced
bronchial reactivity reported previously in sheep (Abraham et al., 1980).
Additional evidence in guinea pigs suggests that 03~induced bronchial
hyperreactivity may be due to airway mucosal injury and mast cell infiltration
(Murlas and Roum, 1985). Specific airway resistance was measured as a function
of increasing intravenous acetylcholine doses for varying periods of from 2 hr
g
up to 4 days after exposure to 5880 pg/m (3.0 ppm) of 03 for 2 hr. The largest
airway response to acetylcholine challenge occurred 2 hr after 0- exposure with
complete remission by the fourth day. Meutrophil infiltration occurred later and
lasted longer despite the remission in airway hyperreactivity suggesting that the
influx of neutrophils was a result of the initial damage and not a direct cause of
increased airway responsivensss.
Increased drug-induced bronchoconstriction is not the only indicator of
airway hyperreactivity following 03 exposure. Animal experiments were designed
to investigate the mechanisms responsible for the abnormal, rapid, shallow
breathing found in human subjects exercising during experimental 0- exposure
compared with subjects exercising in clean air (Chapter 10). Lee et al.
(1979, 1980) showed that abnormal, rapid, shallow breathing in conscious dogs
3
immediately following 2-hr exposures to 1100 to 1666 pg/m (0.56 to 0.85 ppm)
of 0- was a hyperreaetive airway response. This abnormal breathing pattern
was elicited by mild exercise, hi stand ne aerosol inhalation, or breathing air
with reduced oxygen (02) or elevated carbon dioxide (COp) concentrations. The
rapid, shallow breathing observed in dogs following 03 exposure was not affec-
ted by drug-induced bronchodilatation (inhaled isoproteronol aerosols) or by
blocking vagally induced bronchoconstriction with atropine. In all cases,
rapid, shallow breathing was abolished by vagal cooling, which blocked the
transmission of sensory nerves located in the airways. These investigators
(Lee et al., 1979, 1980) suggest that the rapid, shallow breathing observed
9-71
-------
following 0~ exposure in dogs is caused by heightened activity of sensory
nerves located in the airways. The increased reactivity of these sensory
nerves is independent of smooth muscle tone (either bronchodilatation or
bronchoconstri cti on).
Studies of lung morphology following 03 exposure showed damage to the
respiratory epithelium (Section 9,3.1). Damage to the epithelium overlying
sensory receptors may be responsible for the increased receptor reactivity to
mechanical stimulation (increased ventilation with exercise, low Op, or high
COp) or chemical (histamine) stimulation (Nadel, 1977; Boushey et a!,, 1980).
The rapid, shallow breathing observed in guinea pigs during 03 exposures (Amdur
et al. 1978; Yokoyama, 1969; Murphy et al., 1964) may also be related to in-
creased sensory neural activity coming from the lungs, not to an indirect effect
of changes in airway diameter or lung distensibility as previously speculated.
In their study of allergic lung sensitization, Osebold et al. (1980)
showed additional functional evidence for epithelial disruption caused by 03
exposure. These investigators studied the anaphylactic response of mice to
intravenous ovalbumin injection following repeated inhalations of aerosolized
3
ovalbumin. Mice were continuously exposed to 980 or 1568 |jg/m (0.5 or 0.8 ppm)
of 0~ for four periods of 3 to 5 days each, separated by 3 to 8 days of ambient
air exposure. During periods of 03 exposure, mice were removed from the exposure
chambers for short periods, and they inhaled ovalbumin aerosols for 30 min.
Mice exposed to 03 and ovalbumin aerosols developed more severe anaphylactic
reactions and had a higher incidence of fatal anaphylaxis than air-exposed
mice receiving the same exposure to aerosolized ovalbumin. In mice exposed to
3
aerosolized ovalbumin, 34 percent of the 03~exposed mice (1568 ug/m ; 0.8 ppm)
developed fatal anaphylaxis following intravenous ovalbumin injection, compared
with 16 percent of the air-exposed animals. This study also showed some
indication of an interaction between 0- exposures and exposures to sulfuric
3 3
acid aerosols. Of the mice exposed to 980 pg/m (0.5 ppm) of 03 plus 1 mg/m
of sulfuric acid aerosols, 55 percent died of anaphylactic shock following
intravenous ovalbumin injection, compared with 20-percent mortality in mice
exposed to 0., alone and zero mortality in mice exposed to sulfuric acid aerosols
alone. The authors propose that these data may indicate that pollutants can
increase not only the total number of clinical asthma attacks, but also the
number of allergically sensitized individuals in the population. Matsumura
9-72
-------
(1970) observed a similar increase in the allergic response of sensitized
2
guinea pigs to inhaled antigen following 30 min of exposure to 3920 ug/m
(2.0 ppm) of 03.
9.3.3 Biochemically Detected Effects
9.3.3.1 Introduction. This section will include some studies involving con-
centrations above 1960 ug/m (1 ppm) of 0~, because the direction of the
effect is opposite that for lower 0, concentrations (decrease vs. increase in
many parameters). An extensive body of data on this topic has been reviewed
by Menzel (1983), Mustafa et al. (1977, 1980, 1983), Mustafa and Lee (1979),
Cross et al. (1976), and Chow (1983). To facilitate presentation of this
information, it has been categorized by broad classes of metabolic activity.
This results in some degree of artificial separation, particularly because
many patterns of response to 0"3 are similar across the classes of metabolism.
Lung permeability is discussed in this section, because it is typically detected
biochemically. The final subsection presents hypotheses about the molecular
mechanism(s) of action of 0~, relying on the data presented earlier by metabolic
class.
9.3.3.2 Antioxidant Metabolism. Antioxidant metabolism of the lungs is
influenced by 03 exposure. As shown in the schematic diagram below (Figure
9-3), this system consists of a number of enzymes. As shall be discussed, 03
has been shown to produce several reactive oxidant species jm vitro from com-
-^—G-6-P——^v—-NADP+-*->«*•** GSH-
G-6-PD GSH GSH
HMP shunt reductase peroxidase
ROH
Figure 9-3. Intracellular compounds active in antioxidant metabolism of
the lung. (G-6-P = glucose-6-phosphate; 6-PG = 6-phosphogluconate;
G-6-PD = glucose-6-phosphate dehydrogenase; HMP shunt = hexose
monophosphate shunt; NADP+ = nicotinamide adenine dinucleotide
phosphate; NADPH = reduced NADP; GSH = glutathicne; GSSG =
glutathione disulfide; [O] = oxidizing moiety [i.e., hydrogen peroxide,
free radical, lipid peroxide]; GSH peroxidase = glutathione perioxidase;
GSH reductase = glutathione reductase; and ROH = reduced form of
[O]).
Source: U.S. Environmental Protection Agency (1978).
9-73
-------
pounds found in the lung, as well as in other organs. It is reasonably certain
that Og can produce such reactive species in the lung after jji vivo exposure.
Many of these oxidant species are metabolized by the glutathione peroxidase
system, rendering them less toxic. Thus, this system is involved in the
toxicology of On.
3
Typically, following exposures to levels of (U below 1960 ug/m (1 ppm),
the activities of most enzymes in this system are increased (Table 9-5).
Whether this increase is due to direct mechanisms (e.g., de noyo synthesis
resulting in greater enzyme activity), indirect mechanisms (e.g., an increased
number of type 2 cells that naturally have a higher enzymic activity than type
1 cells), or a combination of both has not been proven. The increase in type
2 cells is most likely to be the mechanism, because with similar exposure
regimens, the effects (increased enzyme activities and increased numbers of
type 2 cells) increase, reaching a maximum at 3 to 4 days of exposure and a
steady state on day 7 of exposure. Whatever the mechanism, the increase
occurs at low 0^ levels in several species under varying exposure regimens
(Table 9-5). The net result is that the antioxidant metabolism of the lung
is increased. Whether this is a protective or a toxic response is often
debated. To resolve this debate scientifically will require more knowledge of
the mechanisms involved. However, even attributing it to be a protective re-
sponse implies a physiological need for protection (e.g., an initial toxic
response occurred which required protection). A more detailed discussion of
these effects follows.
Acute exposures to high concentrations of 0^ generally decrease anti-
oxidant metabolism, whereas repeated exposures to low levels increase this
metabolism. For example, DeLucia et al. (1975a) compared the effects of acute
(2 to 8 hr) exposures to high 03 levels (3920 and 7840 H9/m3, 2 and 4 ppm) and
short-term (8 .or 24 hr/day, 7 days) exposures to lower Q~ levels (392, 980,
3
1568 ug/m , 0.2, 0.5, 0.8 ppm) on rats. For'nohproteTn sulfhydryllevels
(principally glutathione, GSH), decreases in the level of GSH were progressive
3
with time of exposure (2 to 6 hr) to 7840 |jg/m (4 ppm). For glutathione
disulfide (6SSG), decreases were less and had returned to normal by 6 hr of
exposure. These exposure regimens also decreased the activities of GSH reduc-
tase and glucose-6-phosphate dehydrogenase (G-6-PD). After the first day of a
q
7-day continuous exposure to 1568 pg/m (0.8 ppm) of 03, no significant change
was seen in the nonprotein sulfhydryl or GSH content or in the activities of
G-6-PD, GSH reductase, or disulfide reductase. However, the levels/ activities
9-74
-------
TABLE 9-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE
f
-J
cn
Ozone
concentration
ug/m3 ppm
196 0.1
196 0.1
392 0.2
196 0.1
392 0.2
392 0. 2
980 0. 5
1568 0.8
1568 0.8
. Exposure
Measurement ' duration and
method protocol
NBKI Continuous
. ' for 7 days
I Continuous
for 7 days
MAST, Continuous
NBKI for 7 days
I Continuous
for 7 days; or
8 hr/day for
7 days
Continuous
for 1 to 30
days
Observed effect(s)c Species Reference
With vit E-deficient diet, increased levels of GSH and Rat Chow et a!., 1981
activities of GSH peroxidase, GSH reductase, and G-6-PD;
no effect on malic dehydrogenase. With 11 ppm vit E
diet, increased levels of GSH peroxidase and G-6-PD.
With 110 ppm vit E diet, no change.
With 66 ppm vit E- supplemented diet, increase in Rat Mustafa, 1975
oxygen consumption of lung homogenates only at Mustafa and Lee,
0.2 ppm. With 11 ppm vit E-supplemented diet, 1976
increase in 02 consumption at 0.1 and 0.2 ppm.
Increase due to increased amount of mitochondria
in lungs.
Increased activities of GSH peroxidase, GSH reduc- Rat Plopper et al.,
tase, and G-6-PD and of NPSH levels with (66 mg/kg) 1979
or without (11 mg/kg) vit E supplementation; at
0.2 ppm, effects less with vit E supplementation.
Morphological lesions unaffected by vit E supple-
mentation.
For the continuous exposure to the two higher con- Rat Mustafa and
centrations, increased activities GSH peroxidase, Lee, 1976
GSH reductase, and G-6-PD. At the lower concen-
tration (continuous), increased activities of GSH
peroxidase and GSH reductase. A linear concentration-
related increase in all three enzyme activities. In-
creased 02 consumption using succlnate-cytochrome C
reductase activity fairly proportional to 03 level.
Similar results for intermittent exposure groups.
Increased rates of 02 consumption, reaching a peak
at day 4 and remaining at a plateau for the re-
mainder of the 30 days. Also an initial decrease
(day 1) and a subsequent increase (day 2) in
activity of succinate-cytochrome C reductase
which plateaued between days 3 to 7.
-------
TABLE 9-5. CHANGES IN THE LUNG AHTIOXIDANT HCTABOLISM AND OXYGEH CONSUMPTION BY OZONE (continued)
f
Ozone
concentration
pg/a3
392
686
980
1568
392
980
1568
392
980
1568
392
980
1960
392
980
1960
2352-
16,072
ppm
0,2
0.35
0.5
0.8
0.2
0.5
0.8
0,2
0.5
0.8
0.2
0.5,
1.0
0.2
0.5
1.0
1.2-
8.2
b Exposure
Measurement ' duration and
method protocol
I 8 hr/day for
7 days
N8KI Continuous
for 8 days or
8 hr/day for
7 days
HAST, 8 or 24 hr/day
NBKI for 7 consecu-
tive days
NBKI 3 hr/day for
4 days
NO 4 hr/day for
up to 30 days
4 hr
Observed effect(s)c Species
Increased concentration-related activities of Monkey,
G-6-PD, NADPH-cytochrome c reductase, and
succinate oxidase. Significant increases
occurred in the bonnet monkey at 0.35 and
0.5 ppm; in the rhesus monkey at 0.8 ppn.
However, actual data were only reported for
succinate oxidase.
For continuous exposure to two higher concentrations, Rat
increased activities of GSH peroxidase, GSH reduc-
tase, and G-6-PD. At the lower concentration (con-
tinuous), increased activities of GSH peroxidase
and GSH reductase. A concentration-related linear
Increase in all three enzyme activities. Similar
results obtained for intermittent exposure groups.
Activities of G-6-PD and NADPH-cytochrome C reduc- Rat
tase and succinate oxidase increase- in a concen-
tration-dependent fashion. No significant
differences between the intermittent and continuous
exposure groups.
Reduced glutathione levels increased in a linear House
concentration-dependent manner. No effect at
0.2 ppm in the no-exercise. group. Exercise en-
hanced effect.
GSH content increased directly with 03 concentration Mouse
and exposure duration. Increase in activities of
G-6-PD, GSH reductase, and GSH peroxidase after 7
days of exposure to 0.5 and 1 ppm.
Decrease 1n GSH content after exposure to 8.2 ppm.
No change below 4.0 ppm. Two days postexposure to
4 ppm, GSH content increased, lasting for several
days.
Reference
Mustafa and
Lee, 1976
Chow et al . , 1974
Schwartz et al . ,
1976
Fukase et al . , 1978
Fukase et al., 1975
-------
TABLE 9-5. CHANGES IN THE LUNG ANTIOXIOANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
Ozone
concentration
M9/m3
392
980
1568
1568
3920
7840
392
980
15S8
627
882
ppm
0.2
0.5
0.8
0.8
2
4
0.2
0.5
0.8
0.32
0.45
b Exposure
Measurement ' duration and
method protocol
MAST, 8 or 24 hr/day
NBKI for 7 days
8 hr/day
for 7 days
2 to 8 hr
I Continuous
for 7 days
UV 6 hr
UV Continuous
for 5 days
Observed effect(s)c
All 03 levels: increase in NPSH levels; increased
activities of G-6-PD, GSH reductase, NADH cyt. c
reductase. At 0.5 and 0,8 ppm, increased activity
of succinate cyt. c reductase. At 0.8 ppm, continuous
increase began at day 2 of exposure.
Increased NPSH, GSH, and G-6-PD; no change in other
enzymes.
Loss of GSH; loss of SH from lung
nitochrondrial and microsomal frac-
tions and inhibition of marker
enzyme activities from these
fractions.
Concentration- related increase in 02 consumption.
In both vit E-supplemented and nonsupplemented
groups: increased G-6-PD activities and GSH
levels; decreased ACHase activities.
Mice: increased levels/activities of TSH, NPSH,
GSH peroxidase, GSH reductase, G-6-PD, 6-P-GD,
isocitrate dehydrogenase, cytochrome c oxidase,
Species Reference
Rat DeLucia et al. ,
1975a
Monkey
Rat
Rat Mustafa et al . ,
1973
House Moore et al . , 1980
Mouse, Mustafa et al.,
3 strains 1982
of rats
and succinate oxidase,
Rats: increased levels/activities of NPSH, GSH
peroxidase, and G-6-PD in several strains. Gene-
rally mice were more responsive. For both species,
no change in DNA or protein levels or activity of
GSH-S-transferase.
882
0.45
UV
8 hr/day Oa and 4.8 ppin of N0a alone produced no significant
for 7 days effects but 03 + NOZ produced synergistic effects:
increased total and nonprotein sulfhydryls; increased
activities of succinate oxidase and cytochrome c
Mouse
Mustafa et al.,
1984
-------
PRELIMINARY DRAFT
TABLE 9-5, CHANGES IH THE LUNG AMTIOXIDAHT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
Ozone
concentration
pg/«3 ppi
882 0.45
980 0.5
1372 0.7
1568 0.8
U3 1470 0.75
-J
CO-
1S68 0.8
1568 0.8
1568 0.8
3920 2
u Exposure
Measurement « duration and
Method protocol
ND Continuous
for 7 days
NBKI 8 hr/day for
7 days
NBKI Continuous
for 5 days
Continuous
for 7 days
NBKI Continuous
for up to
30 days
Conti nuous
for 7 days
NBKI Continuous for
3 days
I Continuous for
10 to 20 days
8 hr
Observed effect(s) Species Reference
Increased SOD activity at days 3 and 5, but not Rat Bhatnagar et al.,
days 2 and 7 of exposure. 1983
Increases in activities of GSH peroxidase, GSH Rat Chow et al., 1975
reductase and G-6-PD and In GSH levels of rats. Monkey
No effect In iionkeys.
Increased activities of GSH peroxidase, G-6-PD, Rat Chow and Tappel,
and GSH reductase. Malonaldehyde observed. 1972
The increases in the first two enzymes were partially
inhibited as a logarithmic function of vitanin E
levels in diet.
Increase in activities of GSH peroxidase, GSH Rat Chow and Tappel,
reductase, G-6-PD, 6-P-GQ, and pyruvate kinase 1973
at day 3, reaching a peak at day 10, at which
time beginning of a slight decrease (except for
GSH peroxidase which continued to increase). At
day 30 still elevated over controls.
Increased activities of hexose monophosphate shunt
and glycolytic enzymes of lung.
Increased activities of GSH peroxidase, GSH reductase, Rat Chow et al., 1976b
and G-6-PD; levels of NPSH; general protein synthesis;
and rate of mitochondria! succinate oxidation.
Decrease to control values 6 to 9 days postexposure.
Re-exposure using same reginen (S, 13, or 27 days
postexposure) resulted in similar elevations.
At the higher concentration: increase in the lung Rat Mustafa et al.,
mitochondria! 03 consumption in oxidation of 2- 1973
oxyglutarate and glycerol-1-phosphate and the number
of type 2 alveolar cells which are rich in Mito-
chondria. No change in malonaldeliyde. At the
lower concentration: increase in 02 consumption.
-------
TABLE 9-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
Ozone
concentration
jig/ni3 ppm
1568
3920
0.8
Mea'surement'
method
a,b
Exposure
duration and
protocol
Observed effect(s)
Species
Reference
1568
2940-
7840
0.8 I;
1.5-
4
< 24 hr
< 8 hr
NPSH level unaffected at 0.8 ppm of 03 for < 24 hr or Rat
or 1.5 ppm for < 8 hr; decreased at 2 ppm of 03 for
8 hr or 4 ppm for 6 hr. At 4 ppm of 03 for 6 hr,
decreased level of GSH; no change in GSSG level.
De Lucia et
1975b
al.,
10 days Lung SH levels unchanged. Increase in G-6-PD and
and cytochrome c reductase activities. No change
in malonaldehyde levels.
4 to 8 hr Decrease in lung SH levels and in G-6-PD, GSH
reductase, and cytochrome c reductase activi-
ties. No change in malonaldehyde levels.
High mortality in 7- and 12-day-old rats.
Rat
DeLucia et al.,
1972
3920-
5880
1568
1568
2-
3
0.8 NO
0.8 UV
30 m1n
Continuous
for 7 days
72 hr
In vitro; decrease in SH levels; increase in
•alonaldehyde levels.
Increased activity of superoxide dismutase.
Succinate oxidase, cytochrome c oxidase, and iso-
citrate dehydrogenase: No effect at 24 days old,
increased in 90-day-old rats. G-6-PD, 6-PGH:
increased at 24 and 90 days of age, latter had
greater increase. Succinate oxidase and G-6-PD
decreased in 7- and 12-day-old rats and increased
in 18-day-old rats.
Rat
Rat
(7 to 90
days old)
Mustafa et al . ,
1977
Elsayed et al. ,
1982a
1568 0.8
UV
Continuous Diet was constant vitamin E and deficient or sup-
for 5 days plsmented (2 levels) with selenium (Se). No change
in GSH peroxidase. With 0 Se, decreased GSH reduc-
tase activity; no change with low or high Se.
Progressive increase in activities of G-6-PD and
6-P-GD with increasing Se, beginning at low Se level.
Mouse
Elsayed et al.,
1982b
-------
TABLE 9-5. CHANGES IN THE LUNG ANTOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
vo
•o
Ozone
concentration
ug/m3 ppm
1568 0.8
1568 O.B
k Exposure
Measurement * duration and
nethod protocol
UV Continuous
for 5 days
NBKI continuous
for 7 days
Observed effect(s)c Species
Diet was constant vitamin E and 0 ppm of Se or 1 ppm Mouse
in Se. +Se: increased G-6-PD, 6-P-GD; no change
in GSH reductase or GSH peroxidase. -Se: decreased
GSH reductase.
Both diet groups had increase in TSH and NP.SH, and
lung Se levels after 03.
Vitamin E partially prevented increased activities Rat
of G-6-PD, 6-P-GD, and malic enzyme. Activities
of phosphofructokinase and pyruvate kinase increased.
No effect on aldolase and nalate dehydrogenase.
Reference
El sayed et al . ,
1983
Chow and Tappel ,
1973
1764 0.9
CHEM
96 hr
Trend towards decreased activities of GSH reductase
GSH peroxidase, G-6-PD before 18 days of age, followed
by increases thereafter. For G-6-PD: no change at 5
and 10 days of age; decrease at 15 days, and increase
at 25 and 35 days.
Rat Tyson et al.,
(5-180 1982
days old)
1764 0.9
MAST
>, 96 hr 96 hr: No effect below 20 days of age; G-6-PD in-
creases thereafter up to 35 days, after which (40
and 50 days old) 1t decreases. When exposure started
at 25 or 32 (but not 10 to 15) days of age, the maxi-
mal increase in G-6-PD occurred at about 32 days of
age under continuous exposure conditions.
Rat Lunan et al.,
(10-50 1977
days old)
Measurement method:
MAST = Kl-coulonetric (Mast meter); NBKI = neutral buffered potassium iodide; CHEM = gas solid cheniluminescence; UV = UV photometry;
I =;iodometric; NO = not described.
Calibration method: NBKI = neutral buffered potassium iodide.
cAbbreviations used: GSH = glutathione; GSSG = reduced glutathione; G-6-PD = glucose-6-phosphate dehydrogenase; LDH = lactate dehydrogenase;
NPSH = non-protein sulfhydryls; SH = sulfhydryls; 6-P-GD = 6-phosphogluconate dehydrogenase.
-------
of these constituents increased by day 2 and remained elevated for the remainder
of the exposure period. Comparable results were reported with similar (but
not identical) exposure regimens by DeLucia et al. (1972, 1975b) using rats
and Fukase et al. (1975) using mice.
Investigators (Table 9-5) have found that for lower levels of 0~, in-
creases in antioxidant metabolism are linearly related to 0, concentration.
Most such studies were conducted by using intermittent and continuous exposures.
No differences between these regimens were found, suggesting that concentration
of exposure is more important than time of exposure.
Chow et al. (1974) exposed rats continuously or intermittently (8 hr/day)
3
for 7 days to 392, 980, or 1568 \ig/m (0.2, 0.5, or 0.8 ppm) of 0- and found a
concentration-related linear increase in activities of GSH peroxidase, GSH
reductase, and G-6-PD. Significant increases occurred for all measurements,
3
except G-6-PD at continuous exposure to 392 |jg/m (0.2 ppm). Although the
difference between continuous and intermittent exposure was not examined sta-
tistically, no major differences appeared to exist. Schwartz et al. (1976)
made similar observations for G-6-PD activity when using identical exposure
regimens and found concurrent morphological changes (Section 9.3.1). Mustafa
and Lee (1976), also by using identical exposure regimens, found similar effects
for G-6-PD activity. DeLucia et al. (1975a) found similar changes and increased
nonprotein sulfhydryls at all three concentrations of 0^. A similar study was
performed in mice by using a longer exposure period of 30 days (Fukase et al.,
1975). The increase in GSH level was related to concentration and time of
exposure. Fukase et al. (1978) also observed a linear concentration-related
increase in GSH levels of mouse lungs exposed 3 hr/day for 4 days to 392, 980,
3
or 1960 |jg/m (0.2, 0.5, or 1.0 ppm) of 0,. Exercise enhanced the effect- At ..
the lower 0- level, the increase in GSH was significant only in the exercising
mice.
The influence of time of exposure was examined directly by Chow and
-Tappel (1973). Rats'were exposed continuously to 1470 |jg/m (0.75 ppm) for 1,
3, 10, or 30 days, at which times measurements of GSH reductase, GSH peroxi-
dase, G-6-PD, pyruvate kinase, and 6-phosphogluconate dehydrogenase activities
were made. No statistical tests or indications of data variability were
presented. A few of the enzyme activities (GSH peroxidase and 6-phosphogluconate
dehydrogenase) may have decreased at day 1 of exposure. All enzyme activities
except GSH peroxidase increased by day 3 and reached a pe-ak at 10 days and
then began to return toward control values. GSH peroxidase activity continued
9-81
-------
to increase over this time of exposure. In a similar study (Mustafa and Lee,
1976), G-6-PD activity was measured after rats were exposed for 7 days to 1568
3
ug/m (0.8 ppm) continuously. No effect was detected on day 1, but by day 2
the activity had increased. The peak response was on day 4; the activity
remained elevated to an equivalent degree on day 7. In a similar experiment,
DeLucia et al. (1975a) obtained equivalent results.
The tolerance phenomenon has also been investigated for lung antioxidant
3
metabolism. Rats were exposed continuously for 3 days to 1568 ug/m (0.8 ppm)
of Og, allowed to remain unexposed for 6, 13, or 27 days, and then re-exposed
for 3 days to the same 03 level (Chow et al., 1976b). Immediately after
the first 3 days of exposure, the activities of GSH peroxidase, GSH reductase,
and G-6-PD were increased, as was the nonprotein sulfhydryl content. By 2
days after this exposure ceased, recovery had begun; control values were
completely reached by 9 days postexposure. Following a 30-day recovery period,
no changes were observed. When re-exposure commenced on day 6 of recovery (at
which time incomplete recovery was observed), the metabolic activities returned
to levels equivalent to those of the original exposures. Similar findings
were made when re-exposure commenced on days 13 and 27 days of the recovery
period.
The influence of vitamin E, an antioxidant, on 03 toxicity has been
extensively studied, because it typically reduces the toxicity of 03 in animals.
This topic has been recently reviewed by Chow (1983). Early studies centered
on mortality. For example, vitamin E-deficient rats are more susceptible to
3
continuous exposure to 1960 vg/m (1 ppm) of 03 than rats fed supplements of ,
vitamin E (LT50, the time at which a 50 percent mortality is observed, 8.2
days versus 18.5 days) (Roehm et al., 1971a, 1972). Vitamin E protected
animals from mortality and changes in the wet to dry weight ratios of the lung
o
(lung edema) on continuous exposure to 1568 ug/m (0.8 ppm) of 0- or higher
for 7 days (Fletcher and Tappel, 1973). Vitamin E protection against 03 is
positively correlated to the log concentration of dietary vitamin E fed to the
rats. Rats maintained on vitamin E-supplemented diets and exposed to 1568
o
MS/"1 (0-8 ppm) of 0- continuously for 7 days also had changes in 6-phospho-
3
gluconate dehydrogenase activity. Rats were exposed to 1372 to 31,360 jjg/m
(0.7 to 16 ppm) of 03 while being fed diets containing ascorbic acid, dl-
methionine, and butylated hydroxytoluene (Fletcher and Tappel, 1973). This
combination was supposed to be a more potent antioxidant mixture than vitamin
E alone. Animals fed diets with the highest level of this antioxidant mixture
9-82
-------
had the greatest survival rate. Animals fed ortocopherol (vitamin E) in the
range of 10 to 150 mg/kg of diet had a survival rate slightly lower than those
fed the combination of antioxidants.
Donovan et al. (1977) fed mice 0 (deficient diet), 10.5 (minimal diet),
or 105 (supplemental diet) mg/kg of vitamin E acetate. The diet was also
altered to increase the peroxidilability of the lung by feeding either low or
high polyunsaturated fats (PUFA). Mice were continuously exposed to 1960
3
|jg/m (1 ppm) of 0^. The mortality (LT50 of 29 to 32 days) was the same,
regardless of the large differences in peroxidizability of the lungs of animals
fed high- or low-PUFA diets. High supplemental levels (105 mg/kg) of vitamin E
acetate were protective and delayed the LT50 to 03 by an average of 15 days.
Although these experiments demonstrate clearly the protective effect of vitamin
E against Og toxicity, they do not support the hypothesis that changes in
fatty acid composition of the lung will increase Oj toxicity. The results
could be interpreted to indicate that the scavenging of radicals by vitamin E
is more important than the relative rate of oxidation of PUFA. These findings
led to biochemical studies that used graded levels of dietary vitamin E.
Plopper et al. (1979) correlated biochemical and morphological (Section
9.3.1) effects in rats maintained on a synthetic diet with 11 mg kg/vitamin E
(equivalent to the average U.S. adult intake) or commercial rat chow having 66
mg/kg vitamin E. The 11 mg/kg vitamin E group was exposed continuously for 7
3
days to 196 or 392 M0/m (0.1 or 0.2 ppm) of 0.,, and measurements were made at
the end of exposure. The rats on the commercial diet were exposed to only the
higher concentration. All exposures increased activities of GSH peroxidase,
GSH reductase, and G-6-PD, and the amount of nonprotein sulfhydryl. Although
statistical comparisons between the dietary groups were not made, greater
increases appear to have occurred in the 11 mg/kg vitamin E group; the magnitude
3
of the responses in the higher vitamin E group at 392 |jg/m (0.2 ppm) of 0™
was roughly equivalent to the magnitude of the responses of the low vitamin E
3
group exposed to 196 (jg/m (0.1 ppm).. The 2 dietary groups showed little
variation in morphological effects.
These studies were expanded to include three vitamin E dietary groups:
3
0, 11, or 110 ppm (Chow et al., 1981). Rats were exposed to 196 yg/m (0.1
ppm) of 03 continuously for 7 days. In the 0-ppm vitamin E group, 03 increased
the level of GSH and the activities of GSH peroxidase, GSH reductase, and
G-6-PD. Increases of similar magnitude occurred in the 11-ppm vitamin E
group, with the exception of GSH reductase activity, which was not affected.
9-83
-------
In the highest vitamin E group, no significant effects were observed. Ozone
caused no changes in the activity of malic dehydrogenase in any of the dietary
groups. Morphologically (Section 9.3.1}, only 1 of 6 rats of the 110-ppm
vitamin E group had lesions, whereas more rats of the two other groups had
lesions. These lesions became more severe as the vitamin E level decreased.
They occurred at the bronchio-alveolar junction and were characterized by
disarrangement of the bronchiolar epithelium and an increase in the number of
alveolar macrophages.
3
Chow and Tappel (1972) exposed rats continuously to 1372 ug/m (0.7 ppm)
of 0., for 5 days. The animals had been maintained on diets with different
levels of dl-crtocopherol acetate (vitamin E) (0, 10.5, 45, 150, and 1500
mg/kg diet). Ozone exposure increased GSH peroxidase,'GSH reductase, and
G-6-PD activities. For GSH peroxidase and G-6-PD activities, the increase was
reduced as a function of the logarithmic concentration of vitamin E. Vitamin
E did not alter the magnitude of the effect on GSH reductase, a finding in
contrast to the results of others (Chow et al., 1981; Plopper et al., 1979).
Malonaldehyde, which is produced by lipid peroxidation, increased; this increase
was also partially inhibited as a logarithmic function of vitamin E concentra-
tion. However, others (DeLucia et al.s 1972; Mustafa et al., 1973) have not
observed the presence of malonaldehyde in exposed lungs. The increase in
malonaldehyde and activity of GSH peroxidase were linearly correlated, leading
Chow and Tappel (1972) to propose a compensatory mechanism in which the in-
crease in GSH peroxidase activity increases lipid peroxide catabolism.
Chow and Tappel (1973) observed the typical protection of vitamin E (0
3
and 45 mg/kg diet crtocopherol) from the effect of 03 (1568 ug/m > 0.8 ppm; 7
days, continuous) on increasing G-6-PD activity in rat lungs. Similar findings
occurred for 6-phosphogluconate dehydrogenase and malic enzyme activities.
The activities of two glycolytic regulating enzymes, phosphofructokinase and
pyruvate kinase, were increased by 0~ exposure but were not influenced by
vitamin E levels in the diet. Aldolase and malate dehydrogenase activities
were not affected.
Elsayed et al. (1982b, 1983) examined the influence of selenium (Se) in
the diet on GSH peroxidase activity in the lung. Selenium is an integral part
of one form of the enzyme GSH peroxidase. Mice were raised on a diet contain-
ing 55 ppm vitamin E with either 0 ppm or 1 ppm of Se and exposed to 1568 ± 98
3
fjg/fli (0.8 ppm) of 0- continuously for 5 days. In these mice, Se deficiency
9-84
-------
caused a sevenfold decline in Se level and a threefold decline in GSH peroxi-
dase activity in the lung. Other enzyme activities (e.g., GSH reductase,
G-6-PD, 6-phosphogluconate dehydrogenase) were not affected by dietary Se.
After 03 exposure, the GSH peroxidase activity in the Se-deficient group
remained unstimulated and was associated with a lack of stimulation of GSH
reductase, G-6-PD, and 6-phosphogluconate dehydrogenase activities. In con-
trast, the 0--exposed Se-supplemented group exhibited increases in 6-phosphoglu-
conate dehydrogenase and G-6-PD activities. Dietary deficiency or supplementa-
tion of Se, vis-a-vis alteration of GSH peroxidase activity, did not appear to
influence the effects of Q3 exposure as assessed by other parameters. Although
the animals received the same level of dietary vitamin E, after air or 0«
exposure, the Se-deficient group showed a two-fold increase in lung vitamin E
levels relative to the Se-supplemented group, suggesting a complementary
relationship between Se and vitamin E in the lung. This sparing action between
Se (i.e., GSH peroxidase activity) and vitamin E might explain similar effects
of 03 exposure in Se-deficient and supplemented mice.
Several investigators have studied the responsiveness of different species
to the effect of 0- on antioxidant metabolism. DeLucia et al. (1975a) exposed
3
both Rhesus monkeys and rats for 7 days (8 hr/day) to 1568 ug/m (0.8 ppm).
The nonprotein sulfhydryl and GSH content were increased, as was G-6-PD activity.
Activity of GSH reductase was affected in the rats but not the monkeys. No
statistical comparisons were made between the rats and monkeys. In the only
parameter for which sufficient data were presented for comparison, G-6-PD, the
increase in monkeys was about 125 percent of controls; for rats, it was about
130 percent of controls.
Rats and Rhesus monkeys were compared more extensively by Chow et al.
3
(1975). Animals were exposed to 980 jjg/m (0.5 ppm) of 03 8 hr/day for 7
days. The nonprotein sulfhydryl content and the activities of GSH peroxidase,
GSH reductase, and G-6-PD increased in rats but not in monkeys. The magnitude
of the increases in rats was 20 to 26 percent. The increases in monkeys were
between 10 and 15 percent and statistically insignificant, "because of relative-
ly large variations," according to the authors. The variation in the monkeys
was approximately double that of the rats. The sample size of the monkeys (6)
was lower than that of the rats (8). Statistical tests of the Type II error
(e.g., false negative error) rates were not reported. Thus, the monkeys
apparently were not affected to the same degree as the rats. However, the
experiments with monkeys were apparently not conducted with as much statistical
9-85
-------
power as those with rats. Thus, under the actual study designs used, ozone
would have had to have substantially greater effects on monkeys than rats for
a statistically significant effect to be detected. This did not happen,
leading to the conclusion of the investigators that monkeys are not more
responsive. Studies of improved experimental design would indicate more
definitively whether monkeys are less responsive. Mustafa and Lee (1976) also
alluded to different G-6-PD responses of rats and Bonnet and Rhesus monkeys
Q
after exposures for 8 hr/day for 7 days to levels as low as 392 Mfl/m
(0.2 ppm). However, no data for G-6-PD were presented, and the description of
these results was incomplete.
Mice (Swiss Webster) and 3 strains of rats (Sprague-Dawley, Wistar, and
3
Long Evans) were-compared after a 5-day continuous exposure to 882 pg/m (0.45
ppm) of OQ (Mustafa et a!., 1982). Total sulfhydryl content increased only in
mice. However, nonprotein sulfhydryl content increased in both rats and mice
to a roughly equivalent degree. GSH-S-transferase was not affected in any of
the animals. Mice exhibited the typical increases in the activities of GSH
peroxidase, GSH reductase, G-6-PD, 6-phosphogluconate dehydrogenase, and
isocitrate dehydrogenase. Rats were less affected; no changes were seen in
the activities of GSH reductase or isocitrate dehydrogenase, and not all
strains of rats showed an increase in the activities of GSH peroxidase, G-6-PD,
and 6-phosphogluconate dehydrogenase. For GSH reductase and G-6-PD, the
increased activities in exposed mice were significantly greater than those
in exposed rats.
At present, it is not possible to determine whether these apparent species
differences in responsiveness were due to differences in the total deposited
dose of Og, an innate difference in species sensitivity, or differences in
experimental design (e.g., small sample sizes, insufficient concentration-
response studies).
Age-dependent responsiveness to Q-Hnduced changes in GSH systems has
been observed. Tyson et al. (1982) exposed rats (5 to 180 days old) to 1764
a
ug/m (0.9 ppm) of 03 continuously for 96 hr, except for suckling neonates (5
to 20 days old) which received an intermittent exposure (4 hr of exposure, 1.5
hr no exposure, 4 hr exposure). Given that others (Mustafa and Lee, 1976;
Chow et al., 1974; Schwartz et al., 1976) have observed no differences between
continuous and intermittent exposures for these enzymatic activities, this
difference in regimen can be considered inconsequential. All ages given are
ages at the time of initiation of exposure. They were calculated from those
9-86
-------
given in the report to facilitate comparisons with other reports on age sensi-
tivity. Weanlings (25 and 35 days old) and nursing dams (57 and 87 days old)
had higher lung to body weight ratios. Generally, the DNA content of the
younger animals was unchanged. When the activities of G-6-PD, GSH reductase,
and GSH peroxidase were measured after 0^ exposure, the trend was a decrease
in activities at and before 18 days of age, followed by increases thereafter.
For G-6-PD, this trend was most pronounced; at 5 and 10 days of age, no signi-
ficant changes were seen; at 15 days of age, a decrease was seen; and at 25
and 35 days of age, progressively greater increases were seen.
3
Ten- to 50-day-old rats were exposed continuously to 1764 |jg/m (0.9 ppm)
of 0- (Lunan et al», 1977). Ages of rats reported are presumably ages at ini-
tiation of exposure. In rats (10 to 40 days old) exposed for 3 days, G-6-PD
activity was measured periodically during exposure. No statistical analyses
were reported. Ozone caused a possible increase (20 percent of control) in
the activity of G-6-PD in the 20-day-old group, and the magnitude continued to
increase as age increased up to about 35 days (~ 75 percent of control), after
which (40 and 50 days of age) the effect became less (40 percent of control at
50 days of age). In another experiment, a complex design was used in which
rats at 10, 15, 25, and 32 days of age were exposed up to 32 to 34 days of
age; thus, the duration of exposure for each group was different. When the
animals were younger than 20 days, .no effect was observed. When older mice
were used, the greatest magnitude of the increased activity occurred at about
32 days of age, regardless of the absolute length of exposure.
•2
Elsayed et al. (1982a) exposed rats of various ages to 1568 \ig/m (0.8
ppm) of 0~ continuously for 72 hr. Ages given are those at initiation of
exposure. Ozone increased lung weights, total lung protein, and total lung
DNA in an age-dependent fashion, with the older (90-day-old) rats being more
affected than 24-day-old animals. For isocitrate dehydrogenase, activity, no
effect was seen in the 24-day-old rats,, but an increase was observed in the
90-day-old animals. For G-6-PD and 6-phosphogluconate dehydrogenase, increases
were observed in the 24- and 90-day-old rats, with a greater magnitude of the
effect occurring in the 90-day-old group. Younger rats (7 to 18 days old)
were also examined. The exposure caused >60 percent mortality to the 7- and
12-day-old rats. Glucose-6-phosphate dehydrogenase activity decreased in both
the 7-and-12-day old groups, with the younger rats being more affected. The
18-day-old rats had an increase in this activity. These trends were similar
9-87
-------
to those observed by Tyson et al. (1982), although the exact age for signifi-
cant changes differed slightly.
The reason for these age-dependent changes is not known. The younger
animals (< 24 days of age} have lower basal levels of the studied enzymes than
the older animals examined (> 38 days of age) (Tyson et al., 1982; Elsayed et
al., 1982a). It is conceivable that age influenced the dosimetry of 03. The
decreased activities observed in the neonates are reminiscent of the decreased
activities that occur at higher 03 levels in adults (DeLucia et al., 1972,
1975a; Fukase et al., 1975). The increased activities in the later stages of
weanlings or in young, growing adults is consistent with the effects observed
in other studies of adult rats (Table 9-5).
Mustafa et al. (1984) are the only researchers to report the effects of
combined exposures to 0- and nitrogen dioxide on the GSH peroxidase system.
q
Mice were exposed 8 hr/day for 7 days to either 9024 ug/m (4.8 ppm) of nitro-
3
gen dioxide, or 882 ug/m (0.45 ppm) of 0~, or a mixture of these. Ozone and
nitrogen dioxide alone caused no significant effects on most of the endpoints;
however, synergistic effects were observed in the mixture group. The total
sulfhydryl and nonprotein sulfhydryl contents were increased. The activities
of GSH peroxidase, G-6-PD, 6-phosphogluconate dehydrogenase, and isocitrate
dehydrogenase increased, but the activities of GSH reductase and GSH S-trans-
ferase were unchanged. Tissue 0« utilization was also increased, as shown by
the increase in succinate oxidate and cytochrome c oxidase activities.
Superoxide dismutase (SOD) catalyzes the dismutation of (and therefore
destroys) superoxide (02~), a toxic oxidant species thought to be formed from
0-, exposure, and is thus involved in antioxidant metabolism. Rats exposed
3
continuously for 7 days to 1568 ug/m (0.8 ppm) of 0., exhibited an increased
activity of SOD in cytosolic and mitochondria! fractions of the lungs (Mustafa
et al., 1977). In a more complex exposure regimen in which rats were exposed
o
for 3 days to 1568 pg/m (0.8 ppm) and then various days of combinations of
2940 ug/m3 (1.5 ppm) and 5880 ug/m3 (3 ppm) of 03, SOD activity also increased.
Bhatnager et al. (1983) studied the time course of the increase in SOD activity
o
after continuous exposure of rats to 882 ug/m (0.45 ppm) of On. On day 2 of
exposure, there was no effect. On days 3 and 5, activity had increased; by
day 7 of exposure, values were not different from control.
9.3.3.3. Oxidative and Energy Metabolism. Mitochondrial enzyme activities
are typically studied to evaluate effects on 02 consumption, which is a funda-
mental parameter of cellular metabolism. Mitochondria are cellular organelles
9-88
-------
that are the major sites of Op utilization and energy production. Many of the
enzymes in mitochondria have functional sulfhydryl groups, which are known to
be affected by 0», and mitochondria! membranes have unsaturated fatty acids
that are also susceptible to CL. The patterns of Ck effects on Op consumption,
as will be discussed, are quite similar to effects on antioxidant metabolism
(Section 9.3.3.2).
Mustafa et al. (1973) showed in rats that acute (8-hr) exposure to a high
3
concentration of 0^ (3920 |jg/m , 2 ppm) decreases Op consumption using the
substrates succinate, a-oxoglutarate, and glycerol-1-phosphate. Similar
findings were made by DeLucia et al. (1975a). Decreases in mitochondria!
total sulfhydryl levels were also observed (Mustafa et al., 1973). Equivalent
changes occurred in whole-lung homogenate and the mitochondria! fraction. No
change in malonaldehyde levels was found. When rats were exposed to high 0-
3
levels (5880 |jg/m , 3 ppm; 4 hr), the immediate depression in succinate oxidase
activity was followed by an increase that peaked about 2 days postexposure and
returned to normal by 20 days postexposure (Mustafa et al., 1977). A 10- or
3
20-day continuous exposure to a lower 0., concentration (1568 |ag/m , 0.8 ppm)
caused an increase in Op consumption of lung homogenate which was greater at
20 days (Mustafa et al., 1973). When the activity of the mitochondrial fraction
per mg of protein was measured, the increased actfvity was less than that of
the lung homogenate per mg of protein. Morphological comparisons indicated
that the exposed lungs had a threefold increase in type 2 cells, which contain
more mitochondria than type 1 cells. Thus, the increase in Op consumption
appears to reflect changes in cell populations.
Schwartz et al. (1976) exposed rats for 7 days continuously or intermit-
tently (8 hr/day) to 392, 980, or 1568 (jg/m3 (0.2, 0.5, or 0.8 ppm) of 03.
Succinate oxidase activity increased linearly with 03 concentration. No major
differences were apparent between continuous and intermittent exposures. No
statistical analyses were reported. Concentration-dependent morphological
effects were also observed (Section 9.3.1). When using rats and an identical
exposure regimen, Mustafa and Lee (1976) found similar responses for succinate
oxidase and succinate-cytochrome ,c reductase activity. These increases were
statistically significant. Mustafa et al. (1973), when using 7 days of con-
tinuous exposure, also showed that Op consumption of rats increased with in-
creasing 03 level (392, 980, 1568 yg/m3, 0.2, 0.5, 0.8 ppm).
Although concentration appears to be a stronger determinant of the effect,
time of exposure also plays a role (Mustafa and Lee, 1976). Rats were exposed
9-89
-------
o
to 1568 ug/m (0.8 ppm) of 0, continuously for 30 days, and 0^ consumption was
measured as the activities of succinate oxidase, 2-oxoglutarate oxidase, and
glycerol-1-phosphate oxidase. On day I, the" effect was not significant.
However, at day 2 and following, these enzyme activities increased. The peak
increase occurred on day 4 and remained at that elevated level throughout the
30 days of exposure. Mitochondrial succinate-cytochrome c reductase exhibited
a similar pattern under similar 07 levels for 7 days of exposure. Equivalent
3
results occurred in rats during a 7-day continuous exposure to 1568 ug/m
(0.8 ppm) of 0. (DeLucia et a!., 1975a).
3
In rats, recovery from an ozone-induced (1568 ug/m , 0.8 ppm; 3 days,
continuous) increase in succinate oxidase activity occurred by 6 days post-
exposure (Chow et a!., 1976b). When the rats were re-exposed to the same
exposure regimen at 6, 13, and 27 days of recovery, the increased activity was
equivalent to that of the initial exposure. Thus, no long-lasting tolerance
was observed.
Dietary vitamin E can also reduce the effects of 0,, on 0« consumption.
3
After 7 days of continuous exposure to 196 or 392 ug/m (0.1 or 0.2 ppm) of
Og, the lung homogenates of rats maintained on diets with either 11 or 66 ppm
of vitamin E were examined for changes in 02 consumption (succinate oxidase
activity) (Mustafa, 1975; Mustafa and Lee, 1976). In the 11-ppm vitamin E
group, increases in 00 consumption occurred at both 0., levels. In the 66-ppm
3
vitamin E group, only 392 ug/m (0.2 ppm) of 03 caused an increase. Mitochon-
dria were isolated from the lungs and studied. Neither dietary group had
Og-induced changes in the respiratory rate of mitochondria (on a per mg of
protein basis). However, the amount of mitochondria (measured as total protein
content of the mitochondria! fraction of the lung) from the 0~-exposed rats of
the 11-ppm vitamin E group did increase (15-20 percent).
Similar to earlier discussions for antioxidant metabolism (Section 9.3.3.2),
responsiveness to effects of 0, on 09 consumption is age-related (Table 9-5).
3
Elsayed et al. (1982a) exposed rats of various ages to 1568 ug/m (0.8 ppm) of
0- continuously for 72 hr. The 0.,-induced increase in the activities of
succinate oxidase and cytochrome c oxidase increased with age (from 24 to 90
days of age), with no significant change at 24 days of age. When younger rats
were examined, succinate oxidase activity decreased in both the 7- and 12-day-
old animals, with the younger ones more affected. The 18-day-old rats had an
increase in this activity.
9-90
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Species and strain differences were observed after a 5-day continuous
exposure to 882 ug/m (0.45 ppm) of 03 (Mustafa et al., 1982). Mice and
Sprague Dawley rats (but not other strains of rats) had an increase in activi-
ty of succinate oxidase. Cytochrome c oxidase was increased in mice, Long-
Evans rats, and Sprague Dawley rats, but not Wistar rats; the increase in the
mice was greater than that in the rats.
Rats have also been compared to two strains of monkeys after a 7-day (8
hr/day) exposure to various concentrations of 0., (Mustafa and Lee, 1976).
o
Rats were exposed to 392, 980, or 1568 M9/m (0.2, 0.5, or 0.8 ppm) of 03 and
exhibited increases in succinate oxidase activity. Rhesus monkeys exposed to
o
either 980 or 1568 ug/m (0.5 or 0.8 ppm) of 03 had an increase in this enzyme
activity only at the higher exposure concentration. When Bonnet monkeys were
exposed to 392, 686, or 980 ug/m (0.2, 0.35, or 0.5 ppm) of 03, succinate
oxidase activity increased at the two higher 0« levels. The number of animals
3
used was not specified, which makes interpretation difficult. At the 392-ug/m
(0.2 ppm) level, the increase in rats was to 118 percent of controls (signifi-
cant); in Bonnet monkeys, it was to 113 percent of controls (not significant).
2
At the 980-(jg/m (0.5 ppm) of 03 level, the magnitude of the significant
increases was not different between rats (133 percent of controls) and Bonnet
monkeys (130 percent of controls). Rhesus monkeys may have been slightly less
responsive than rats, but no statistical analyses were performed to assess
this question.
The increase in levels of nonprotein sulfhydryls, antioxidant enzymes,
and enzymes involved in 02 consumption is typically attributed to concurrent
morphological changes (Section 9.3.1) in the lungs, principally the loss of
type 1 cells and the increase of type 2 cells and the infiltration of alveolar
macrophages. Several investigators have made such correlated observations in
rats (Plopper et al., 1979; Chow, et a!., 1981; Schwartz et a!., 1976; DeLucia
et a!., 1975a). Type 2 cells are more metabolically active than type 1 cells
and have more abundant mitochondria and endoplasmic reticula. This hypothesis
is supported by the findings of Mustafa et al. (1973), Mustafa (1975), and
DeLucia et al. (1975a). For example, succinate oxidase was studied in both
lung homogenates and isolated mitochondria of rats after a 7-day exposure to
1568 ug/m (0.8 ppm) of 03 (DeLucia et al., 1975a). The increase in the homo-
genate was about double that of the isolated mitochondria (on a per mg of
protein basis). As mentioned previously, this indicates that an increase in
9-91
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the number of mitochondria, rather than an increased activity within a given
mitochondrion, in exposed lungs is the probable dominant cause.
9.3.3.4 Honooxygenases. Multiple microsomal enzymes function in the metabo-
lism of both endogenous (e.g., biogenic amines, hormones) and exogenous (xeno-
biotic) substances. These substrates are either activated or detoxified,
depending on the substrate and the enzyme. Only a few of the enzymes have
been studied subsequent to G"3 exposure (Table 9-6).
Monoamine oxidase (MAO) activity has been investigated (Mustafa et a!.,
1977) in view of its importance in catalyzing the metabolic degradation of
bioactive amines like 5-hydroxytryptamine and norepinephrine. Although MAO
activity is located principally in the mitochondria, it also is found in
microsomes. Activity levels of MAO in rats were determined after exposure to
3 3
3920 ug/m (2 ppm) for 8 hr or 1568 ug/m (0.8 ppm) continuously for 7 days.
Substrates used included n-amylamine, benzylamine, tyramine, and 3-hydroxyty-
ramine; three tissue preparations were used (whole lung homogenate, mitochon-
dria, and microsomes). The acute high-level exposure reduced MAO activity in
2
all tissue preparations. The longer exposure to 1568 ug/m (0.8 ppm) increased
MAO activity in all tissue preparations. This pattern is similar to that
found for mitochondria! enzymes and antioxidant metabolism (Sections 9.3.3.2
and 9.3.3.3).
The cytochrome P-450-dependent enzymes have been studied because of their
function in drug and carcinogen metabolism. Palmer et al. (1971S 1972) found
Q
that hamsters exposed to 1,470 ug/m (0.75 ppm) of 0, for 3 hr had lower
benzo(a)pyrene hydroxylase activity in the lung. Goldstein et al. (1975)
q
showed that rabbits exposed to 1,960 ug/m (1 ppm) of 03 for 90 min had
decreased levels of lung cytochrome P-450. Maximal decreases occurred 3.6 days
following exposure. Recovery to control values occurred somewhere between 8
days and 45 days. Cytochrome P-450-mediated activity of benzphetamine N-
3
demethylase in the lung was lowered by a 24-hr exposure to 1,960 ug/m (1 ppm)
of 0, in rats (Montgomery and Niewoehner, 1979). The cytochrome P-450 dependent
activity began to recover by 4 days postexposure but was still decreased.
Complete recovery occurred by I we.ek. Cytochrome bn-mediated lipid desaturation
was stimulated by Og 4, 7, and 14 days postexposure. Immediately after expos-
ure, the desaturase activity was quite depressed, but this was attributed to
anorexia in the rats, and not to 03. Cytochrome P-450-dependent enzymes exist
in multiple forms, because they have different substrate affinities that
overlap. Measuring activity with only one substrate does not characterize a
9-92
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TABLE 9-6. MQNQOXYGENASES
Ozone
concentration
ug/«a
392
980
1568
1470
5880
19,600
1470
5880
19,600
1568
3920
1568
3920
392
1568
1568
1568
1568
ppni
0.2
0.5
0.8
0.75
3.0
10.0
0.75
3.0
10.0
0.8
2.0
0.8
2.0
0.2
0.5
0.8
0.8
0.8
b Exposure
Measurement ' duration and
method protocol
MAST Continuous or
N8KI 8 hr/day for 7
days
I 3 hr
I 3 hr
NO Continuous for
7 days
8 hr
I Continuous
for 7 days
8 hr
I Continuous or
8 hr/day
8 hr/day
I Continuous for
7 days
Observed effect(s) Species
Concentration-related 'linear Rat
increase in NADPH cytochrome c
reductase activity. No
difference between continuous
and intermittent.
Decreased activity of benzpyrene Hamster
hydroxylase in lung parenchyma.
Decreased activity of benzpyrene Rabbit
hydroxylase in tracheobronchial
inucosae.
High 03 level reduced monoamine Rat
oxidase activity; low 03 level
increased it.
High 03 level decreased activity Rat
of NADPH cytochrome c reductase;
low level increased it.
Increased activity of NADPH cyto- Rat
chrome c reductase. At 0.8 ppm,
increase began at day 2 of exposure.
No change in NADPH cytochrome c Monkey
reductase activity.
Increased activity of NADPH cyto- Rat
chrome c reductase on days 2 through
7. Maximal increase on day 4.
Reference
Mustafa and Lee,
1976; Schwartz
et al. , 1976;
Mustafa et al . ,
1977
Palmer et al., 1971
Palmer et al., 1972
Mustafa et al. ,
1977
DeLucia et al. ,
1972, 1975a
DeLucia et al. ,
1975a
Mustafa and Lee,
1976
-------
TABLE 9-6. MONOOXYGENASES (continued)
i
UD
Ozone
concentration
ug/nr1 ppm
1960 1. 0
1960 1. 0
5880 3
b Exposure
Measurement ' duration and
method protocol
NO 90 min
MAST 24 hr
NBKI 10 min before
lung perfusion
and continuous
throughout
experiment.
Observed effect(s) Species
Decreased levels of lung cyto- Rabbit
chrome P-450. Maximal decrease
at 3.6 days postexposure.
50% decrease in benzphetamine Rat
N-demethylase activity 1 day
postexposure; return to control
levels by 1 wk postexposure.
Stimulation of cytochrome
bs-mediated lipid desaturation.
Decreased enzymatic conversion of Rat
arachidonic acid to prostaglandins
when using isolated ventilated per-
fused lung.
Reference
Goldstein et al. ,
1975
Montgomery and
Niewoehner, 1979
Menzel et al . , 1976
Measurement method:
MAST = Kl-coulometric (Mast meter); CHEM = gas solid chemiluminescence; NBKI = neutral buffered potassium iodide;
I = lodometric; ND = not described.
Calibration method: UKI = unbuffered potassium iodide.
-------
single enzyme. More importantly, the relatively long time for recovery suggests
that cell injury, rather than enzyme destruction, has occurred, Benzo(a)pyrene
hydroxylase is the first major enzyme in the activation of benzo(a)pyrene and
several other polycyclic hydrocarbons to an active carcinogen. However,
additional enzymes not studied after 0- exposure are involved in the activa-
tion, which makes full interpretation of the effect of 0, on this metabolism
impossible. The impact of the decrease in cytochrome P-45Q depends on the
activation or detoxification of the metabolized compound by this system.
Also involved in mixed function oxidase metabolism is NADPH cytochrome c
reductase. As with other classes of enzymes (Sections 9.3.3.2; 9.3.3.3),
3
acute exposure to a high 03 level (3920 ug/m , 2 ppm; 8 hr) reduced NADPH
cytochrome c reductase activity (DeLucia et a!., 1972, 1975a). After a con-
tinuous or 8 hr/day exposure of rats for 7 days, the activity of NADPH cyto-
chrome c reductase increased linearly in a concentration-related fashion (392,
980, and 1568 ug/m3; 0.2, 0.5, and 0.8 ppm) (Mustafa and Lee, 1976; Schwartz
et al., 1976; Mustafa et al.s 1977). Continuous and intermittent exposures
2
were not different. The time course of the response to 1568 |jg/m (0.8 ppm)
was an increase in activity that began at day 2, peaked at day 4, and was
still increased at day 7 of continuous exposure in the rat (Mustafa and Lee,
1976). The rat, but not the Rhesus monkey, is apparently affected after
o
exposure for 8 hr/day for 7 days to 1568 ug/m (0.8 ppm) of 03 (DeLucia et al.,
1975a). However, monkey data were not reported in any detail.
9.3.3.5 Lactate Dehydrogenase and Lysosomal Enzymes. Lactate dehydrogenase
(LDH) and lysosomal enzymes are frequently used as markers of cellular damage
if levels are observed to increase in lung lavage or serum/plasma, because
these enzymes are released by cells upon certain types of damage. Effects of
0- on these enzymes are described in Table 9-7. No lung lavage studies have
been reported; whole-lung homogenates were used. Therefore, it is not possible
to determine whether the observed increases reflect a leakage into lung fluids
and a compensatory resynthesis in tissue or cellular changes (See Section
9.3.1), such as an increase in type 2 cells and alveolar macrophages and poly-
morphonuclear leukocytes rich in lysosomal hydrolases. In some instances, cor-
relation with plasma values was sought. They are described briefly here; more
detail is given in Section 9.4.3.
Lactate dehydrogenase is an intracellular enzyme that consists of two
subunits combined as a tetramer. Various combinations of the two basic subunits
change the electrophoretic pattern of LDH so that its various isoenzymes can
9-95
-------
TABLE 9-7. LACTATE DEHYDRQGEHASE AND LYSQS0HAL EHZYHES
vo
Ozone
concentration
{JgTP ppl
196
392
980
1568
980
1568
1568
1372
1568
1372-
1568
1568
1568
0.1
0.2
0.5
0.8
0.5
0.8
0.8
0.7
0.8
0.7-
0.8
0.8
0.8
h Exposure
Heasurenent ' duration and
nethod protocol
NBKI
HAST,
NBKI
NBKI
NBKI
MAST,
NBKI
HAST,
NBKI
HAST,
NBKI
Continuous
for 7 days
Continuous
for 8 days or
8 hr/days for
7 days
8 hr/day for
7 days
Continuous
for 5 days
Continuous
for 7 days
Continuous
for 7 days
Continuous
for 7 days
Continuous
for 7 days
Observed effect(s) Species
Increase in total LDH activity 1n Rat
diet group receiving 0 ppm
vitamin E. Groups with 11 or
110 ppu of vitamin E had no effect.
Increased lung lysozytne activity only Rat
after continuous exposure to 0,8 ppn.
Increased LDH activity in lungs. Rat
Change in LDH isoenzyme distri-
bution at 0.8 ppffl.
No change in total LDH activity or Monkey
isoenzyne pattern in lungs.
Specific activities of various Rat
lysosomal hydrolases increased.
Increase in lung acid phosphatase Rat
activity; no observed increases
in B-glucuronidase activity.
Bronchiolar epithelium had decreased Rat
NADH and NADPH activities and In-
creased ATPase activity.
Increase in LDH activity not Rat
affected by vitamin E (0 or
45 mg/kg diet).
Reference
Chow et al . ,
Chow et al . ,
Chow et al . ,
Dillard et al
1972
Castleman et
1973a
Castleman et
1973b
1981
1974
1977
* 1
al.,
al.,
Chow and Tappel ,
1973
Measurement method:
Calibration method:
MAST = Kl-couloiiietric (Mast meter); NBKI = neutral buffered potassium iodide.
NBKI = neutral buffered potassium iodide.
-------
be detected. Chow and Tappel (1973) found an increase in LDH activity in the
3
homogenate of rat lungs exposed to 1568 ug/m (0.8 ppm) of 0, continuously for
3
7 days. Lower levels (196 [jg/m , 0.1 ppm; 7 days continuous) only increased
LDH activity of lung homogenate when rats were on a diet deficient in vitamin" E
(Chow et al., 1981). Vitamin E levels in the diet did not significantly in-
fluence the response. In following up this finding, Chow et al. (1977) studied
LDH activity and isoenzyme pattern (relative ratios of different LDH isoenzymes)
3
in the lungs, plasma, and erythrocytes of (L-exposed rats (980 or 1,568 pg/m ,
3
0.5 or 0.8 ppm) and monkeys (1568 M9/m > 0.8 ppm). Exposure was for 8 hr/day
for 7 days. In monkeys, no significant changes in either total LDH activity or
isoenzyme pattern in lungs, plasma, or erythrocytes were detected. The total
LDH activity in the lungs of rats was increased after exposure to 1,568 or
o
980 ug/m (0.8 or 0.5 ppm), but no changes in the plasma or erythrocytes were
detected. The isoenzyme pattern of LDH following 0- exposure was more com-
plex, with the LDH-5 fraction significantly decreased in lungs and plasma of
3
rats exposed to 1,568 ug/m (0.8 ppm). The LDH-4 fraction in lungs and plasma
and the LDH-3 fraction in lungs were increased. No changes were discernible
3
in rats exposed to ,980 ug/m (0.5 ppm) of 0-. The changes in LDH isoenzyme
pattern appeared to be due to a relative increase in the LDH isoenzymes contain-
ing the H (heart type) subunits. Although the increase in LDH suggests cyto-
toxicity after 0- exposure, no clear-cut interpretation can be placed on the
importance of the isoenzyme pattern. Some specific cell types in the lung may
contain more H-type LDH than others and be damaged by 0- exposure. Further
studies of the fundamental distribution of LDH in lung cell types are needed
to clarify this point.
Lysosomal enzymes have been found to increase in the lungs of animals
exposed to 0~ at concentrations of 1,372 ± 294 ug/m3 (0.70 ± 0.15 ppm) for 5
3
days and 1,548 ± 274 |jg/m (0.79 ± 0.14 ppm) for 7 days, whether detected by
biochemical (whole-lung homogenates and fractions) or histochemical means
(Dillard et al., 1972). Dietary vitamin E (0 to 1500 mg/kg diet) did not in-
fluence the effects. These increased activities were attributed to the infil-
tration of the lung by phagocytic cells during the inflammatory response phase
from 0~ exposure. Similarly, Castleman et al. (1973a,b) found that activity
of lung acid phosphatase was increased in young rats that had been exposed to
3
1,372 to 1,568 ug/m (0.7 to 0.8 ppm) of 03 continuously for 7 days. Increases
in p-glucuronidase activity were not observed. The histochemical and cytochem-
ical localization suggested that 03 exposure results in damage to the lung's
9-97
-------
lysosomal membranes. Castleman et al. (1973b) also found that the bronchiolar
epithelium in infiltrated areas had lower NADPH- and NADH diaphorase activ-
ities and higher ATPase activities than similar epithelium of control lungs.
They discussed in greater detail the enzymatic distribution within the lung
and suggested that some of the pyridine nucleotide-dependent reactions could
represent an enzymatic protective mechanism operating locally in the centri-
acinar regions of 03-exposed lungs. Chow et al. (1974) also observed an
increase in lysozyme activity (lung homogenate) in rats exposed continuously
3
to 1568 pg/m (0.8 ppm) for 8 days but not in rats exposed intermittently (8
hr/day, 7 days). No effect was seen in continuous or intermittent exposure of
•3
rats to 392 or 980 pg/m (0.2 or 0.5 ppm) of Oo.
Lysosomal acid hydrolases include enzymes that digest protein and can
initiate emphysema. The contribution of the increases in these enzymes obser-
ved by some after 0, exposure to morphological changes has not been demon-
strated.
9.3.3.6 Protein Synthesis. The effects of 0, on protein synthesis can be
divided into two general areas: (1) the effects on the synthesis of collagen
and related structural connective tissue proteins, and (2) the effects on the
synthesis or secretion of mucus. The studies are summarized in Table 9-8.
Hesterberg and Last (1981) found that increased collagen synthesis caused
o
by continuous exposure to 03 at 1568, 2352, and 2940 ug/m (0.8, 1.2, and 1.5
ppm) for 7 days could be inhibited by concurrent treatment with methylpred-
nisol one (1 to 50 mg/kg/day).
Hussain et al. (1976a,b) showed that lung prolyl hydroxylase activity and
3
hydroxyproline content increased on exposure of rats to 980 and 1568 ug/m
3
(0.5 and 0.8 ppm) of 0™ for 7 days. At 392 ug/m (0.2 ppm), 03 produced a
statistically insignificant increase in prolyl hydroxylase activity. Prolyl
hydroxylase is the enzyme that catalyzes the conversion of proline to hydroxy-
proline in collagen. This conversion is essential for collagen to form the
fibrous conformation necessary for its structural function. Hydroxyproline is
3
an indirect measure of collagen content. When rats were exposed to 980 ug/m
(0.5 ppm) of 0,, for 30 days, the augmentation of activity seen earlier at
£ -C?
7 days of exposure had diminished, and by 60 days, the enzyme activity was
within the normal range despite continued 0» exposure. When rats were exposed
3
to 1568 ug/m (0.8 ppm) of 03> the prolyl hydroxylase activity continued to
rise for about 7 days; hydroxyproline content of the lung rose to a maximum
value at about 3 days after exposure began and remained equivalently elevated
9-98
-------
TABLE 9-8. EFFECTS OF OZONE ON LUNG PROTEIN SYNTHESIS
vo
Ozone
concentration
pgTi3
392
784
1176
1568
980
392
980
1568
392
784
1176
1568
392
1568
3920
882
1568
980-
3920
ppm
0.2
0,4
0.6
0.8
0.5
0.2
0.5
0.8
0.2
0.4
0.6
0.8
0.2
0.8
2.0
0.45
0.8
0.5-
2
b Exposure
Measurement ' duration and
method protocol
ND 8 hr/day for
3 days
Continuous for
1 through 90 days
Continuous for
3 or 14 days and
combined with
H2S04
MAST Continuous for
7 days
UV 8 hr/day for
1 to 90 days
UV, 6 hr/day, 5 days/
NBKI wk, 12.4 wk (62
days of exposure)
ND Conti nuous
for 7 days
NO Continuous
for 90 days
UV 1, 2, or 3 wk
Observed effect(s) Species
Decreased rate of glycoprotein secretion by Rat
trachea] explants at 0.6 ppm.
Decreased rate of glycoprotein secretion.
Increased rate of glycoprotein secretion.
Concentration-dependent increase in lung prolyl Rat
hydroxylase activity. No effect at 0.2 ppm. Meta-
bolic adaptation suggested at 980 ng/m8 (0.5 ppm)
At 0.8 ppm, collagen and noncollagenous protein
synthesis increased; effect on prolyl hydroxylase
returned to normal by about 10 days postexposure,
but hydroxyproline was still increased at 28 days.
At 0.8 ppm, tracheal explants had decreased rate Rat
of glycoprotein secretion for up to 1 wk, followed
by increased rate up to 12 wks. Three day exposure
to three lower concentrations caused decrease at
only 0.6 ppm.
Decrease in collagen and elastin at 0.2 and Rat
and 0.8 ppm; increase at 2 ppm.
Increased collagen synthesis at 5 and 7, but Mouse
not 2 days of exposure. Similar pattern
for increase in superoxide dismutase activity.
Increased prolyl hydroxylase activity at 2, 3,
5, and 7 days of exposure; maximal effect
at day 5.
Increase in prolyl hydroxylase activity through Rat
7 days. No effect 20 days and beyond.
Increased rate of collagen synthesis; fibrosis Rat
of alveolar duct walls; linear concentration
response.
Reference
Last and Kaizu,
1980; Last and
Cross, 1978
H us sain et al. ,
1976a,b
Last et al., 1977
Costa et al., 1983
Bhatnagar et al . ,
1983
Last et al., 1979
-------
TABLE 9-8. EFFECTS Of OZONE ON LUNG PROTEIN SYNTHESIS (continued)
Ozone
concentration Measurement '
ug/»3 pjS nethod
vo
H
O
O
980
980-
2940
5000
980
1000
1254
5000
1882
5000
1254
1254
1882
0.5 W
0.5- NBKI
1.5
(NH4)2S04
0.5 NBKI
H2S04
0.64 . W
0.96 OM
(NH4)2S04
0.64 (N
0.64 UM
0.96
. Exposure
duration and
protocol
Continuous for
up to 180 days
Continuous for
7 days
Continuous for
3 to 50 days
Continuous
for 3 days
Continuous for
7 to 14 days
8 hr/day,
361 days
Continuous for
90 days; inter-
Observed effect(s)
Increase 1n protein and hydroxyproline content of
lungs. No change 2 no postexposure.
03 caused linear, concentration-related increases
in collagen synthesis; (HH4)2S04 combined with
03 Increased collagen synthesis rates by 180% at
1.2 and 1.5 ppn 03.
03 increased collagen synthesis at 3, 30, and 50
days; H2S04 combined with 03 increased collagen
synthesis rates by 220%.
No significant effect of 03 or (NH4)2S04 on
collagen synthesis; (NH4)2 S04 + 03 increased
collagen synthesis rates by 230%.
Interstitial edema and inflammation of proximal
alveolar ducts; (NH,t)2 S04 increased the severity
of OB effects at lesion sites without Increasing
the number of lesions.
Increase in collagen content
Equivalent increase in collagen in all but the
0.64 ppn continuous group which only had a
Species Reference
Rat Last and Greenberg,
1980
Rat Last et al., 1983
Rat Last et al., 1984a
Monkey Last et al . , 1984b
Rat Last et al., 1984b
(young
aiittent units of
5 days (8 hr/
day) of 03, and
9 days of air,
repeated 7 times
with i total of
35 exposure
days over a 90-day
Interval
•arginal (p <0.1) increase.
adult)
-------
TABLE 9-8. EFFECTS OF OZONE ON LUNG PROTEIN SYNTHESIS (continued)
Ozone
concentration
VD
H
O
H
|jg/m3
1254
1882
1882
2352
1882
1882
1254
1568
1568
1568
2352
2940
ppm
,64
,96
0.96
1.2
.96
.96
0.64
0,8
0.8
0.8
1.2
1.5
. Exposure
Measurement * duration and
method protocol
8 hr day
7 days/wk,
6 wk
Continuous
for 3 wk
Continuous for
1 wk, then
2 wk of air
Intermittent
units of 3 days
(8 hr/day) of 03,
and 4 days of air,
repeated 6 times
7 wk, 8 hr/day,
7 days/wk
UV Continuous
for 7 days
NO Continuous
for 7 days
NBKI Continuous
for 3 days
UV Continuous
for 7 days
Observed effect(s)
Increased collagen content at 0.96 ppm 03.
At 6 wk post-exposure, both 03 levels in-
creased collagen. Suggestion of progressive
effects.
Both groups had an equivalent increase in lung
collagen content.
No effect on collagen content.
Increase in collagen content.
Increased lung collagen and protein synthesis
rates; results of statistical analyses were not
reported.
Decreased protein synthesis on day 1; increased
synthesis day 2 and thereafter; peak response on
days 3 and 4.
Increased protein synthesis; recovery by 6 days
later; after re-exposure 6, 13, or 27 days later,
protein synthesis increased.
Net rate of collagen synthesis by lung minces
increased in concentration-dependent manner;
methyl prednisolone administered during 03
exposure prevented increase.
Species Reference
Rat
(wean-
ling)
Rat,
(young
adult)
Rat,
(wean-
ling)
Rat,
(young
adult)
Rat Myers et al . , 1984
Rat Mustafa et al . ,
1977
Rat Chow et al . , 1976b
Rat Hesterberg and Last,
1981
aMeasurement method:
Calibration method:
MAST = Kl-coulometric (Mast meter); UV = UV photometry; NBKI = neutral buffered potassium iodide; ND = not described.
NBKI = neutral buffered potassium iodide.
-------
through day 7 of exposure. Incorporation of radiolabeled amino acids into
collagen and noncollagenous protein rose to a plateau value at about 3 (colla-
genous) or 4 to 7 (noncollagenous) days after exposure. Synthesis of collagen
was about 1.6 times greater than that of noncollagenous proteins during the
first few days of exposure; no major differences were apparent by 7 days of
exposure. After the 7-day exposure ended, about 10 days were required for
recovery to initial values of prolyl hydroxylase. However, hydroxyproline
levels were still increased 28 days postexposure. This suggests that although
collagen biosynthesis returns to normal, the product of that increased synthesis,
collagen, remains stable for some time.
The shape of the concentration-response curve was investigated by Last et
al. (1979) for biochemical and histological responses of rat lungs after
3
exposure to ozone for 1, 2, or 3 weeks at levels ranging from 980 to 3920 ug/m
(0.5 to 2 ppm). A general correlation was found between fibresis detected
histologically and the quantitative changes in collagen synthesis in minces of
Og-exposed rat lungs. The stimulation of collagen biosynthesis was essen-
tially the same, regardless of whether the rats had been exposed for 1, 2 or 3
weeks; it was linearly related to the 03 concentration to which the rats were
exposed.
Protein deficiency and food restriction do not have a major influence on
the effects of 03 on lung hydroxyproline, lung elastin, or apparent rates for
lung collagen synthesis and elastin accumulation (Myers et al., 1984). In
3
this study weanling or young adult rats were exposed continuously to 1254 ug/m
(0.64 ppm) Oo for 7 days. It appears that 0~ caused an increase in apparent
lung collagen and protein synthetic rates and no major change in elastin
accumulation, but the results of statistical analyses were not reported.
3
Continuous exposure of mice for 7 days to 882 |jg/m (0.45 ppm) of 03
caused an increase in collagen synthesis after 5 or 7, but not 2 days of expo-
sure (Bhatnagar et al., 1983). The 5- and 7-day results showed little if any
difference. The effect on synthesis of noncollagen protein was not significant.
Prolyl hydroxylase activity was also increased at 2t 3, 5, and 7 days of
exposure, with the maximal increase at day 5. The day 7 results were only
\
slightly different (no statistical analysis) from the day 2 data. Superoxide
dismutase activity was investigated, because it has been observed (in other
studies) to prevent a superoxide-induced increase in collagen synthesis and
prolyl hydroxylase. Activity of superoxide dismutase increased in a pattern
parallel to that for collagen synthesis.
9-102
-------
Bhatnagar , et al . (1983) also studied rats exposed continuously for 90
days to 1568 ug/m (0.8 ppm) of 0,,. Prolyl hydroxylase activity continued to
increase through 7 days of exposure. By 20, 50, and 90 days of exposure, no
significant effects were observed.
Last and his colleagues performed a series of studies to evaluate the
effects of subchronic exposure to Q3 on rats. In one experiment (Last et al.,
19845), young adult (60-65 days old) rats were exposed continuously for 90 days
to 1254 ug/m (0.64 ppm) or 1882 ug/m (0,96 ppm) 03; the higher level increased
lung collagen content, while the lower level only caused a marginal (p <0.1)
increase. Rats were exposed to these same concentrations in an intermittent
regimen consisting of 5 days of exposure (8 hr/day), 9 days of air, 5 days of
exposure, 9 days of air, etc., for a total of 35 days of 0- and 54 days of air
C/
within the 90-day experiment. Both concentrations of 03 caused an equivalent
increase in collagen content. The magnitudes of the effects after continuous
or intermittent exposure were not statistically different.
Weanling rats (28 days old) were examined after a 6-wk exposure (8 hr/day,
7 days/wk) to 1254 yg/m (0.64 ppm) or 1882 ug/m3 (0.96 ppm) 0-, (Last et al.,
3
1984b). Immediately after exposure, only those animals exposed to 1882
(0.96 ppm) 03 exhibited an increase in lung collagen content. However, 6 wk
postexposure, animals exposed to both the high and low concentrations of Q~
had increases in collagen content. It appears that the collagen content
increased during this postexposure period, but no statistical comparisons were
reported.
Q
Young adult rats exposed continuously for 3 weeks to 1882 ug/m (0.96 ppm)
DO had an increase in lung collagen (Last et al., 1984b). Rats exposed to
3
2352 ug/m (1-2 ppm) 0, for I wk and examined 2 wk later had an increase in
3
collagen equivalent to that of the 3-wk exposure group (1882 ug/m , 0.96 ppm).
2
Weanling rats were also studied after intermittent exposure to 1882 |jg/m
(0.96 ppm) 03 (Last et al. ,.. 1984b).. -The regimen.-was 3 days of exposure for
8 hr/day, followed by 4 days of air; this unit was repeated 6 times. No
significant changes in collagen content occurred.
Lung collagen of juvenile cynomolgus monkeys (6-7 mo old at start of
3
exposure), exposed to 1254 ug/m (0.64 ppm) 03 for 8 hr/day, 7 days/wk for
I yr (361 days), was studied by Last et al. (1984b). Collagen content was
increased. Collagen type ratios were determined, and there were no apparent
shifts in collagen types; however, the authors report that given the surgical
variation, small shifts in collagen types would not be likely to be detected.
9-103
-------
3
Rats were exposed to 980 jjg/m (0.5 ppm) continuously for up to 180 days
and examined at various times during exposure and 2 months after exposure
ceased (Last and Greenberg, 1980). The total protein content of the lungs
increased during exposure, with the greatest increase occurring after 88 days
of exposure. By 53 days postexposure, values had returned to control levels.
Hydroxyproline content of the lungs also increased following 3, 30, 50, or 88,
but not 180, days of exposure. No such effect was observed at the 53-day
postexposure examination. The rates of protein and hydroxyproline synthesis
were also measured. Protein synthesis was not affected significantly. Although
the authors mentioned that hydroxyproline synthesis rates "appeared to be
greater," statistical significance was not discussed and values appeared to be
only slightly increased, considering the variability of the data. In dis-
cussing their data, the authors referred to a concurrent morphological study
(Moore and Schwartz, 1981) that showed an increase in lung volume, mild thick-
ening of the interalveolar septa and alveolar interstitium, and an increase in
collagen (histochemistry) in these areas. Different results for collagen
levels were observed after another longerrterm exposure (6 hr/day, 5 days/wk,
12.4 wk) to 392, 1568, or 3920 ug/m3 (0.2, 0.8, or 2.0 ppm) of 03 (Costa et
a!., 1983). At the two lower concentrations, rats exhibited an equivalent
decrease in hydroxyproline. At the highest concentration, an increase was
observed. Similar findings were made for elastin levels.
Most reports, such as those described above, are on the effects of Q3 on
collagen synthesis. Very little is known about the effects of 03 on collagen
turnover (i.e., the integration of synthesis and degradation). Curran et al.
(1984) found that in vitro exposure to high levels of 0,, (19600 ug/m3, 10 ppm
^
for 1-4 hr) caused degradation of collagen. A lower level, 490 ug/m (0.25 ppm)
did not cause degradation, but the collagen became more susceptible to proteoly-
tic degradation.
Last (1983) and Last et al. (1983) investigated the interaction between
0~ and aerosols of ammonium sulfate [(NH.^SO.] and sulfuric acid (H^SO^) in
rats. Rats were exposed continuously for 7 days to four concentrations of 0,,
OO O
ranging from 980 ug/m to 2940 ug/m (0.5 to 1.5 ppm) Og. According to the
authors, these levels were determined by the KI method and should be multiplied
by 0.8 to compare to UV photometric methods (i.e., 0.4 to 1.2 ppm). The
authors' actual measured values are noted here. The 03 exposure resulted in a
linear, concentration-related increase in collagen synthesis rate. A 7-day
3
continuous exposure to 5 mg/m (NH»)2SO. (0.8-1.0 urn mass median aerodynamic
9-104
-------
diameter, MMAD) had no effect. However, when mixtures of 0-. from 1568 to
2940 (jg/m3 (0.8 to 1.2 ppm) and 5 mg/m3 (NH4)2$04 were used, the collagen
synthesis rate increased to about 180% over the rates of 07 for the higher 07
Q O O
levels (apparently 2352 and 2940 (jg/m , 1.2 and 1.5 ppm).
Mixtures of 03 and H2$04 were also reported (Last et al. , 1983). Rats
were exposed to 980 (jg/m (0.5 ppm) 0, or this 0, level in combination with
1000 (jg/m3 H2S04 (0.38 (jm, MMAD) continuously for 3 to 50 days. As expected,
Oo exposure increased collagen synthesis rates at the three times of examina-
tion (3, 30, and 50 days). The mixture of 0- and H,,S04 caused greater effects.
Examination of the slope ratio of regression lines indicated that hUSO. in the
mixture caused a 220 percent enhancement. The authors state that FLSO. alone
had no effects.
The statistical procedures applied in these collagen studies (Last et al.,
1983) were questioned by Krupnick and Frank (1984) in a letter to the editor.
The general criticism was that too few statistical tests were applied to test
the hypothesis of synergism and that too few pollutant data points were used
in the design to develop robust regression lines. The authors (Last et al.
1983) responded that they did apply most of the statistical tests and found
significant differences between regressions comparing 03 only to 03 plus
aerosols, but the journal would not accede to publishing these analyses.
The proximal acinar regions of rats from the above-mentioned collagen
3
studies, exposed for 1 days to 2352 (jg/m (1.2 ppm) 0Q alone or in combination
3
with 5000 ug/m (NH4)2S04, were also evaluated. Ammonium sulfate alone caused
no morphological or morphometric effects. Ozone exposure resulted in a thicken-
ing of the interstitium and an influx of inflammatory cells. The mixture of
0- and (NH4)2$04 caused the same response plus an apparent deposition of
fibrous material. These lesions were then examined morphometrically. In the
03 and 03 plus (NH4)2$04 groups, there were no changes in the volume ratio of
the lesion per lung or the volume ratio of the extracellular connective tissue
per lesion. In examining volume density of different cell types in the lung
lesions, both groups had an increased percentage of fibroblasts and smooth
muscle. When the number of cells of each type present per area of lesion was
calculated, there was a 340 percent increase in the number of fibroblasts in
the 0- plus (NH.)pSO, group compared to the 0-, group. These findings correlate
well with the biochemical effects.
These studies were expanded by Last et al. (1984a) to better evaluate the
influence of length of exposure. All exposures were continuous to 03, (NH4)2S04,
9-105
-------
3
or a mixture of the two; the ammonium sulfate concentration was about 5000 ug/m
(0.5 |jm MMAD), the precise concentration depending upon the experiment. After
3
3 days of exposure to either 1254 ug/m (0.64 ppm) 03 or (NH4)2S04, no significant
effects on collagen synthesis of rats were observed; the mixture of 03 and
(NH*)2SO» more than doubled the collagen synthesis rate over controls. The
pulmonary lesions (i.e., aggregations of interstitial inflammatory cells in
terminal bronchioles) of the proximal acinus in these rats were examined
histologically and morphometrically. Ammonium sulfate alone caused no effects.
In the 0, and 03 plus (NH4)2S04 groups, there was no fibrosis, but thickening
by edema and inflammatory cell infiltrate was observed. Morphometrically,
(NH4)2S(L caused no effects. However, 03 and 0~ plus (NH4)2S04 increased
total cell numbers in lesions, with the mixture producing a significantly
greater increase over 03 alone. This increase was principally due to an
increase in the numbers of macrophages, monocytes, and fibroblasts. The
increase in the number of fibroblasts is consistent with the biochemical
findings.
Similar measurements were made on rats exposed continuously for 7 days to
1882 ug/m (0.96 ppm) 03, 5000 ug/m (NH4)2S04, or a mixture of the two (Last
et a!., 1984a). Control data were not presented and all statistical comparisons
reported were in reference to the 0~ alone group. Ammonium sulfate exposure
resulted in fewer, numbers of cells than Q3 alone. For macrophage and monocyte
numbers, 0~ and (NH4)2S04 were apparently additive. For fibroblast and total
cell numbers, 03 and (NFL)2SO, were apparently synergistic.
The frequency of occurrence of lesions was also examined in the 3-day and
the 7-day studies described above (Last et al., 1984a). There was no difference
in the frequency of lesion occurrence between 0, and Q3 plus (NH»)2SO,. The
individual lesions were larger in the 03 plus (NH4)2S04 group; the (NH4)2S04
group had almost no lesions. Using collagen staining procedures, the 0, plus
(NH^pSO, group had a greater volume of collagen than did lesions in the 0,
alone group. The authors summarized these studies (Last et al., 1983; Last
et al., 1984a) by stating that (NH.)2SO, increases the severity of 03 effects
at lesion sites without^ increasing the number of lesions; and the cellular
changes correlate with biochemical and histological indications of potential
later fibrosis. From all these studies it is apparent that synergism occurs.
However, it further appears that the synergism is dependent on the concentration
of 03. For instance, Last et al. (1983) demonstrated that 5000 ug/m (NH4)2SQ4
9-106
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3 3
increased collagen synthesis rates in rats exposed to 2352 [jg/m or 2940 ug/m
3
(1.2 or 1.5 ppm) 0,, but not in those exposed to 1568 ug/m (0.8 ppm) CU.
Protein synthesis (incorporation of radiolabeled leucine) was increased
3
in rats after 3 days of continuous exposure to 1568 |jg/m (0.8 ppm) of Q,
(Chow et al. , 1976b). Recovery had occurred by 6 days postexposure. No
adaptation was observed, because when animals were re-exposed to the same Qg
regimen 6, 13 or 27 days after the first exposure, protein synthesis increased
as it had earlier. Mustafa et al. (1977) investigated the time course of the
increased jjn vivo incorporation of radioactive ami no acids. Rats were exposed
2
continuously for 7 days to 1568 ug/m (0.8 ppm) of 0,. No statistics were re-
ported. One day of exposure caused a decrease in protein synthesis. However,
by day 2, an increase occurred, which peaked on days 3 and 4 of exposure. On
day 7, the effect had not diminished. The authors attributed this finding to
synthesis of noncollagenous protein. They also found no radioactive incorpora-
tion into blood or alveolar macrophages. Hence, the observed increases were
due to lung tissue protein synthesis, and not a concurrent influx of alveolar
macrophages or serum into the lungs.
The production of mucous glycoproteins and their secretion by trachea!
explants have been reviewed by Last and Kaizu (1980). Mucous glycoprotein
synthesis and secretion were measured by the rate of incorporation of H-glucos-
amine into mucous glycoproteins and their subsequent secretion into supernatant
fluid of the trachea! explant culture medium. This method has been found to
be a reproducible index of mucous production, and these authors maintain that
this measurement ex y 1 vo following exposure |ri vivo is representative of
3
injuries occurring in vivo. When rats were exposed to 1568 ug/m (0.8 ppm) of
0., for 8 hr/day for 1 to 90 days, Last et al. (1977) found a depression of
glycoprotein synthesis and secretion into the tissue culture medium for the
initial week that was statistically significant only on days 1 and 2 of exposure.
Rebound occurred subsequently, with increased glycoprotein secretion for at
least 12 weeks of continued exposure to 03 (only significant at 1 and 3 months
of exposure). Rats were also exposed intermittently for 3 days to 1176, 784,
o
and 392 ug/m (0.6, 0.4, 0.2 ppm) of O^. Glycoprotein secretion decreased
only at the higher concentration. Tracheal explants from Bonnet monkeys
exposed to 0, 980, or 1568 ug/m3 (0, 0-5 or 0.8 ppm) Og for 7 days appeared to
have increased rates of secretion of mucus (Last and Kaizu, 1980). However,
few monkeys were used and statistical analysis was not reported.
9-107
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3
A combination exposure to 0- (980 |jg/m ; 0.5 ppm) and sulfuric acid
3
(HgSO*) aerosol (1.1 mg/m ) caused complex effects on mucous secretions in
rats (Last and Kaizu, 1980; Last and Cross, 1978). A 3-day exposure to
3
980 ug/m (0.5 ppm) of 0, decreased mucous, secretion rates, but HpSO, had no
effect. In rats exposed to the combination of H-SO, aerosol and 0~, mucous
secretion significantly increased. After 14 days of continuous exposure, the
rats receiving a combined exposure to both H?SO. aerosol and 03 had elevated
values (132 percent) over the control group of animals. Because mucous secretion
and synthesis are intimately involved in diminishing the exposure of underlying
cells to 03 and removing adventitiously inhaled particles, alterations in the
mucous secretory rate may have significant biological importance. Experiments
reported to date do not clearly indicate what human health effects may be
likely, nor their importance.
9.3.3.7 Lipid Metabolism and Content of the Lung. If 0™ initiates peroxi-
dation of unsaturated fatty acids in the lung, then changes in the fatty acid
composition of the lung indicative of this process should be detectable.
Because the fatty acid content of the lung depends on the dietary intake,
changes in fatty acid content due to 0. exposure are difficult to determine in
the absence of rigid dietary control. Studies on lipid metabolism and content
of the lung are summarized in Table 9-9. Generally, the unsaturated fatty
3
acid content decreased in rats exposed to 980 |jg/m (0.5 ppm) of 03 for up to
6 weeks (Roehm et a!., 1972).
Peroxidation of polyunsaturated fatty acids produces pentane and ethane
to be exhaled in the breath of animals (Donovan and Menzel, 1978; Downey et
al., 1978). A discussion of the use of ethane and pentane as indicators of
peroxidation is presented by Gelmont et al. (1981) and Filser et al. (1983).
Normal animals and humans exhale both pentane and ethane in the breath.
Dumelin et al. (1978b) found that exhalation of ethane decreased and exhalation
of pentane increased when rats were deficient in vitamin E and exposed to
3
1960 ug/m (1 ppm) of 0., for 60 min. The provision of 11 (minimum vitamin
requirement) or 40 IU (supplemented level) of vitamin E acetate per kg of diet
resulted in a decrease in expired ethane and pentane after 03 exposure.
Dumelin et al. (1978a) also measured breath ethane and pentane in Bonnet
a
monkeys exposed to 0, 980, or 1568 M9/m (0, 0.5, or 0.8 ppm) of 03 for 7, 28,
or 90 days. They failed to detect any additional ethane or pentane in the
breath of these monkeys and attributed the lack of such additional evolution
to be due to the high level of vitamin E provided in the food.
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TABLE 9-9. EFFECTS OF OZONE EXPOSURE ON LIPID METABOLISM AND CONTENT OF THE LUNG
H
O
Ozone
concentration
ng/m3
980
980
1568
1960
1960
ppm
0.5
0.5
0.8
1.0
1.0
Exposure
Measurement duration and
method protocol
I Continuous for
2, 4, or 6 wk
UV 8 hr/day for 7,
28, or 90 days
NBKI . 60 min
ND 4 hr
Observed effect(s) Species
Increase in arachidonic and palmitic Rat
acids; decrease in oleic and lino-
leic acids.
No effect on ethane and pentane Monkey
production in animals fed diets
supplemented with high levels of
vitamin E.
With vitamin E-deficient diet, in- . Rat
creased pentane production and de-
creased ethane production. With
vitamin E supplementation of 11 or
40 IU vitamin E/kg diet, decreased
ethane and pentane production.
Decreased incorporation of fatty Rabbit
acids into lecithin.
Reference
Roehm et al . , 1972
Dumelin et al. ,
1978a
Dumelin et al. ,
1978b
Kyei-Aboagye
et al. , 1973
Measurement method: NBKI = neutral buffered potassium iodide; UV = UV photometry; I = iodometric; ND = not described.
-------
Kyei-Aboagye et al, (1973) found that the synthesis of lung surfactant in
rabbits, as measured by dipalmitoyl lecithin synthesis, was inhibited by
3
exposure to 1960 pg/m (1 ppm) of 03 for 4 hr. Pulmonary lavage showed an
increase in radiolabeled lecithins. The authors proposed that 0~ may decrease
lecithin formation while simultaneously stimulating the release of surfactant
lecithins. This may suggest the presence of a larger disarrangement of lipid
metabolism following 03 exposure. However, although changes in lipid com-
position of lavage fluid occur, the changes apparently do not alter the sur-
face tension lowering properties of the fluid, as shown by Gardner et al.
3
(1971) and Huber et al. (1971) when using high levels of 03 (> 9800 ug/m ;
5 ppm).
9.3.3.8 Lung Permeability. Table 9-10 summarizes studies of the effects
on lung permeability of exposures to different concentrations of 0,,.
The lung possesses several active-transport mechanisms for removal of
substances from the airways to the capillary circulation. These removal mech-
anisms have been demonstrated to be carrier-mediated and specific for certain
ions. Williams et al. (1980) studied the effect of 0- on active transport of
3
phenol red in the lungs of rats exposed to 1176 to 4116 (jg/m (0.6 to 2.1 ppm)
of 03 continuously for 24 hr. Ozone inhibited the carrier-mediated transport
of intratracheally instilled phenol red from the lung to the circulation and
increased the nonspecific diffusion of phenol red from the lung. These changes
in ion permeability may also explain in part the effects of 03 on the respira-
tory response of animals to bronchoconstrictors (Lee et al., 1977; Abraham et
al., 1980).
As another index of increased lung permeability following Q~ exposure,
the appearance of albumin and immunoglobins in airway secretions has been
3
examined. Reasor et al. (1979) found that dogs breathing 1960 to 2940 ug/m
(1.0 to 1.5 ppm) of 0,, had increased albumin and immunoglobjn G content of
their airway secretions. Alpert et al. (1971a), using rats exposed for 6 hr to
3 .
490 to 4900 |jg/m (0.25 to 2.5 ppm) 0_, also found increased albumin in lung
3
lavage in animals exposed to 980 ug/m (0.5 ppm) or more.
In a series of experiments, Hu et al. (1982) exposed guinea pigs to 196,
510, 1000, or 1960 ug/m3 (0.1, 0.26, 0.51, or 1.0 ppm) of 03 for 72 hr and
found increased lavage fluid protein content sampled immediately after exposure
to concentrations > 510 ug/m (0.26 ppm) as compared with controls. Ozone-
exposed guinea pigs had no accumulation of proteins when the exposure time was
9-110
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TABLE 9-10. EFFECTS OF OZONE ON LUNG PERMEABILITY
H
H
Ozone
concentration
pg/m3
196
510
1000
1960
353
510
980
1000
490
880
1960
4900
1176
2156
3136
4116
1960-
2940
ppm
0.1
0.26
0.51
1.0
0.18
0.26
0.5
0.51
0.25
0.5
1.0
2.5
0.6
1.1
1.6
2.1
1.0-
1.5
k Exposyre
Measurement ' duration and
method protocol
CHEM, 3 hr or 72 hr
UV
3 hr; 8 hr/day
for 5 or 10 days
3, 24, or 72 hr
3 hr
MAST, 6 hr
NBKI
NBKI 24 hr
HAST 2 hr
Observed effect(s) Species Reference
Increased levels of lavage fluid protein Guinea Hu et al., 1982
>_ 0.26 ppm immediately after 72-hr exposure pig
or 15 hr after a 3-hr exposure. Vitamin C
deficiency did not influence sensitivity.
No effect on protein levels.
Lavage was 15 hr postexposure. At 0.26 ppm in-
creased protein only after 24 hr exposure; at
6.5 ppn, increased protein after 24 hr
of exposure.
Increase in protein levels at 10- and 15- (but not
0, 5, or 24) hr postexposure.
Increased alveolar protein accumulation at 0.5 ppm Rat Alpert et al.,
and above. 1971a
Concentration-dependent loss of carrier-mediated Rat Williams et al.,
transport for phenol red. 1980
Increased albumin and immunoglobin G in airway Dog Reasor et al.,
secretions. 1979
Measurement method
b,
MAST = Kl-coulometric (Mast meter); CHEM = gas phase chemiluminescence; UV = UV photometry; NBKI = neutral buffered
potassium iodide.
Calibration method: KBKI = neutral buffered potassium iodide; UV = UV photometry.
-------
reduced from 72 to 3 hr, unless the time of lavage was delayed for 10 to 15 hr
following exposure. The protein content of the lavage fluid determined 10 to
15 hr following'a 3-hr exposure increased in a concentration-related manner
c
3
from 500 to 1470 pg/m3 (0.256 to 0.75 ppm). Again 196 pg/m3- (0.1 ppm) had no
effects. The lavage fluid protein content of guinea pigs exposed to 353 pg/nf
(0.18 ppm) of DO for 8 hr per day for 5 or 10 consecutive days was not dif-
ferent from air controls. No effect-of vitamin C deficiency could be found on
the accumulation of the lavage fluid protein in guinea pigs exposed to 196 to
1470 pg/m3 (0.10 to 0.75 ppm) 03 for 3 hr (Hu et al.s 1982). In contrast,
vitamin Odeficient guinea pigs have increased sensitivity to NO,, (Selgrade et
a!., 1981). Polyacrylamide gel electrophoresis of lavage fluid proteins from
3
animals exposed for 3 hr to 196 to 1470 |jg/m (0.1 to 0.75 ppm) QS showed the
appearance of extra protein bands which co-migrated with serum proteins and of
increased intensity of bands that also occur in air-exposed controls. This
led the authors to conclude that the main source of the increased protein was
serum.
Prostaglandins are intermediates in tissue edema resulting from a wide
variety of mechanisms of injury. Increased lung permeability and edema pro-
duced by 03 might also be mediated by prostaglandins. Non-steroidal anti-in-
flammatory drugs (aspirin and indomethacin) at appropriate doses inhibit lung
edema in rats from exposure to 7890 pg/m (4 ppm) of 0, for 4 hr (Giri et al.,
1975). Prostaglandins F2 and £„ were markedly increased in plasma and lung
lavage of rats exposed to 7840 pg/m (4 ppm) for up to 8 hr (Giri et al.,
1980). Ozonolysis of arachidonic acid jm vitro produces fatty acid peroxides
and other products having prostaglandin-like activity (Roycroft et al., 1977).
Fatty acid cycloperoxides are produced directly by ozone-catalyzed peroxidation
3
(Pryor, 1976; Pryor et al., 1976). Acute exposure to 5880 ug/m (3 ppm) 03
inhibits uncompetitively rat lung prostagl-andin cyclooxygenase (Menzel et al.3
1976).
Earlier reports that prostaglandin synthesis inhibitors exacerbated
Q3-produced edema (Dixon and Mountain, 1965; Matzen, 1957a,b), as did methyl
prednisolone (Alpert et al., 1971a), tend to confuse the interpretation of the
role of prostaglandin in 03~produced injury. Prostaglandin synthesis inhibitors
clearly inhibit the degradation of prostaglandins and alter the balance between
alternative pathways of fatty acid peroxide metabolism. Thus, while prostanoids
are highly likely to be involved in Q3~produced edema, their exact role is
still unexplained.
9-112
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9.3.3.9 Proposed Molecular Mechanisms of Effects. Experts generally agree
that the toxicity of 0,, depends on its oxidative properties. The precise
mechanism of O3's toxicity at the subcellular level is unclear, but several
theories have been advanced. These theories include the following:
1. Oxidation of polyunsaturated lipids contained mainly in cell
membranes;
2. Oxidation of sulfhydryl, alcohol, aldehyde, or amine groups in
low molecular weight compounds or proteins;
3. Formation of toxic compounds (ozonides and peroxides) through
reaction with polyunsaturated lipids;
4. Formation of free radicals, either directly or indirectly,
through lipid peroxidation; and
5. Injury mediated by some pharmacologic action, such as via a
neurohormonal mechanism, or release of histamine.
These mechanisms have been discussed in several reviews: U.S. Environmen-
tal Protection Agency (1978); National Air Pollution Control Administration
(1970); North Atlantic Treaty Organization (1974); National Research Council
(1977); Shakman (1974); Menzel (1970, 1976); Nasr (1967); Cross et al. (1976);
Pryor et al. (1983); and Mudd and Freeman (1977). From these reviews and
recent research detailed here or in previous biochemistry sections, two hypo-
theses are favored and may in fact be related.
9.3.3.9.1 Oxidation of polyunsaturated lipids. The first hypothesis is that
03 initiates peroxidation of polyunsaturated fatty acids (PUFA) to peroxides,
which produces toxicity through changes in the properties of cell membranes.
Ozone addition to ethylene groups of PUFA can take place in membranes yet give
rise to water-soluble products that can find their way to the cytosol. Alde-
hydes, peroxides, and hydroxyl radicals formed by peroxidatien all can react
with proteins. In addition to the direct oxidation of amino acids by 0,,
*i j
secondary reaction products from O^-initiated PUFA peroxidation can also
oxidize amino acids or react with proteins to alter the function of the proteins.
Since large numbers of proteins are embedded with lipids in membranes and rely
on the associated lipids to maintain the tertiary structure of the protein,
alterations in the lipid surrounding the protein can result in structural
9-113
-------
changes of the membrane-embedded protein. At present, methods are not avail-
able to differentiate effects on membrane proteins from effects on membrane
lipids.
Some of the strongest evidence that the toxic reaction of 03 can be
associated with the PUFA of membranes is the protective effect of dietary
vitamin E on 03 toxicity (Roehm et al., 1971a, 1972; Chow and Tappel, 1972;
Fletcher and Tappel, 1973; Donovan et al., 1977; Sato et al., 1976a; Chow and
Kaneko, 1979; Plopper et al,, 1979; Chow et al., 1981; Chow, 1983; Mustafa et
al., 1983; Mustafa, 1975). Generally, vitamin E reduced the CU-induced increase
in enzyme activities of the glutathione peroxidase system (Section 9.3.3.2)
and those involved in oxygen consumption (Section 9.3.3.3). More details are
provided in Table 9-5. Morphological effects due to CL exposure are also
lessened by dietary vitamin E (Section 9.3.1.4.1.1). Although this is not
the strongest evidence, vitamin E supplementation also prevented 0~-indueed
changes in red blood cells (Chow and Kaneko, 1979).
These data consistently show that vitamin E has a profound effect on the
toxicity of 0- in animals. They also support indirectly lipid peroxidation as
a toxic lesion in animals. However, although the influence of dietary vitamin
E is clear, its relation to vitamin E levels in the lung, where presumably
most lipid peroxidation would occur, is poorly understood. Rats of various
ages (5, 10 and 90 days old and 2 yrs old) were fed normal diets; and 90-day-
old rats were fed diets containing 0, 200, and 3000 mg of vitamin E/kg diet
(Stephens et al., 1983). (Most of the biochemical studies of vitamin E protec-
tive effects were conducted with diets having far less than 200 mg/kg diet.)
3
Rats were exposed to 1764 pg/m (0.9 ppm) 03 continuously for 72 hr, and
periodic morphological observations were made. Those animals on normal diets
had equivalent levels of vitamin E in the lung, but responses of the different
age groups differed. Animals of a given age (90 days) maintained on the three
vitamin E diets had different levels of vitamin E in the lungs (5.3 to 325 yg
of vitamin E/g of tissue), but morphological responses were very similar.
Stephens et al. concluded that 03~induced responses in the lung are indepen-
dent of the vitamin E content of the lung. Independent interpretation of this
study is not possible, since very minimal descriptions of responses were pro-
vided and the number of animals was not given. Also, others (Chow et al.,
1981; Plopper et al., 1979) found that for a given age of rat, different
dietary levels of vitamin E (and perhaps different lung levels as shown by
Stephens et al., 1983) influenced the morphological responses of rats to 03-
9-114
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9.3.3.9.2 Oxidation of sulfhydryl or amine groups. The second hypothesis is
that ozone exerts its toxicity by the oxidation of low-molecular-weight compounds
containing thiol, amine, aldehyde, and alcohol functional groups and by oxida-
tion of proteins. Mudd and Freeman (1977) present a summary of the arguments
for oxidation of thiols, amines, and proteins as the primary mechanism of 03
toxicity based upon i_n vitro exposure data. Ami no acids are readily oxidized
by 03 (Mudd et al., 1969; Mudd and Freeman, 1977; Previero et a!., 1964). In
the following descending order of rate, 0~ oxidizes the ami no acids cysteine >
methionine >_ tryptophan > tyrosine > histidine > cystine > phenylalanine. The
remaining common ami no acids are not oxidized by 03, Thiols are the most
readily oxidized functional groups of proteins and peptides (Mudd et al.,
1969; Menzel, 1971). Tryptophan in proteins is also oxidized in vitro by 0~
"" '"'"' O
as shown by studies of avidin, the biotin-bitiding protein. Oxidation of
tyrosine in egg albumin by 0, occurs in vitro, converting the 0.,-oxidized egg
*5 «—- , IJTTT,,„,, -».,- m,., J
albumin to a form immunologically distinct from native egg albumin (Scheel et
al., 1959). Ozone inactivated human alpha-1-protease inhibitor i_n vitro (Johnson,
1980). When treated with 0^, alpha-1-protease inhibitor lost its ability to
inhibit trypsin, chymotrypsin, and elastase.
Meiners et al. (1977) found that 0- reacted in vitro with tryptophan,
J —-~• T~ """
5-hydroxytryptophan, 5-hydroxytryptamine, and 5-hydroxyindolacetic acid. One
mole of 0~ was rapidly consumed by each mole of indole compound. Oxidation of
tryptophan by Q? a^so generates hydrogen peroxide. Hydrogen peroxide is a
toxic substance in itself and initiates peroxidation of lipids (McCord and
Fridovich, 1978). Other active Op species such as HO- and Op~ are formed from
hydrogen peroxide (McCord and Fridovich, 1978).
The results of the oxidation of functional groups in proteins can be
generally observed by reduction of enzyme activities at high concentrations of
3
0, (viz., 1960 to 7840 M9/m , 1 to 4 ppm for several hours). Many enzymes
examined (Tables 10-5 to 10-10) in tissues have decreased activities (in many
cases not statistically significant) immediately following even lower level
3
(1960 p:g/m , 1 ppm) 0, exposures. The enzymes decreased,include those cataly-
zing key steps supplying reduced cofactors to other processes in the cell,
such as glucose-6-phosphate dehydrogenase (DeLucia et al., 1972) and succinate
dehydrogenase (Mountain, 1963). Cytochrome P--450 (Goldstein et al., 1975),
the lecithin synthetase system (Kyei-Aboagye et al., 1973), lysozyme (Holzman
et al., 1968), and the prostaglandin synthetase system (Menzel et al., 1976)
are decreased by 0™ exposure. Respiratory control of mitochondria is lost,
9-115
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and mitochondria! energy production is similarly decreased (Mustafa and Cross,
1974).
Mudd and Freeman (1977) point out that proteins are the major component
of nearly all cell membranes, forming 50 to 70 percent by weight. The remainder
of the weight of the cell membranes is lipids (phospholipids, glycolipids,
glycerides, and cholesterol). Polyunsaturated fatty acids are components of
membrane phospholipids, glycolipids, and glycerides. Mudd and Freeman contend,
however, that proteins are far more easily oxidized by 03 than are lipids.
Indirect evidence in support of the idea that amines in particular are oxidized
preferentially by 03 is the protective effect of p-aminobenzoic acid (Goldstein
et al.» 1972a). Rats injected with p-aminobenzoic acid were partially
protected from the mortality due to high concentrations- of 03. Presumably,
the added p-aminobenzoic acid is oxidized by 03 in place of proteins. Goldstein
and Balchum (1974) later suggested that the protection of p-aminobenzoic acid,
allylisopropy!acetamide, and chlorpromazine was due to the induction of mixed
function oxidase systems rather than a direct free radical scavenging effect.
However, they also recognized that chlorpromazine can mask free radicals and
result in membrane stabilization, which could account for the protective
effects of these compounds preventing edema and inflammation. Acetylcholines-
terase found on red blood cells is protected from inhibition by jm vitro
o
exposure to 78,400 ug/m (40 ppm) 03 through p-aminobenzoic acid treatment
(Goldstein et a!., 1972a). Amines are also efficient lipid antioxidants,
t
so the results of these experiments could be interpreted in favor of the
theory of peroxidation of the cell membrane as a mechanism of toxicity, as
we! 1.
9.3.3.9.3 Formation of toxic compounds through reaction with polyunsaturated
lipids. The effects of 03 could be due to the elaboration of products of
peroxidation as well as peroxidation of the membrane itself. Menzel et al.
(1973) injected 10 pg to 10 ug of fatty acid ozonides from oleic, linoleic,
linolenic, and arachidonic acids into animals and found increased vascular
permeability measured by extravascularly located Pontamine Blue dye bound to
serum proteins. Extravascularization of serum proteins could be blocked by
simultaneous antihistamine injection or by prior treatment with compound 48/80,
a substance that depletes histamine stores. Cortesi and Privett (1972) injected
methyl linoleate ozonide into rats or gave the compound orally; in each case
acute pulmonary edema resulted. The pulmonary edema and changes in fatty acid
9-116
-------
composition of the serum and lung lipids were similar to those occurring after
03 exposure (Roehm et al., 1971a,b, 1972; Menzel et a!., 1976).
Peroxidation of lung lipids could lead to cytotoxic products. Phosphatid-
yl choline liposomes (spheres formed by emulsifying phosphatidyl choline in
water) were lysed on exposure to 0- (Teige et al., 1974). Liposomes exposed
to On were more active than 0, alone in lysing red blood cells. The products
of ozonolysis of phosphatidyl choline could be stable, yet toxic, intermediates.
9.3.3.9.4 Formation of free radicals and injury mediatedby pnarmacologic
action. The other theories may be linked to the consequences of peroxidation
of PUFA. Ozonation of PUFA results in the formation of peroxides (e.g., ROOH
or RQOR) rather than oxidation of alkenes to higher oxidation states (e.g., RON,
RCHO or RC02H). Peroxides (ROOH or ROOR) are chemically reactive and may be the
ultimate toxicants, not simply products of oxidation. During the process of
peroxidation or direct addition of 0, to PUFA, free radicals may be generated
(Pryor et al., 1983), and these free radicals may be the ultimate toxicants.
Because the metabolism of PUFA peroxides in the lungs is intimately
linked with the metabolism of thiol compounds (such as glutathione, GSH),
direct oxidation of thiols by 0, (hypothesis B) may link the two major hypothe-
ses A and B with hypothesis C, formation of toxic products. Ozone depletion
of GSH could render the peroxide detoxification mechanism ineffective. However,
q
at levels of Oo below 1960 |jg/m (1 ppm), increases in glutathione have been
observed (Plopper et al., 1979; Fukase et al., 1975; Moore eta!., 1980;
Mustafa et al., 1982). The rat lung is sensitive to the increase of glutathione
peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase
activities at levels as low as 196 yg/m (0.1 ppm) 0- continuously for 7 days
(Plopper et al., 1979; Chow-et al., 1981; Mustafa, 1975; Mustafa and Lee,
3
1976) in vitamin E-deficient rats. With vitamin E-supplemented rats, 392 pg/m
q
(0.2 ppm) caused similar effects. After acute exposures to >1960 jjg/m (1 ppm)
Oq, decreases are observed in these enzyme activities and glutathione levels.
Other species (mice and monkeys) exhibit similar effects at different concentra-
tions (Table 9-5). These changes at the lower levels of Q, appear to be
coincidental with the initiation of repair and proliferative phases of lung
injury (Cross et al., 1976; Dungworth et al., 1975a,b; Mustafa and Lee, 1976;
Mustafa et a!., 1977, 1980; Mustafa and Tierney, 1978). The parallel increase
in the number of type 2 cells having higher levels of metabolic activity would
be expected to cause an overall increase in the metabolic activity of the
9-117
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lung. This was substantiated by Mustafa (1975) and DeLucia et al. (1975a),
who found that the 02 consumption per mitochondrion was not increased in
Og-exposed lungs but that the number of mitochondria was increased. Chow and
Tappel (1972) proposed that alterations of enzymatic activity were due to
stimulation of glutathione peroxidase pathway. Chow and Tappel (1973) also
proposed that the changes in the pentose shunt and glycolytic enzymes in lungs
of the Oj-exposed rats were due to the demands of the glutathione peroxidase
system for reducing equivalents in the form of NADPH. Cross et al. (1976) and
DeLucia (1975a,b) reported that 03 exposure oxidized glutathione and formed
mixed disulfides between proteins and non-protein sulfhydryl compounds.
The general importance of glutathione in preventing lipid peroxidation in
the absence of 0- has been shown by Younes and Siegers (1980) in rat and mouse
liver where depletion of glutathione by treatment with vinylidene chloride or
diethylmaleate led to increased spontaneous peroxidation. These authors
suggest that glutathione prevents spontaneous peroxidation by suppression of
radicals formed by the enzyme cytochrome P-450 or already produced hydroperox-
ides.
Chow and Tappel (1973) suggest that peroxides formed via lipid peroxida-
tion increase glutathione peroxidase activity and, in turn, increase levels of
the enzymes necessary to supply reducing equivalents (NADPH) to glutathione
reductase. Vitamin E suppresses spontaneous formation of lipid peroxides and,
therefore decreased the glutathione peroxidase activity in mouse red cells
(Donovan and Menzel, 1975; Menzel et al., 1978). The supplementation of rats
with vitamin E could, therefore, decrease the utilization of glutathione by
spontaneous reaction or by ozone-initiated peroxidation. The two mechanisms
could then interact in a concerted fashion to decrease ozone cytotoxicity.
Eliminating vitamin E from the diet increases the chances of increases of this
system.
In support of hypothesis E, Wong and Hochstein (1981) found that thyroxin
enhanced the osmotic fragility of human erythrocytes exposed to 0- jjn vitro.
They found also that 125I from radio!abeled thyroxin was incorporated into the
major membrane glycoprotein, glycophorin, of red cells. When these events had
occurred, the cation permeability of the human red cells was enhanced without
measurable inhibition of ATPase or membrane lipid peroxidation. They suggested
that thyroid hormones play an important role in 03 toxicity. Fairchild and
Graham (1963) found that thyroidectomy, thiourea, and antithyroid drugs protec-
ted animals from lethal exposures to 03 and nitrogen dioxide. Fairchild and
9-118
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Graham ascribed the mortality following 03 to pulmonary edema. Wong and
Hochstein (1981) suggested that Q3 toxicity in the lung may be altered through
a free radical mechanism involving iodine transfer from thyroxin to lung
membranes. The hormonal status of animals could alter a variety of defense
mechanisms and 03 sensitivity.
9.3.3.9.5 Summary. The actual toxic mechanism of CL may involve a mixture
of all of these chemical mechanisms because of the interrelationships between
the peroxide detoxification mechanisms and glutathione (See Section 9.3.3.2)
and the complexity of the products produced from ozonation of PUFA. A single
chemical reaction may not be adequate to explain 0-. toxicity. The relative
importance of any one reaction, oxidation of proteins, PUFA, or small molecular
weight compounds, will depend upon a number of factors such as the presence of
enzymatic pathways of decomposition of products formed (peroxides), pathways
for regeneration of thiols, the presence of non-enzymatic means of terminating
free radical reactions (vitamins E and C), and differences in membrane composi-
tion of PUFA (relative ease of attack of 03), for example.
9,3.4 Effects on Host Defense Mechanisms
The mammalian respiratory tract has a number of closely coordinated pul-
monary .defense mechanisms that, when functioning normally, provide protection
from the adverse effects of a wide variety of inhaled microbes and other par-
ticles. A variety of sensitive and reliable methods have been used to assess
the effects of (L on the" various components of this defense system to provide
a better understanding of the health effects of this pollutant.
The previous Air Quality Criteria Document for Ozone and Photochemical
Oxidants (U.S. Environmental Protection Agency, 1978) provided a review and
evaluation of the scientific literature published up to 1978 regarding the
effects of 03 on host defenses. Other reviews have recently been written that
provide valuable references to the complexity of the host defense system and
the effects of environmental chemicals such as 0, on its integrity (Gardner,
1981; Ehrlich, 1980; Gardner and Ehrlich, 1983; Goldstein, 1984).
This section describes the existing data base and, where appropriate,
provides an interpretation of the data, including an assessment of the different
microbial defense parameters used, their sensitivity in detecting abnormalities,
and the importance of the abnormalities with regard to the pathogenesis of
infectious disease in the exposed host. This section also discusses the
9-119
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various components of host defenses, such as the mucociliary escalator and the
alveolar macrophages, which clear the lung of both viable and nonviable parti-
cles, and integrated mechanisms, which are studied by investigating the host's
response to experimentally induced pulmonary infections. The immune system,
which defends the overall host against both infectious and neoplastic diseases,
is also discussed.
9.3.4.1 Mucociliary Clearance. The mucociliary transport system is one of
the lung's primary defense mechanisms against inhaled particles. It protects
the conducting airways by trapping and quickly removing material that has been
deposited on the mucociliary escalator. The effectiveness of mucociliary
clearance can be determined by measuring such biological activities as the
rate of transport of deposited particles; the frequency of ciliary beating;
structural integrity of the ciliated cells; and the size, number, and distribu-
tion of mucus-secreting cells. Once this defense mechanism has been altered,
a buildup of both nonviable and possibly viable inhaled substances can occur
on the epithelium and may jeopardize the health of the host, depending on the
nature of the uncleared substance. As an example, regardless of the severity
of the influenza infection, the virus concentrates initially in the lining of
the airways. The virus then spreads to alveolar cells of the lung parenchyma.
It is suspected that the macrophage might be the site of replication of this
virus (Yilyma et a!., 1979; Nayak et a!., 1964; Raut et a!., 1975).
A number of studies with various animal species have reported morpho-
logical damage to the cells of the tracheobronchial tree from acute and sub-
3
chronic exposure to 490 to 1960 ug/m (0.20 to 1.0 ppm) of 03. (See Section
9.3.1.) The cilia were either completely absent or had become noticeably
shorter or blunt. By removing these animals to a clean-air environment, the
structurally damaged cilia regenerated and appeared normal. Based on such
morphological observations, related effects such as ciliostasis, increased
mucus secretions, and a slowing of mucociliary transport rates might be ex-
pected. However, no measurable changes in ciliary beating activity have been
reported due to 0~ exposure alone. Assay of isolated trachea! rings from
3
hamsters immediately after a 3-hr exposure to 196 ug/m (0.1 ppm) of 03 showed
no significant loss in ciliary beating activity (Grose et a!., 1980). In the
3
same study, when the animals were subsequently exposed for 2 hr to 1090 ug/m
HSO* (0.30 urn volume median diameter), a significant reduction in ciliary
9-120
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beating frequency occurred. The magnitude of this effect was, however, signi-
ficantly less than that observed due to the effect of H?SCL expbsure alone.
In either case, the animals completely recovered within 72 hr when allowed to
remain in a clean-air environment. These authors found only a slight decrease
in beating frequency with a simultaneous exposure to 196 ug/m (0.1 ppm) of 0_
3
and 847 yg/m H^SCL (Grose et a!., 1982). These data indicate that 0, appears
to partially protect against the effects of H-SO^ on ciliary beating frequency.
Grose et al. (1982) proposed that the ciliary cells may be partially protected
due to the increase in mucus trachea! secretion of glycoproteins (Last and
Cross, 1978) resulting from exposure to these chemicals.
Studies cited in the previous' criteria document (U.S. Environmental
Protection Agency, 1978) gave evidence on the effect of 0,, on the host's
* O
ability to physically remove deposited particles (Table 9-11). The slowing
of mucus transport in both rat and rabbit trachea as a result of Oj exposure
was reported in the early literature (Tremer et al., 1959; Kensler and Battista,
1966). Goldstein, E., et al. (1971a,b, 1974) provided evidence that the
primary effect of 0., on the defense mechanism of the mouse lung was to diminish
*5 *
bactericidal activity but not to significantly affect physical removal of the
deposited bacteria. In these studies, mice were exposed to an aerosol of
32
P-labeled Staphylococcus aureus either after a 17-hr exposure to 0^ or
before a 4-hr exposure. Concentrations of Q, were 1180, 1370, 1570, or
3
1960 ug/m (0.6, 0.7, 0.8, or 1.0 ppm). The physical clearance and bacteri-
cidal capabilities of the lung were then measured 4 to 5 hr after bacterial
exposure. Exposure 17 hr before infection caused a significant reduction in
bactericidal activity beginning at 1960 jjg/m (1.0 ppm) of 0_. When mice were
exposed to 0, for 4 hr after being infected, there was a significant decrease
in bactericidal activity for each 0., concentration, and with increasing 0,
concentration, there was a progressive decrease in bactericidal activity. The
investigators proposed that because mucociliary clearance was unaffected by
subsequent 0,, exposure, the bactericidal effect was due to dysfunction of the
alveolar macrophage. Warshauer et al. (1974) reported that a deficiency in
vitamin E would further reduce the lungs' bactericidal activity.
Friberg et al. (1972) studied the effect of a 16-hr/day exposure to 980
•3
H9/m (0.5 ppm) of 0, on the lung clearance rate of radiolabeled monodisperse
polystyrene and iron particles in the rabbit and of bacteria in the guinea
9-121
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TABLE 9-11, EFFECTS OF OZONE OH HOST OEFEHSE MECHANISMS: DEPOSITION AND CLEARANCE
Ozone
concentration Measurement Exposure
jjgTi3 pjS method duration and protocol
196
784
784
*P 785, 1568,
H 1960
NJ
K)
1764
980
980
980, 1960
784-3979
0.1 CHEM
0.4 NBKI
0.4 NO
0.4, 0.8, UV
1.0
0.5 HAST
0.5 NBKI
0.5 NBKI
0.5, 1.0 CHEM
0.57-2.03 M
3 hr
3 hr
4 hr
4 hr
1.4 hr
16 hr/day,
7 months
2 months
2 hr
17 hr before
bacteria
Observed effects
No effect on ciliary beating frequency.
Initially lowers deposition of inhaled bacteria,
but subsequently a higher number are present
due to reproduction.
Bactericidal activity inhibited. Silicosis
did not enhance 03 effect.
Delay in nueociliary clearance, acceleration
in alveolar clearance.
Increases nasal deposition and growth of
virus; no effect in the lungs.
No effect on clearance of polystyrene and
iron particles.
•Reduced clearance of viable bacteria.
Reduced tracheal mucus velocity at 1.0 pprn.
Ho effect at O.S ppn.
• Physical clearance not affected, but bactericidal
activity affected at 0.99 ppm. Decrease in
deposition of inhaled organisms at 0.57 ppm.
Species Reference
Hamster Grose et a!
Mouse Coffin and
., 1980
Gardner, 1972b
Mouse Goldstein et al. , 1972b
Rat Kenoyer et
Mouse Fairchild,
Rabbit Friberg et
Guinea pig Friberg et
Sheep Abraham et
al., 1981
1974, 1977
al., 1972
al., 1972
al., 1980
Mouse Goldstein et al., 1971a
-------
TABLE 9-11. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: DEPOSITION AND CLEARANCE (continued)
Ozone
concentration Measurement
Exposure
ug/ms ppm method duration and protocol
y3
H
NJ
W
1176 0.62-4.25 M
1372 0.7 H
1372 0.7 G
1S68 0.8 UV
1960 1.0 NBKI
2352 1.2 UV
4 hr after
bacteria
7 days
3-4 hr
4 hr
3 hr
4 hr
Observed Effects
No effect on bacterial deposition and
clearance; reduced bactericidal activity
at each exposure level.
Deficiency of Vitamin E further reduced
bactericidal activity after 7 days.
Reduced bactericidal activity in lungs.
Slowed tracheobronchial clearance and
accelerated alveolar clearance. Effects
greater with higher humidity.
Bacteria clear lung and invade blood.
Delayed mucociliary clearance of particles.
Species Reference
Mouse Goldstein
Rat Warshauer
Mouse Bergers et
Rat Phalen et
et al . , 1971b
et al., 1974
al . , 1982
al., 1980
Mouse Coffin and Gardner, 1972b
Rat Frager et
al., 1979
Measurement method: NO = not described; CHEM = gas phase chemiluminescence; UV = UV photometry; NBKI = neutral buffered potassium iodide;
MAST = Kl-coulonetric (Mast meter); M = microcoulomb sensor; G = galvanic meter.
-------
pig. *The results from the guinea pig studies showed a reduced clearance of
viable bacteria. The rabbit's lung clearance rate was not affected by 0~. In
this latter study, however, a large number of the test animals died during
exposure from a respiratory disease, and the results must be viewed with
caution.
Recent studies have continued to examine the effects of 03 on mucociliary
transport in the intact animal. Phalen et al. (1980) attempted to quantitate
the removal rates of deposited material in the upper and lower respiratory
tract of the rat. In this study, the clearance rates of radio!abeled monodis-
perse polystyrene latex spheres were followed after 0~ exposure. A 4-hr
3
exposure to 1568 |jg/m (0.8 ppm) of 03 significantly slowed the early (tracheo-
bronchial) clearance and accelerated the late (alveolar) clearance rates at
both low (30 to 40 percent) and high (> 80 percent) relative humidity. These
effects were even greater at higher humidity, which produced nearly additive
effects. Combining 03 with various sulfates [Fe2(SO.)2, H^SO., (NH^gSO^]
gave clearance rates very similar to those for 03 alone. Accelerating the
long-term clearance from the alveoli may not in itself be harmful; however,
because this process may result from an influx of macrophages into the alveolar
region, the accumulation of excess numbers of macrophages might present a
potential health hazard because of their high content of proteolytic enzymes
and Oy free radicals, which have the capability for tissue destruction.
Essentially no data are available on the effects of prolonged exposure to 0,
on ciliary functional activity or on mucociliary transport rates measured in
the intact animal.
Frager et al. (1979) deposited insoluble, radioactive-labeled particles
via inhalation and monitored the clearance rate after a 4-hr exposure to 2352
3
ug/m (1.2 ppm) of 0~. This exposure caused a substantial delay in rapid
(mucociliary) clearance in the rat. However, if the animals were exposed 3
3
days earlier to 1568 ug/m (0.8 ppm) of 03 for 4 hr, the pre-exposure elimi-
nated this effect, resulting in a clearance rate that was essentially the same
as for controls. Thus, the pre-exposure to a lower level 3 days before rechal-
lenging with a higher concentration of 03 appeared to afford complete protec-
tion at 3 days. After a 13-day interval between the pre-exposure and the
challenges, this adaptation or tolerance was lost.
9-124
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These results were confirmed when Kenoyer et al. (1981) repeated these
2
studies, with three different concentrations of 0-, 784, 1568, and 1960 jjg/m
(0.4, 0.8, and 1.0 ppm). At each of the three concentrations, a delay was ob-
served in early (0 to 50 hr postdeposition of particles) clearance, and an
acceleration was seen in long-term (50 to 300 hr postdeposition) clearance, as
compared to controls. Concentration-response curves showed that clearance was
affected more by the higher concentrations of 0,.
The velocity of the trachea! mucus of sheep was not significantly altered
3
from a baseline value of 14.1 mm/min after a 2-hr exposure to 980 ug/m (0.5 ppm)
of 03 (Abraham et al., 1980). The authors state that 1960 ug/m3 (1 ppm) of 03
for 2 hr did significantly reduce, both immediately and 2 hr postexposure, the
trachea! mucus velocity.
9.3.4.2 AlveolarMacrophages. Within the gaseous exchange region of the
lung, the first line of defense against microorganisms and nonviable insoluble
particles is the resident population of alveolar macrophages. These cells are
responsible for a variety of important activities, including detoxification
and removal of inhaled particles, maintenance of pulmonary sterility, and
interaction with lymphoid cells for immunological protection. In addition,
macrophages act as scavengers by removing cellular debris. To adequately
fulfill their purpose, these defense cells must maintain active mobility, a
high degree of phagocytic activity, an integrated membrane structure, and a
well-developed and functioning enzyme system. Table 9-12 illustrates the
effects of 03 on the alveolar macrophage (AM).
Under normal conditions, the number of free AMs located in the alveoli is
relatively constant when measured by lavage (Brain et al., 1977, 1978).
Initially, Q-, through its cytolytic action that is probably mediated through
its action on the cell membrane, significantly reduces the total number of
these defense cells immediately after exposure (Coffin and Gardner, 1972b).
The host responds with an immediate influx of cells to aid the lung in
combating this assault. Little is known about the mechanisms of action that
stimulate this migration or about the fate of these immigrant cells. The
source of these new cells may be either (1) the influx of interstitial macro-
phages, (2) the proliferation of interstitial macrophagic precursors with
subsequent migration of the progeny into the air space, (3) migration of blood
monocytes, or (4) division of free AMs. The rapid increase in the number of
macrophages is evidently a biphasic response, arising from an early phase
9-125
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TABLE 9-12. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: HACROPHAGE ALTERATIONS
Ozone
concentration
{jg/B3
1%
1960
392
490
980
K m
a\
980
980
980
1313
980
1960
1058
PP»
0.1
1.0
0.2
0.25
0.50
0.5
0.5
0.5
0.5
0.67
0.5
1
0.54
Measurement3 ' Exposure
tnethod duration and protocol
NBKI
MAST
NBKI
NBKI
NBKI
NBKI
CHEN
NBKI
NBKI
UV
2.5 hr or
30 nin in vitro
8 hr/day for
7 days
3 hr
(in vivo and
Tn vitro)
8 hr/day for
7 days
3 hr
3 hr
3 hr
2 hr
(in vitro)
23 hr/day for
34 days
Observed effects0
Lung protective factor partially inactivated,
increasing fragility of utacrophages (concen-
tration-related).
Increased number of macrophages in lungs
(norphology).
Decreased activity of the lysosomal
enzymes lysozyme, acid phosphatase,
and p-glucuronidase.
Increased osmotic fragility.
Decreased enzyme activity and increased influx
of PMHs.
Decreased red blood cell rosette binding to
macrophages.
Decreased ability to ingest bacteria.
Decreased agglutination in the presence of
concanavalin A,
Increased number of raacrophages (morphological).
Species
Rabbit
Monkey
Rat
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Rat
Mouse
Reference
Gardner et al . , 1971
Castleaan et al., 1977
Dungworth, 1976
Stephens et al., 1976
Hurst et al., 1970
Hurst and Coffin, 1971
Dowel 1 et al., 1970
Alpert et al., 1971b
Hadley et al., 1977
Coffin et al., 1968
Coffin and Gardner, 1972b
Goldstein et al., 1977
Zitnik et al., 1978
-------
TABLE 9-12. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: MACROPHAGE ALTERATIONS (continued)
Ozone
concentration
ug/ma
1168
1568
1568
1568
& I960
1 9800
— i
SJ
^ 1960
3136
6860
ppn
0.8
0.8~
0.8
0.8
1
5
1
1.6 to
3.5
Measurement ' Exposure
method duration and protocol
NO 11 days
NO 90 days
UV 7 days
MAST 3, 7, 20 days
CHEM 3 hr
UV 4 hr
N8KI 2 hr to 3 hr
Observed effects0 Species
No effect on in vitro interferon production Mouse
with alveolar macrophages but did inhibit
the production of interferon by tracheal
epithelial cells.
Eightfold increase in number of macrophages at Rat
7 days, reducing to fourfold after 90 days.
Decreased number of migrating macrophages Monkey
and total distance migrated.
Increased phagocytosis. Rat
Decreased ability to produce interferon Rabbit
in vitro.
Decreased in vitro niarational ability, as Rat
evidenced by decreased number of macrophages able
to migrate.
Decreased superoxide anion radical production. Rat
Reference
Ibrahim et al., 1976
Booraan et al . , 1977
Schwartz and Christman, 1979
Christman and Schwartz, 1982
Shlngu et al., 1980
McAllen et al., 1981
Amoruso et al . , 1981
Witz et al., 1983
4900
2.5
5 hr
Loss of p-glucuronidase and acid phosphatase in
. PAH with ingested bacteria; decreased rate of
bacterial ingestion.
Rat
Goldstein et al., 1978b
4900
2.5
5 hr
Diminished rate of bacterial killing, increased
numbers of intracellular staphylococcal clumps;
lack of lysozyme in macrophages with staphylococcal
clumps.
Rat
Kimura and Goldstein, 1981
aMeasure«ent method: NO = not described; CHEM = gas phase chemiluminescence; UV = UV photometry; NBKI = neutral buffered potassium iodide;
MAST = Kl-coulometrlc (Mast meter); H = nicrocoulomb sensor.
Calibration method: NBKI = neutral buffered potassium iodide.
Abbreviations used: PMN = polymorphonuclear leukocytes; PAM = pulmonary alveolar macrophage.
-------
apparently correlated to a local cellular response and a later phase of inter-
stitial cell proliferation, which is responsible for the maintenance of the
high influx of macrophages (Brain et al., 1978).
Morphological studies have supported the observation that exposure to 03
can result in a raacrophage influx in several animal species. Exposure of mice
3
to 1058 ug/m (0.54 ppm) of 03 23 hr/day for a maximum of 34 days resulted in
an increased number of macrophages within the proximal alveolar ducts (Zitnik
et al., 1978). These cells were highly vacuolated and contained many secondary
i
phagocytic vacuoles filled with cellular debris. The effect was most prominent
after 7 days of exposure and became less evident as the exposure continued.
This observation correlated with the finding in rats of an eightfold increase
q
in the number of pulmonary free cells after exposure to 1568 pg/m (0.8 ppm)
of 0- for 7 days, but only a fourfold increase after exposure for 90 days
(Brummer et al., 1977; Boorman et al., 1977). In similar studies, other
authors found that exposure of both monkeys and rats to concentrations as low
g
as 392 ug/m (0.2 ppm) of 0, for 8 hr/day on 7 consecutive days resulted in an
accumulation of macrophages in the lungs of these exposed animals (Castleman
et al., 1977; Dungworth, 1976; Stephens et al., 1976). The data from these
studies suggest that these two species of animals are approximately equal in
susceptibility to the short-term effects of CU.
Thus, the total available data would indicate that, after short periods
of Og insult, there is a significant reduction in the number of free macrophages
available for pulmonary defense, and that these macrophages are more fragile,
are less phagocytic, and have decreased enzymatic activity (Dowel! et al.,
1970; Coffin et al., 1968; Coffin and Gardner, 1972b; Hurst et al., 1970).
However, histological studies have reported that with longer exposure periods,
there is an influx and accumulation of macrophages within the airways. Such a
marked accumulation of macrophages within alveoli may appear to be a reasonable
response to the immediate insult, but it has been speculated that the conse-
quences of this mass recruitment may also be instrumental in the development
of future pulmonary disease due to the release of proteolytic enzymes by the
AMs (Brain, 1980; Menzel et al., 1983).
A number of integrated steps are involved in phagocytosis processes, the
first being the ability of the macrophage to migrate to the foreign substance
on stimulus. McAllen et al. (1981) studied the effects of 1960 ug/m3 (1.0
ppm) of 03 for 4 hr on the migration rate of AMs. Migration was measured by
9-128
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determining the area macrophages could clear of gold-colloid particles that
had been previously precipitated onto cover slips. In this study, it was not
clear whether the gold was actually ingested or merely adhered to the outer
surface of the cell. Nevertheless, the cells from 0~-exposed rats appeared
less mobile, in that they migrated 50 percent less than the sham-exposed
group.
A decrease in the ability of macrophages to phagocytize bacteria after
3
exposure to concentrations as low as 980 jjg/m (0.5 ppm) of 03 for 3 hr was
demonstrated by Coffin et al. (1968) using rabbits. However, Christman and
Schwartz (1982) may have demonstrated that with longer exposure periods, the
effects may be different (i.e., the phagocytic rate may increase). In this
study, rats were exposed to 1568 (jg/m (0.8 ppm) of 0, for 3, 7 or 20 days; at
those times the macrophages were isolated, allowed to adhere to glass, and
incubated with carbon-coated latex microspheres. The percentages of phagocytic
cells were determined at 0.25, 0.5, I, 2, 4, 8 and 24 hr of incubation. At
all exposure time periods tested, the number of spheres engulfed had increased.
The greatest increase in phagocytic activity was observed after 3 days of
exposure. The exposed cells engulfed a greater number of spheres than controls,
and a larger percentage of macrophages from exposed animals was phagocytic.
This enhancement correlated well with a significant increase in cell spreading
of AMs from exposed rats as compared to controls. If longer-term 0-, exposure
enhances macrophage function and causes a migration of macrophages into the
lung, the comparisons of function of these new cells with controls may not be
valid, because these new cells are biochemically younger. Another significant
problem with this study is that the cells examined were a selected population
because only the cells that adhered to the glass surface were available for
study. The cells that did not adhere were removed by washing. In this study,
only 51 percent of the collected cells adhered after 3 days of Q3 exposure,
compared to 85 percent of the controls. Although the effects of 0- on cell
attachment have not been studied directly, there is evidence that 03 affects
AM membranes involved in the attachment process (Hadley et al., 1977; Dowell
et al., 1970; Aranyi et al., 1976; Goldstein et al., 1977). The cells most
affected by the cytotoxic action of the 03 exposure might never have been
tested, because they were discarded.
Goldstein et al. (1977) studied the effect of a 2-hr exposure on the
ability of AM to be agglutinated by concanavalin-A, a parameter reflecting
9-129
-------
membrane organization. A decrease in agglutination of rat AMs was found after
o
exposure to 980 or 1960 ug/m (0.5 or 1.0 ppm) of 03. A decre>ase in concanavalin-
A agglutinability of trypsinized red blood cells obtained from rats exposed
3
for 2 hr to 1960 ug/m (1 ppm) was also noted. Hadley et al. (1977) investigated
o
AM membrane receptors from rabbits exposed to 980 ug/m (0.5 ppm) of 0, for
3 hr. Following 0_ exposure, lectin-treated AMs have increased rosette forma-
tion with rabbit red blood cells. The authors hypothesized that the O^induced
response indicates alterations of macrophage membrane receptors for the wheat
germ agglutinin that may lead to changes in the recognitive ability of the
cell.
Ehrlich et al. (1979) studied the effects of 0, and N02 mixtures on the
activity of AMs isolated from the lungs of mice exposed for 1, 2, and 3 months.
3
Only after a 3-month exposure to the mixture of 196 ug/m (0.1 ppm) of 03 and
0.5 ppm of NOp (3 hr/day, 5 days/week) did viability in macrophages decrease
significantly. Iji vitro phagocytic activity was also not affected by a 1-month
exposure to this level of pollutants, but after 2- and 3-month exposures the
percentage of macrophages that had phagocytic activity decreased significantly.
It has been reported that the acellular fluid that lines the lungs also
plays an important role in defense of the lung through its interaction with
pulmonary macrophages (Gardner et al., 1971; Gardner and Graham, 1977). These
studies demonstrated that the protective components of this acellular fluid
o
can be inactivated by a 2.5-hr exposure to 03 levels as low as 196 ug/m
(0.1 ppm). When normal AM's are placed in fluid lavaged from 03-exposed
animals, they showed an increase in lysis (10 percent over control). A similar
effect was seen when normal AM's were placed in protective fluid that had been
exposed ui vitro to 03. The data indicate that some of the effects of 03 on
lung cells may be mediated through this lung lining fluid. Schwartz and
Christman (1979) provided evidence that normal lung lining material enhanced
macrophage migration, but the macrophages obtained from rhesus monkeys after
o
exposure for 7 days to 1568 ug/m (0.8 ppm) of 03 demonstrated both a decrease
in the number of cells that migrated (28 percent of control value) and in the
total distance they traveled (71 percent of control value). Adding normal
lining fluid to isolated 03~exposed macrophages did enhance the migration, but
it was still significantly less than controls.
Macrophages are rich in lysosomal enzymes. Because these enzymes are
crucial in the functioning of the macrophage, perturbation of the metabolic
9-130
-------
or enzymatic mechanisms of these cells may have Important consequences on the
abilities of the lung to defend itself against disease. Enzymes that have
been identified include acid phosphatase, acid ribonuclease, beta galactosidase,
beta glucuronidase, cytochrome oxidase, lipase, lysozyme, and protease. Ozone
decreased significantly the activity levels of lysozyme, p-glucuronidase, and
acid phosphatase in macrophages after a 3-hr exposure of rabbits to concentra-
q
tions as low as 490 |jg/m (0.25 ppm) of 0- (Hurst et al,, 1970). Such enzymatic
reductions were also observed in AMs exposed if} vitro (Hurst and Coffin, 1971).
The ability of 0, to alter macrophages1 enzyme activity was also studied by
means of unilateral lung exposure of rabbits (Alpert et af., 1971b). A sig-
nificant reduction in these same three intracellular enzymes was found to be
specific to the lung that breathed 0,, rather than a generalized systemic re-
3
sponse. These effects were concentration-related, beginning at 980 ug/m (0.5
ppm) of 0™. The extracellular release of such enzymes may occur either as a
result of direct cytotoxic damage and leakage of intracellular contents, or
they may be selectively released without any cell injury. Hurst and Coffin (1971)
showed that the reduction in intracellular lysosomal enzyme activity observed
after ID vitro exposure coincides with the release of the enzyme into the sur-
rounding medium. In these studies, the sum of the intra- plus extracellular
enzyme activity did not equal the total activity, indicating that the pollutant
itself can inactivate the hydrolytic enzyme as well as alter the cell mem-
brane. Recently, Witz et al. (1983) and Amoruso et al. (1981) reported that
in vivo CU exposure affected the production of superoxide anion radicals (CL)
4 t
by rat AMs. This oxygen radical is important in antibacterial activity.
3
Exposure to concentrations above 3136 ug/m (1.6 ppm) of 0™ for 2 hr appears
to result in a progressive decrease in (}„ production. No statistical evaluation
of the data was performed. The type of membrane damage as well as the mecha-
nisms by which this damage is incurred are not well understood. It is not
known .whether the 0,-induced inhibition of CL... production arises from the
direct oxidative damage of the membrane enzyme involved in the metabolism of
Oy to 02, or whether it is a result of oxidative degradation of membrane
lipids that may serve a cofactor function.
Shingu et al. (1980) reported the effects of 0™ on the ability of two
cell types, macrophages and tonsiliar lymphocytes, to produce interferon, a
substance that aids in defending the host organism against viral infections.
3
Macrophages from rabbits exposed to 1960 ug/m (1.0 ppm ) of 0, for 3 hr ex-
hibited a depression in interferon production. Interferon production by
9-131
-------
tonsillar lymphocytes was not significantly depressed by 9800 (jg/m (5.0 ppm)
of 0-. The authors suggested that an impairment of interferon production
might play an important role in the ability of the host to combat respiratory
viral i>fortiops. In neither of the studies were statistical analyses of the
data reported. Ibrahim et al. (1976) also exposed mice to 0- and illustrated
3
that 1568 ug/m (0.8 ppm) of 03 for a period of 11 days inhibited the in vitro
ability of tracheal epithelial cells to produce interferon, but no effect was
observed with alveolar macrophages.
9.3.4.3 Interaction with Infectious Agents. In general, the consequences of
any toxic response depend on the particular cell or organ affected, the severity
of the damage, and the capability of the impaired cells or tissue to recover
from the assault. , Do small decrements in the functioning of these various
host defense mechanisms compromise the host so that it is unable to defend
itself against a wide variety of opportunistic pathogens? It has been sugges-
ted from epidemiological data that exposure to ambient levels of oxidants can
enhance the development of respiratory infection in humans (Dohan et al.,
1962; Thompson et al., 1970). Measurement of the competency of the host's
antimicrobial mechanisms can best be tested by challenging both the toxic-
exposed animals and the clean-air exposed control animals to an aerosol of
viable microorganisms. If the test substance, such as 03, had any adverse
influence on the efficiency of any of the host's many protective mechanisms
(i.e., physical clearance via the mucociliary escalator, biocidal activity
mediated through macrophages, and associated cellular and humoral immunological
events) that would normally function in defense against a microbe, the microbe,
in its attempt to survive, would take advantage of these weaknesses. A detailed
description of the infectivity model commonly used for 03 studies has been
published elsewhere (Coffin and Gardner, 1972b; Ehrlich et al., 1979; Gardner,
1982a). Briefly, animals are randomly selected to be exposed to either clean
air or 03. After the exposure ends, the animals from both chambers are combined
and exposed to an aerosol of viable microorganisms. The vast majority of
these studies have been conducted with Streptococcus sp. At the termination
of this 15- to 20-min exposure, the animals are housed in clean air, and the
rate of mortality in the two groups is determined during a 15-day holding
period. In this system, the concentrations of 03 used do not cause any mortal-
ity. The mortality in the control group (clean air plus exposure to the
microorganism) is approximately 10 to 20 percent and reflects the natural
9-132
-------
resistance of the host to the infectious agent. The difference in mortality
between the 0~-exposure group and the controls is concentration-related (Gardner,
1982a). No studies have yet been conducted to determine the.lowest possible
number of viable microbes that when deposited in the lung will cause a pulmon-
ary infection in the host exposed to 0,. Miller et al. (1978a) examined the
cumulative data from nearly 3,000 control mice used in these infectivity
studies and determined that within the range of 200 to 4,000 colony-forming
units per lung, there is no correlation between number of bacteria deposited
in the lung and the resulting increase in percent mortality. It must be noted
that in these studies, the control animals are capable of maintaining pulmonary
sterility by inactivating the microbes that are deposited in the lung.
If the test agent does not impair the host's defense mechanisms, there is
a rapid inactivation of inhaled microorganisms that have been deposited in the
respiratory system. However, if the chemical exposure alters the ability of
these defense cells to function, i.e., rate of bacterial killing, the number
of microbes in the lungs could increase (Coffin and Gardner, 1972b; Miller et
al., 1978; Gardner, 1982a). This acceleration in bacterial growth has been
attributed to the pollutant's alteration of the capability of the lung to
destroy the inhaled bacteria, thus permitting those with pathogenic potential
to multiply and produce respiratory pneumonia. With this accelerating growth,
there is an invasion of the blood, and death has been predicted from a positive
blood culture (Coffin and Gardner, 1972b).
Coffin et al. (1967) treated mice with 03 (0.08 ppm and higher levels for
3 hr) arid subsequently exposed them to an aerosol of infectious Streptococcus
sp. In this study, 03 increased the animals' susceptibility to infection,
resulting in a significant increase in mortality rate in the O.-treated group.
Ehrlich et al. (1977), when using the same bacteria but a different strain of
mice, found a similar effect at 0.1 ppm for 3 hr. When using CD-I mice and
Streptococcus sp., Miller et al. (1978) studied the effects of a 3-hr exposure
3
to 0-j at 196 MQ/tn (0.1 ppm) in which the bacterial aerosol was administered
either immediately or 2, 4, or 6 hr after cessation of the 0- exposure. For
«5
these postexposure challenges, only the 2-hr time resulted in a significant
increase in mortality (6.7 percent) over controls. However, when the animals
were infected with streptococci during the actual 03 exposure, a significant
increase in mortality of 21 percent was observed. When this latter experimental
3
regimen was used, exposure to 157 ug/m (0.08 ppm) of 0- resulted in a signifi-
cant increase in mortality of 5.4 percent.
9-133
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The differences in results among these studies may have been due to a
variation in the sensitivity of the method of 03 monitoring, a difference in
mouse strain, changes in the pathogenicity of the bacteria, or "differences in
sample size. The results from such studies that use this infectivity model
indicate the model's sensitivity for detecting biological effects at low
pollutant concentrations and its response to modifications in technique (i.e.,
using different mouse strains or varying the time of bacterial challenge).
The model is supported by experimental evidence showing that pollutants
(albeit at different concentrations) that cause an enhancement of mortality in
the infectivity system also cause reductions in essential host defense
systems, such as pulmonary bactericidal capability, the functioning of the
alveolar macrophage, and the cytological and biochemical integrity of the
alveolar macrophage.
The pulmonary defenses in the (^-treatment group were significantly less
effective in combating the infectious agent to the extent that, even at low
concentrations, there were significant increases in mortality over controls.
As the 03 concentration increased, mortality increased. In some studies,
additive effects were reported. These effects, increases in respiratory
infection, are supported by many mechanistic studies discussed in this
section. They indicate that 03 does effectively cause a reduction in a number
of essential host defenses that would normally play a major role in fighting
pulmonary infections. Since 1978, a number of new studies have continued to
confirm these previous findings and improve the existing data base (Table
9-13).
o
When mice were exposed 4 hr to 392 to 1372 pg/m (0.2 to 0.7 ppm) of 03
and then challenged with virulent Klebsiella pneumoniae, a significant in-
3
crease in mortality was noted at 785 (jg/m (0.4 ppm) 0, (Bergers et al.,
1983). Groups of 30 mice inhaled approximately 30, 100, and 300 bacteria/
3
mouse. At 392 ug/m (0.2 ppm) of 03, the 03 group showed an increase in
mortality, but it was not significantly different from controls. At 785 pg/m
(0.4 ppm) of 03 a nearly threefold lowering of the bacterial ID™ value compu-
ted from the three challenge doses of bacteria was found for the 03~exposed
group, indicating a significant increase in mortality. In the same study, the
o
authors also found that 1372 ug/m (0.7 ppm) of 03 for 3 hr resulted in a
decreased ability of the lung to clear (bactericidal) inhaled staphylococci.
9-134
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TABLE 9-13. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: INTERACTIONS WITH INFECTIOUS AGENTS
Ozone
concentration Measurement Exposure
ug/n3 ppm method
157 0.08 NO
157-196 0.08, 0.1 CHEM
196 0.1 CHEM
196 0.1 UV
196, 588 0.1, 0.3 CHEM
T 392-1372 0.2-0.7 G
M
to
Ul
588 0.3 NO
1372-1764 0.7-0.9 NO
1960 1.0 CHEM
2940 1.5 NO
980 0.5 UV
1254 0.64 CHEM
duration and protocol
3 hr
3 hr
3 hr
5 hr/day,
5 days/wk for
103 days
3 hr
3-4 hr
3 hr/day for
2 days
3 hr
3 hr/day,
5 days/wk
for 8 weeks
4 hr/day,
5 days/wk
for 2 months
2-4 wks
4 wks
Observed effects
Increase in mortality to Streptococcus sp.
Significant increase in mortality during 0%
exposure (Streptococcus sp. ).
Increased mortality to Streptococcus sj>.
Increased susceptibility to bacterial infection
(Streptococcus sp. ).
Exercise enhances mortality in
infectivity model system.
Significant increase in mortality following
challenge with aerosol of Klebsiella pneumoniae.
Effect seen at 0.4 ppm.
Enhancement of severity of bacterial pneumonia
(Pasteurella haeinolytica).
Increased susceptibility to infection
(Streptococcus sp. ).
Increase in Mycobacterium tuberculosis lung titers.
No effect on resistance to Hycofaacterium
tuberculosis.
Reduced widespread viral infection of the lung,
resulting in a decrease in disease severity.
No enhancement of the severity of chronic
pulmonary infection with Pseudomonas aeruginosa.
Species
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Sheep
Mouse •
Mouse
Mouse
Mouse
Rat
Reference
Coffin et'al. ,
Miller et al.,
Ehrlich et al.
Aranyi et al . ,
11 ling et al. ,
Bergers et al.
Abraham et al.
1967
1978a
!
,; 1977
1983
1980
, 1983
, 1982
Coffin and Blommer, 1970
Thomas et al . ,
Thienas et al.
Wolcott et al.
Sherwood et al
1981b
, 1965
, 1982
;, 1984
Measurement method: ND = not described; CHEM = gas phase chemiluminescence; UV = UV photometry; G = galvanic meter.
-------
This finding is consistent with that of Goldstein et al. (1971b) and confirms
previous studies reported by Miller and Ehrlich (1958) and Ehrlich (1963).
Sherwood et al. (1984) established a chronic pulmonary infection in rats
by inoculating agar beads containing viable Pseudomonas aeruginosa (PAO-381)
3
and then exposed these infected animals for 4 wks to 1254 ug/m (0.64 ppm) 0^.
The exposure to ozone did not affect the pulmonary antibacterial defense
systems—i.e., no increase in number of organisms cultured from the lung—but
it did cause significant anatomical damages. The lungs of rats exposed to 0~
were larger and heavier when compared with controls, and had an increased
number of macrophages in their terminal bronchioles. The authors state that
the reason why these results are different than those described above for the
"infectivity model" is that in these later studies, the infective organisms
are given by inhalation and are therefore deposited throughout the lung and
the pollutant was able to interfere with the initial phase of the host-parasite
interaction. In this chronic study, the infection was isolated to the distal
area of the lung and it was in its later stage of development when the animals
received the 0« exposure. Thus, the timing of the exposure to 0~ may be a
significant factor in the impairment of the lung's antibacterial defenses.
Exposure to ambient levels of 0, (0.5 ppm) for 2 to 4 wks has been shown
to alter the pathogenesis of respiratory infection of mice with influenza A -
virus (Wolcott et al., 1982). The 03-exposed animals showed a reduction in
severity of the disease (less mortality) and an increase in survival time.
The reduction of disease severity appears to be dependent on the continuous
presence of the DO during the infectious process. This effect did not corre-
late with virus, interferon, or neutralizing antibody titers recovered from
the lung, or with neutralizing antibody titers in the sera. The reduced
disease severity in the 0~-exposed animals appears to be due to significant
alterations in the distribution of viral antigens within the pulmonary tissues,
i.e., less widespread infection of the lung.
Chiappino and Vigiani (1982) also reported that 0^ potentiated pulmonary
infection in rats. In this study, the investigators wanted to know in what
way On modified the reactions to silica in specific pathogen-free (SPF) rats.
3
Silica-treated animals were exposed to 1960 ug/m (1.0 ppm) 0, (8 hr/day, 5
days a week for up to 1 year) and housed in either an SPF environment or in a
conventional animal house, thus being exposed to the bacterial flora normally
present there. The SPF-maintained, 0,-exposed rats showed a complete absence
9-136
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of saprophytes and pathogens in the lungs, whereas the mlcrobial flora of the
lungs of the conventionally kept rats, also exposed, consisted of staphylococci
(2500 to 4000 per gram of tissue) and streptococci (300 to 800 per gram of
tissue). The lungs of these rats showed bronchitis, purulent bronchiolitis,
and foci of pneumonia. The exposure to 03 did not have any effect on particle
retention, nor did it modify the lungs' reaction to silica, but it did increase
the animals' susceptibility to respiratory infections.
Changes in susceptibility to infection resulting from 03 exposure have
also been tested in sheep. In these studies, sheep were infected by an inocu-
lation of Pasteurella haemolytica either 2 days before being exposed to 588
3
ug/m (0.3 ppm) of 03 or 2 days after the CL exposure. In both cases, the Q3
exposure was for 3 hr/day for 2 days. Ozone enhanced the severity of the
disease (volume of consolidated lung tissues), with the greatest effect seen
when the 0, insult followed the exposure to the bacteria (Abraham et a!.,
1982). Unfortunately, only a small number of animals were used in each Q3
treatment (n = 3).
Thomas et al. (l981b) studied the effects of single and multiple expo-
sures to 0, on the susceptibility of mice to experimental tuberculosis.
3
Multiple exposures to 1960 ug/m (1.0 ppm) of 0~ 3 hr/day, 5 days/week for up
to 8 weeks, initiated 7 or 14 days after the infectious challenge with
Mycobacten'um tuberculosis H37RV, resulted in significantly increased bacterial
lung titers, as compared with controls. Exposure to lower concentrations of
03 did not produce any significant effects. In an earlier study, Thienes et
al. (1965) reported that exposure to 2940 ug/m3 (1.5 ppm) of 03 4 hr/day, 5
days/week for 2 months also did not alter the resistance of mice to M._
tuberculosis H37RV, but he did not measure lung titers.
Table 9-14 summarizes a number of studies that used mixtures of pollutants
in their exposure regimes. Ehrlich et al. (1979) and Ehrlich (1983) expanded
the earlier 3-hr-exposure studies to determine the effects of longer periods
of exposure to 0- and N09 mixtures. In the earlier studies (Ehrlich et al.,
3
1977), they reported that exposures to mixtures containing 196 to 980 ug/m
(0.1 to 0.5 ppm) of-03 and 2920 to 9400 ug/m3 (1.5 to 5 ppm) of N02 produced
an additive effect expressed as an increased susceptibility to streptococcal
pneumonia. In the later studies, mice were exposed 3 hr/day, 5 days/week for
3 3
up to 6 months to mixtures of 196 |jg/m (0.1 ppm) of 03 and 940 ug/m (0.5
ppm) of NOp and challenged with bacterial aerosol. The 1- or 2-month exposure
9-137
-------
TABLE 9-14. EFFECTS OF OZONE ON HOST DEFENSE HECBANISHS: MIXTURES
Ozone
concentration
(jg/B3 pprn
98 0.05
3760
98-196 0.05-0.1
100-400
1500
196 0.1
241-483
V, 196 0.1
f 900
H
00
00
196 0.1
1090
196 0.1
940
196 0.1
940
196 0.1
13200
1040
Pollutant
03 +
MOj,
034
W2 +
ZnS04
03 +
H2S04
03 +
H2S04
03 +
H2S04
0,+
H02
03 +
N02
03 +
S02 +
(NH4)2SO«
Keasurenent3 Exposure
•ethod duration and protocol
CHEH 3 hr
CHEM 3 hr
CHEH 3 hr
CHEH 3 hr +
2 hr
CHEH 3 hr +
2 hr
CHEH 3 months
CHEH 3 hr/day for
1-6 months
UV 5 hr/day,
5 days/wk
for 103 days
h
Observed effects Species Reference
Exposure to Mixtures caused synergistic Mouse Ehrlich et al., 1977,
effect after multiple exposures; additive 1979, 1980
effect after single exposure.
Additive effect of pollutant nixtures House Ehrlich, 1983
with infecti vity node!.
Increased susceptibility to Streptococcus Mouse Grose et al., 1982
pyogenes.
Sequential exposure resulted in signifi- House Gardner et al., 1977
cant increase in respiratory infection.
Neither alone produced a significant
effect. .
Sequential exposure resulted in signifi- Hamster Grose et al., 1980
cant reduction in ciliary beating
activity over H2S04 alone.
Significant decrease in viability of Mouse Ehrlich et al., 1979
alveolar macrophages seen with mixtures.
At 3 and 6 months, susceptibility to Mouse Ehrlich, 1980, 1983
pulmonary infection increased sig-
nificantly. Delayed clearance rate.
Highly signifi cant -increase in Mouse Aranyi et al.s 1983
susceptibility to infection. Effects
attributed to 03. Increased 'bactericidal
rate over 03 alone. Mixture showed greater
growth inhibition in leukemia target cells
and an Increase in blastogenic response to
PHA, Con-A, and alloantigens.
-------
TABLE 9-14. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: MIXTURES (continued)
10
H
U)
Ozone
concentration
ug/mj ppm
216-784 0.11-0.4
3760-13720 2-7.3
980 0.5
11-3000
1570 0.8
3500
3500
3500
Pol 1 utant
03 +
N02
03 +
Hj,S04
03 +
Fe2(S04)3 +
H2S04 +
(NH4)2S04
Measurement9 Exposure
method duration and protocol
A 17 hr before
bacteria or
4 hr after
bacterial
exposure
UV 3 and 14 days
UV 4 hr
Observed effects Species Reference
Physical removal of bacteria not Mouse Goldstein, E. , et al.,
affected. Bactericidal activity 1974
reduced at higher concentrations.
Significant increase in glycoprotein Rat Last and Cross, 1978
secretion; synergism reported;
effect reversible in clean air.
Exposure to mixtures produced same Rat Phalen et al., 1980
effect as exposure to 03 alone.
Measurement method: CHEM = gas phase chemiluminescence; UV = UV photometry; A = amperometric.
Abbreviations used: PHA = phytohemagglutinin; Con-A = concanavalin-A
-------
did not induce any significant changes in susceptibility to streptococcal
infection. After 3 and 6 months of exposure, the resistance to infection was
significantly reduced. If the mice were re-exposed to the 03 and NO, mixture
after the infectious challenge, a significant increase in mortality rate could
be detected 1 month earlier. The clearance rate of inhaled viable streptococci
from the lungs also became significantly slower after the 3-month exposure to
this oxidant mixture.
In more complex exposure studies, mice were exposed to a background
concentration of 188 ug/m (0.1 ppm) of N09 for 24 hr/day, 7 days/week with a
3
superimposed 3-hr daily peak (5 days/week) containing a mixture of 196 (jg/m
o
(0.1 ppm) of 0- and 940 ug/m (0.5 ppm) of NOp. Mortality rates from strepto-
coccal infection were not altered by 1- and 2-month exposures, but a marked,
although only marginally significant (p <-0.1), increase was seen after a
6-month exposure.
The same laboratory (Aranyi et al., 1983) recently reported a study in
which mice were exposed 5 hr daily, 5 days/week up to 103 days to 0., and a
3
mixture of 0,, SO,,, and (NH4)2SO,. The concentrations were 196 pg/m (0.1 ppm)
of 03> 13.2 mg/m of SOg, and 1.04 mg/m3 of (NH4)2S04> Both groups showed a
highly significant overall increase in mortality, compared to control mice
exposed to filtered air. However, the two exposure groups did not differ,
indicating that 0, was the major constituent of the mixture affecting the
host's susceptibility to infection. These investigators also measured a
number of specific host defenses to determine if host response td the mixtures
was significantly different from that of the controls or of the 0,-only groups.
Neither the total number, differential, nor the ATP levels of macrophages
differed from controls in either group, but the complex mixture did produce a
significant increase in bactericidal activity over the 03-alone and the control
animals.
Previous studies (Gardner et a!., 1977) indicated that a sequential expo-
3 -3
sure to 196 ug/m (0.1 ppm) 0~ followed by 1000 ug/m H^SO, significantly
increased streptococcal pneumonia-induced mortality rates in mice. Ozone and
HgSth had an additive effect when exposed in this' sequence. However, the
reverse sequence did not affect incidence of mortality. Sulfuric acid alone
caused no significant effect. Grose et al. (1982) expanded these studies and
3
showed that a 3-hr exposure to the mixture of 196 ug/m (0.1 ppm) of 0, and
9-140
-------
3
483 or 241 pg/rn HUSO, also significantly increased the percent mortality, as
compared to control.
The effects of exercise on the response to low levels of 03 were also
3
studied by using the infectivity model. Mice were exposed to 196 |jg/m (0.1 ppm)
3
of 0™ and 588 |jg/m (0.3 ppm) of ()„ for 3 hr while exercising. Each exposure
level yielded mortality rates that were significantly higher than those observed
in the 03 group that was not exercised (Illing et al.f 1980). Such activity
could change pulmonary dosimetry, thus increasing the amount of 0- reaching
the respiratory system. Thus, such studies clearly demonstrate that the
activity level of the exposed subjects is an important concomitant variable
influencing the determination of the lowest effective concentration of the
pollutant.
9.3.4.4 Immunology. In addition to the above nonspecific, nonselective
mechanisms of pulmonary defense, the respiratory system also is provided with
specific, immunologic mechanisms, which can be activated by inhaled antigens.
There are two types of immune mechanisms: " antibody (humoral)-mediated and
cell-mediated. Both serve to protect the respiratory tract against inhaled
pathogens. Much less information is available on how 0, reacts with these
immunological defenses than is known about the macrophage system (see Table
9-15). Most studies have involved the systemic immune system which, to a
degree, is compartmentalized from the pulmonary immune system.
The effects of 2900 ug/m3 (1.48 ppm) of 03 for 3 hr on cell-mediated
immune response were studied by Thomas et al. (1981b), who determined the
cutaneous delayed hypersensitivity reaction to purified protein derivative
(PPD), expressed as the diameter of erythemas. In the guinea pigs infected
with inhaled Mycobacterium tuberculosis, the cutaneous sensitivity to PPD was
significantly affected by 0,,. The diameters of the erythemas from the 0,-exposed
animals were significantly smaller during the 4 to 7 weeks after the infectious
challenge, indicating depressed cell-mediated immune response. Exposure to
980 jjg/m3 (0.5 ppm) of Q3 had no effect.
The systemic immune system was studied by Aranyi et al. (1983), who ob-
served the blastogenic response of splenic lymphocytes to mitogens and allo-
antigens and plaque-forming cells' response to sheep red blood cells after a
chronic exposure to 196 ug/m (0.1 ppm) of 03 5 hr/day, 5 days/week for 90 days.
No alteration in the response to alloantigens or the B-ceTl mitogen lipopoly-
saccharide (IPS) was noted, but a statistically significant suppression in
9-141
-------
TABLE 9-15. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: IMMUNOLOGY
Ozone
concentration Measurement Exposure
pi'/i3 ppl method duration and protocol
1% 0.1 UV 5 hr/day,
5 days/wk for
90 days
980, 2940 0,5, 1,5 NO 4 hr
,1 980, 1568 0.5, 0.8 Hast Continuous
*j 3-4 days
980-2900 0.5-1.48 CHEH 3 hr
1150 0.59 'ND 36 days
1568 0.8 NO 1, 3, 7, and
14 days
* Observed effects
Significant suppression in blastogenesis to
T-cell mitogen, PHA, and Con-A. No effect
on B-cell mitogen , LPS, or alloantigen of
splenic lymphocytes.
Attempt to increase immune activity with drug
Levandsole failed.
Increase in number of IgE- and IgA-containing
cells in the lung, resulting in an increase
in anaphylactic sensitivity.
Depressed cell -mediated immunity. No effect at
0.5 ppra for 5 days. Hemaggluti nation antibody
titers increased over control.
Impaired resistance to toxin stress.
Itnaunosuppression.
Depressed splenic lymphocyte response to
T-cell dependent antigen that correlated with
changes in thymus weights.
Species Reference
House Aranyi et al . , 1983
House Goldstein et al., 1978a
House Osebold et al., 1979, 1980
Gershwin et al. , 1981
Guinea pig Thomas et al,, 1981b
Mouse Campbell and Hilsenroth,
1976
Mouse Fujimaki et al., 1984
Measurement method: ND = not described; CHEH = gas phase chemiluninescence; UV = UV photometry; Mast = Mast meter.
Abbreviations used: PHA = phytohemagglutinin; con-A = concanavalin-A; LPS = lipopolysaccharide; IgE = imnunoglobulin-E', IgA = inraiunoglobulin-A.
-------
blastogenesis to the T-cell mitogens (PHA and Con-A) was detected. The authors
suggested that the cellular mechanisms for recognition and proliferation to
IPS and alloantigens were intact, but CL might have been interfering with the
cellular response to PHA and Con-A through alterations in cell surface receptors
or the binding of specific mitogens to them. Ozone exposure enhances peritoneal
macrophage cytostasis to tumor target cells. There was no effect on the
ability of splenic lymphocytes to produce antibodies against injected antigen
(red blood cells).
The effects of 0™ on the humoral immune response was also studied by
3
Fujimaki et al. (1984). BALB/c mice that were exposed continuously to 1568 pg/m
(0.8 ppm) had an increase in lung weight after 3, 7, and 14 days of exposure.
Spleen weights were decreased only after 1 and 3 days of exposure and then
returned to normal. The thymus weight was decreased at all time periods
tested, i.e., 1, 3, 7, and 14 days. Similar mice exposed to 0.4 ppm failed to
show any marked changes in the organ weights. At 0.8 ppm 03, exposure depressed
the antibody response to sheep red blood cells (T-cell dependent antigen), but
not at the antibody response to the T-cell independent antigen. These changes
were correlated with the changes of thymus weights. The authors also concluded
that 03 affected mainly the T-cell population rather than the B-cell population.
Campbell and Hilsenroth (1976) used a toxoid immunization-toxin challenge
approach to determine if continuous exposure to 1150 |jg/m (0.59 ppm) of 0,
for 36 days impaired resistance to a toxin stress. Mice were immunized with
tetanus toxoid on the fifth day of 0,, exposure and challenged with the tetanus
toxin on day 27. Compared with controls, the 0,,-exposed animals had greater
mortality and morbidity following the challenge. The authors suggested that
the effect was due to immunosuppression.
Because the data indicate that 0~ can alter the functioning of pulmonary
macrophages, Goldstein et al. (1978a) tried to counteract this effect by using
a known immunologic stimulant, Levamisole, which protects rodents against
systemic infections of staphylococci and streptococci. The purpose was to
determine if this drug might repair this dysfunction and improve the bacteri-
cidal activity of the macrophage. In this study, two concentrations were
3
tested, 2940 and 980 pg/m (0.5 and 1.5 ppm) of 03 for 4 hr. In no case did
Levamisole improve the bactericidal activity of the 0~-exposed macrophages.
The cells still failed to respond normally.
9-143
-------
The possibility that 0~ may be responsible for the enhancement of allergic
sensitization has important implications for human health effects. Gershwin
et al. (1981) reported that 03 (0.8 and 0.5 ppm for 4 days) exposure caused a
thirty-fourfold increase in the number of IgE-containing cells in the lungs of
mice that had been previously exposed to aerosolized ovalbumin. In general,
the number of IgE-containing cells correlated positively with levels of anaphy-
lactic sensitivity, Oxidant damage (0.5 to 0.8 ppm for 4 days) also causes an
Increase in IgA-containing cells in the lungs,and a rise in IgA content in
respiratory secretions and accumulation of lymphoid tissue along the airways
(Osebold et al., 1979). The number of IgM and IgG containing cells did not
increase. These authors showed that a significant increase in anaphylactic
sensitivity occurred when antigen-stimulated and O^-exposed animals were
compared to controls (Osebold et al., 1980). Significantly greater numbers of
animals were allergic in experimental groups when 0- exposure ranged from 0.8
ppm to 0.5 ppm for 3 days. The effects observed were most pronounced when the
allergen (ovalbumin) was administered by I.V. injection than by the aerosol
route. Further studies are needed to determine the threshold level of these
effects,
9.3.5 To!erance
Acclimatization, whether it be a long-term or a moment-to-moment response
of the organism to a changing environment, has been a phenomenon of major in-
terest to toxicologists for years. Tolerance, in the broadest sense of the
word, may be viewed as a special form of acclimatization in which exposure to
a chemical agent results in increased resistance, either partial or complete,
to the toxicant (Hammond and Bellies, 1980). Often the terms tolerance and
resistance are used interchangeably. The word, tolerance, is primarily used
when the observed decrease in susceptibility occurs in an individual organism
as a result of its own previous or continuing exposure to the particular toxi-
cant or to some other related stimulus. Resistance generally refers to relative
insusceptibility that is genetically determined (Hayes, 1975).
A third term, adaptation, has been widely used primarily to describe the
diminution of response seen in human subjects who have undergone repeated 0,
exposure (Chapter 10, Section 3). This adaptation might well result from a
different biologic process than that referred to in the various animal tole-
rance studies. It is not yet known whether the laboratory animal develops
9-144
-------
adaptive responses similar to those seen in humans (i.e., respiratory mechani-
cal functions, symptoms of respiratory irritation, and airway reactivity).
Thus, to date, the precise distinction or definition of these two terms,
tolerance and adaptation, are not yet fully understood. There are also
limited data regarding the ability of cells in these "adapted" respiratory
systems to once again return to a pre-exposure condition following termination
of the exposure. However, Plopper et al. (1978) found that after 6 days in
clean air, the rat's lungs were almost recovered from the damage caused by
3 days of exposure to 0.8 ppm 0,, and that these "new recovered" cells have
approximately the same degree of susceptibility to a re-exposure of 0- as
their pre-exposed counterparts, i.e., fully susceptible to re-exposure. These
morphological findings confirm similar conclusions based on concomitant
biochemical studies of Chow et al. (1976b).
In animal oxidant toxicity studies, the term tolerance classically is
defined as the phenomenon wherein a previous exposure to a nonlethal
concentration of 0, will provide some protection against a subsequent exposure
to a concentration of 0^ expected to be lethal. The degree of tolerance
depends considerably on the duration of the exposure and the concentration.
Tolerance occurs rapidly and can persist for several weeks (Mustafa and
Tierney, 1978). The term tolerance should not be considered to indicate
complete or absolute protection, because continuing injury does occur and can
eventually lead to nonreversible morphological changes. This protective
phenomenon seen with oxidants was originally described by Laqueur and Magnus
(1921) in cats undergoing exposure to phosgene.
In the typical experiment, animals are pre-exposed to a lower concentra-
tion of 0, and then challenged at a later time to a higher concentration. As
early as 1956, Stokinger et al. presented data clearly indicating that an
animal could also become tolerant to the lethal effects of 0-. Such tolerance
has also been reported by many investigators, including Matzen (1957a),
Mendenhall and Stokinger (1959), Henschler (1960), and Fairchild (1967). The
observation of this tolerance phenomenon in experimentally exposed animals has
led to the speculation that it may also be a mechanism for protecting environ-
mentally exposed humans. Tolerance to 0, also provides cross-protection
against the pulmonary effects of other chemical agents, such as N02, ketene,
phosgene, and hydrogen peroxide (Stokinger and Coffin, 1968) and recently to
hyperoxia (Jackson and Frank, 1984).
9-145
-------
The previous criteria document for 0, and other photochemical oxidants
cited various studies that examined 0- tolerance and presented some evidence
«3 —
indicating possible mechanisms of action. Review of these earlier data reveals
that pre-exposure to a certain concentration of 0~ can protect test animals
from the acute lethal effects of a second exposure to 0-. This protection has
been attributed to a significant reduction in pulmonary edema in the pre-exposed
animals. Table 9-16 lists the key studies on 03 tolerance.
Because 0, has a marked proclivity to reduce the ability of alveolar
macrophages to function, studies were conducted to determine how the pulmonary
defense system in tolerant animals compared with naive animals. With the
bacterial infectivity model- (Section 9.3.4.3), the pre-exposed (tolerant)
animals were only partially protected from the aerosol infectious challenge
(Coffin and Gardner, 1972a; Gardner and Graham, 1977). The partial protection
was evident at 0, concentrations that had been shown to be edemagenic; however,
3
at the lowest concentration, 200 pg/m (0.10 ppm) of 0-, there was no signifi-
cant difference imparted by the use of the tolerant-eliciting exposure. The
data suggest that at the higher concentrations (> 0.3 ppm), pre-exposure
prevented edema, which prophylactically aided the animals' defenses against
the inhaled microorganisms. Because the protection was only fractional and
did not occur at the lowest level, however, 03 still suppressed specific body
defenses that were not protected by the phenomenon of tolerance.
To further investigate this hypothesis (Alpert and Lewis, 1971; Gardner
et a!., 1972), studies were conducted to evaluate the effects of-tolerance at
the cellular level. These studies indicated that the initial 0, exposure did
induce tolerance against pulmonary edema in the exposed lung; however, there
was no protection afforded against the cytotoxic effects of Q3 at the cellular
level. The cytological toxic injuries measured in this study (including sig-
nificant reductions in enzymatic activities of macrophages and an increase in
inflammation, as measured by the presence of poJlymorphonuclear leukocytes)
showed that there was no protection against these cellular defense mechanisms.
Frager et al. (1979) studied the possibility of tolerance to 0, in mucocil-
iary clearance. Exposure of rats to 1.2 ppm of 0, following particle deposition
caused a substantial delay in mucociliary clearance. The CL effect could be
3 -
eliminated by a pre-exposure to 1600 pg/m (0.80 ppm) of 03 for 4 hr, 3 days
before the deposition of the particles. Thus, the pre-exposure provided
complete protection against the higher 03 level that lasted for about one
9-146
-------
TABLE 9-16. TOLERANCE TO OZONE
Ozone
(ug/m3)
p re-
exposure
196-1960
196
490
M3
jL 98°
*»
588
588
588-980
980-1960
Ozone
(ppm)
pre-
exposure
0.1-1.0
0.1
0.25
0.5
0.3
0.3
0.3-0.5
0.5, 1.0
Ozone Ozone
(Mg/m3) (ppm)
Length of after after
pre- latent latent
exposure period period
3 hr 196- 0.1-1.0
1960
30 min 196 0.1
6 hr 1966 1
6 hr
1 hr 39,200 20
3 hr 588 0.3
4 days 980 0.5
1372 0.7 1.0
1960
3 hr 43,120 22
Length of
exposure
after
latent
period
3 hr
30 min
6 hr
2 hr
3 hr
I, 2, 4
days
3 hr
Observed effect(s) Species Reference
Lower mortality for pre-exposed mice than Mouse Gardner and Graham, 1977
• mice receiving only one 03 dose. Complete
tolerance was not evident.
Tolerance exhibited in the lungs' periphery, Dog Gertner et a!., 1983b
as measured by collateral resistance.
Response < controls in tolerant animals.
No tolerance to edema unless pretreated Rat Alpert et a!,, 1971a
with methylprednisolone.
Edema as measured by recovery of 132I
in pulmonary lavage fluid.
Tolerance to edema effects of Oa did not House Gregory et al. , 1967
develop in thymectomized animals but
developed in sham-operated animals, in-
dicating the thymus may be involved in
tolerance.
20% lower mortality for pre-exposed mice than Mouse Coffin and Gardner, 1972a
mice receiving only one 03 dose. Partial
tolerance probably due to inhibition of edema-
genesis.
Lack of total protection indicated by increased Rat Evans et al., 1971, 1976a,b
numbers of type 2 cells.
With unilateral lung exposure technique, Rabbit • Alpert et al., 1971b
tolerance to edema occurred as a local Alpert and Lewis, 1971
effect (cellular) and was seen only in
the pre-exposed lung.
-------
TABLE 9-16. TOLERANCE TO OZONE (continued)
Ozone Ozone Length of
Ozone Ozone (tig/a3) (pp») exposure
((jg/n3) (pp») Length of after after after
pre- pre- pre- latent latent latent
exposure exposure exposure period period period
980 0.5 3 hr 5880 or 3 and 22 3 hr
1470 0.75 3 days 7840 4.0 8 hr
1490 0.76 3 day 6860-7840 3.5-4 8 hr
I
H
CO
1S70 0.8 3 days 1570 0.8 6 or 27
days
1600 0.8 4 hr 2352 1.2 4 hr
1960 1 " 1'hr NO KO ND
I960 1 1 hr 3920 2 1 hr
Observed effect(s) Species
With unilateral lung exposure technique, Rabbit
tolerance developed only to pulmonary edema.
No tolerance to the chemotaxis of polymorpho-
nuclear leukocytes or decreased lysosomal
hydrolase enzyme activity.
A smaller decrease in activities of glutathione Rat
peroxidase, glutathione reductase, glucose-6-
phosphate dehydrogenase and levels of reduced
glutathione in lungs of tolerant animals, as
compared to nontolerant animals.
When latent period was 11 days, no tolerance Rat
to decrease in 6SH peroxidase system immedi-
ately afttr challenge; 18 hr later, a smaller
decrease occurred. When latent period was
19 days, the decrease in enzyme activities
measured 16 hr post-challenge was less in pre-
exposed animals; 114 hr post-challenge, some
increases in the GSH peroxidase system were
observed.
After 6 days of recovery the lung is again Rat
fully susceptible to re-exposure. Adaptation
lasts only as long as the 0, exposure
continues.
Pre-exposure to 03 caused complete tolerance to Rat
delay in mucociliary clearance at 3 days, but
not 13 days.
All animals X-irradiated to 800 R. 60% of House
Qj-pre-exposed mice survived. 100% of
controls died.
Tolerance to allergic response to inhaled Guinea
acetylcholine. pig
Reference
Gardner et al . , 1972
Chow, 1976
Chow et al . , 1976b
Chow,- 1984
Plopper et al. , 1978
Frager et al. , 1979
Hattori et al., 1963
Matsumura et al . , 1972
ND = not described.
-------
week. The possible mechanism for this protection could be a thickening of the
mucus layer, which would offer the epithelium an extra physical barrier against
Q~. As the secretion returns to normal, the protection is lost. The authors
suggested that another possible mechanism for this protection involves the
ciliated cells and their cilia. In this case, the protection could result
from either the formation of intermediate cilia (Hilding and Hilding, 1966) or
the occurrence of some other temporary change in the regenerating ciliated
cell.
Evans et al. (1971, 1976b) also measured tolerance by studying the kinetics
of alveolar cell division in rats during a period of exposure to an elevated
3
CL concentration of 980 or 1372 ug/m (0.50 or 0.70 ppm, up to four days) that
3
followed initial exposure at a lower concentration of 686 ug/m (0.35 ppm) for
four days. Tolerance in this case was the ability of type 1 cells to with-
stand a second exposure without any increase in the number of type 2 cells,
which would indicate a lack of complete tolerance. Similar to the host defense
studies cited above, these investigations showed that tolerance to the initial
concentration of 03 did not ensure complete protection against re-exposure to
the higher 03 concentration.
Attempts have been made to explain tolerance by examining the morphological
changes that occur due to repeated exposures to 0^. In these studies the
investigators attempt to assess various structural responses with various
exposure profiles and concentrations. Dungworth et al. (1975b) and Castleman
et al. (1980) studied the repair rate of 0,, damage as indicated by DNA synthe-
sis. These effects are fully described in Section 9.3.1.2, and they indicate
that with continuous exposure to 0,,, the lung attempts to initiate the repair
of the 0., lesion, resulting in somewhat reduced or less than expected total
damage. These authors suggest that this is an indication that although the
damage is continuing, it is at a lower rate, and they refer to this phenomenon
as adaptation.
It has been suggested that the tolerance to edema seen in animal studies
can be explained through the indirect evidence that more resistant cells, such
as type 2 cells, may replace the more sensitive, older type 1 cells, or that
the type 2 cells may transform to younger, more resistant cells of the same
type (Mustafa and Tierney, 1978). A number of workers have reported that the
younger type 1 cells are relatively more resistant to the subsequent toxicity
of 0- (Evans et al., 1976a; Dungworth et al., 1975a; Schwartz et al., 1976).
9-149
-------
Thus, there is also the possibility that this reparative-proliterative response
relines the airway epithelium with cells that have a biochemical armamentarium
more resistant to oxidative stress (Mustafa et a!., 1977; Mustafa and Lee,
1976),
Another suggestion is that with CL exposure, there is cellular accumula-
tion within the airways resulting in mounds of cells in the terminal bronchi-
oles that may cause considerable narrowing of the airways (Berliner et a!.,
1978). As the airways become more obstructed, the 0- molecules are le'ss likely
to penetrate to lumen. This may result in a "filtering" system that removes
the 0~ before it reaches the sensitive tissue.
Tolerance to 03 has also been studied by using a variety of biochemical
indicators to measure the extent to which a pre-exposure to 03 protects or
reduces the host response to a subsequent exposure. For example, Jackson and
Frank (1984) found that preexposure to 0, produced cross tolerance to hypoxia.
In these tolerant animals (0.8 ppm 03 x 7 days) there was a significant increase
in total lung superoxide dismutase, glutathione peroxidase, glucose-6-phosphate
dehydrogenase, and catalase. Such an increase in these antioxidant enzymes
occurs with 03 exposure. Chow et al. (1976a,b) compared a variety of metabolic
activities of the lung immediately after an initial 3-day continuous exposure
2
to 1600 |jg/m (0.80 ppm) of 03 with the response after subsequent re-exposure.
At 6, 13, and 27 days after the pre-exposure ended, the animals were once
again treated to the same exposure routine. If tolerant, the animals should
have shown a diminution of response. However, the re-exposed rats responded
similarly to those animals tested after the initial exposure. The lungs of
the naive animals had equivalently higher activities of glutathione peroxidase,
glutathione reductase, glucose-6-phosphate dehydrogenase and higher levels of
nonprotein sulfhydryl than controls and were comparable to the animals that
were exposed and tested immediately after the initial exposure. The authors
state that this indicated that by the time recovery from the pre-exposure is
complete, the lung is as susceptible to the re-exposure injury as a lung that
has never been exposed.
In a follow-up study, Chow (1984) pre-exposed rats for 3 days (apparently
3
continuously) to air or 1490 ug/m (0.76 ppm) 03 and challenged them at various
times with an 8-hr exposure to a higher level of 0~. As expected, the pre-expo-
o
sure protected the rats from the lethal effects of 6860 to 7840 ug/m (3.5 to
4.0 ppm) 0-, whether the challenge was 8, 11, or 19 days later. Generally,
9-150
-------
all 0-, exposures decreased the GSH peroxldase system. When rats were challenged
3
with 7644 ug/m (3.9 ppm) 0~ 11 days after the 3-day pre-exposure, there was .
no tolerance immediately after the challenge exposure; 18 hr after the challenge
exposure, a dampening of the decrease in the GSH peroxidase system was observed.
o
When the pre-exposure and challenge (6860 (jg/m , 3.5 ppm) were separated by
19 days, the decrease in the GSH peroxidase,system measured 16 hr post-challenge
was less in the animals receiving pre-exposure (except for GSH reductase, for
which values were equivalent); 114 hr post-challenge, no tolerance was observed,
but some increases in enzyme activity were observed.
Gertner et al. (1983b) presented data showing that the development of
adaptation and tolerance to pulmonary function changes is rapid and mediated
through the vagus nerve. These investigators used a bronchoscope to expose an
isolated segmental airway of the lung to 03 and study changes in collateral
resistance (Rcoll). During a 30-min exposure to 0.1 ppm, the Rcoll increased
31.5 percent within 2 min and then gradually decreased to control level in
spite of continual exposure to 0~. Fifteen minutes after the 03 exposure
ceased, the Rcoll returned to normal. Subsequent exposure to 0.1 ppm of 0«
did not increase Rcolls indicating that some protection existed. These investi-
gators have tried to distinguish between the terms adaptation and tolerance
based on these studies. They used adaptation to describe the pattern of changes
that occur during continuous exposure to 0., and the term tolerance to describe
resistance to subsequent 0., exposure.
Thus, the available'evidence from animal studies suggests that tolerance
does not develop to all forms of lung injury. The protection described against
edemagenic effects of 03 does not appear to offer complete protection, as
illustrated by the following examples.
1. There is no tolerance (i.e., no protection occurs) on the part of
the specific pulmonary defense mechanisms against bacterial infection
below the edemagenic concentration; whereas above the edema-inducing
concentration the effect of tolerance (i.e., inhibition of pulmonary
edema), can lower the expected mortality rate because the animals do
not have to cope with the additional burden of the edema fluid.
2. Specific cellular functions of the alveolar macrophage (i.e., enzyme
activity) are incapable of being protected by tolerance.
9-151
-------
3. Various biochemical responses were found in both naive and pre-exposed
animals.
4. Tolerance fails to inhibit the influx of polymorphonuclear leukocytes
into the airway.
This last finding is interesting considering the effective tolerance for
edema production. This suggests that the chemotactic effect of 0, may be
separable from the edemagenic effect. This may also explain why chronic
morphological changes in the lung may occur after long-term exposure, even
though there may not be any edema.
The possible explanations for this tolerance phenomenon have been proposed
by Mustafa and Tierney (1978). The primary mechanism of tolerance may not be
due to hormonal or neurogenic pathways, because unilateral lung exposure does
not result in tolerance of the nonpre-exposed lung (Gardner et a!., 1972;
Alpert and Lewis, 1971). But it should be noted that Gertner et al. (1983b)
have evidence that local tolerance may involve a neural reflex. Changes in
Rcoll may be mediated through the vagus nerve. After bilateral cervical
vagotomy, the resistance did not increase during Q~ exposure but did after
challenging with histamine, indicating that the parasympathetic system may
play a role in response to Q~ in the periphery of the lung. There is some
evidence that 03 may cause a decrease in cellular sensitivity, an increased
capacity to destroy the test chemical, or the repair of the injured tissue
(Mustafa and Tierney, 1978). In addition, 03 could possibly cause anatomic
changes, such as an increase in mucus thickness, that may, in effect, reduce
the dose of 0- reaching the gas-exchange areas of the lung.
It should be mentioned that the term tolerance carries with it the conno-
tation that some form of an insult and/or damage has occurred and there has
been an overt response at the structural and/or functional level. The response
may be attenuated or undetectable, but the basis for the establishment of the
tolerance still persists. It is possible that the cost for tolerance may be
minor, such as a slight increase in mucus secretion; however, one must also be
aware that changes in response to diverse kinds of insults to a host's system,
such as the immune system, are adaptations that might even suggest an undesirable
effect of ambient oxidant air pollution.
9-152
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9.4 EXTRAPULMONARY EFFECTS OF OZONE
9.4.1 Central Nervous System and Behavioral Effects
Despite reports of headache, dizziness, and irritation of the nose,
throat, and chest in humans exposed to 03 (see Chapter 10), and the possible
implications of these and other symptoms as indications of low- level Q3 effects,
few recent reports were found on behavioral and other central nervous system
(CNS) effects of CL exposure in animals. Table 9-17 summarizes studies on
avoidance and conditioned behavior, motor activity, and CNS effects.
Early investigations have reported effects of 0- on behavior patterns in
animals. Peterson and Andrews (1963) attempted to characterize the avoidance
behavior of mice to 0- by measuring "their reaction to a 30-min exposure on one
. O
side of an annular plastic mouse chamber. A concentration-related avoidance
3
of the 0- side was reported at 1176 to 16,660 |jg/m (0.60 to 8.50 ppm) of 0-.
However, the study had serious shortcomings, including a lack of positionT
reversal controls (Wood, 1979), considerable intersubject variability, and
other design flaws (Doty, 1975). Tepper et al. (1983) expanded on the design
by using inhalant escape behavior to assess, directly the aversive properties
of Og. Mice were individually expo.sed to 03 for a maximum of 60 sec, followed
by a chamber washout period of 60 sec. The animals could terminate exposure
*
by poking their noses into only one of two brass conical recesses containing a
3
photobeam. The delivery of 980 |jg/m (0.50 ppm) of 03 was reliably turned off
for a greater proportion of experimental trials, compared to control trials
3
with filtered air. At 19,600 (jg/m (10 ppm) of O, all animals turned off
100 percent of the trials with an average latency of approximately 10 sec.
Studies by Murphy et al. (1964) demonstrated that wheel-running activity
decreased by approximately 50 percent when mice were exposed to 392 to 980 pg/m
(0.20 to 0.50 ppm) of 0- for 6 hr and decreased to 60 percent of pre-exposure
3
values during the first 2 days of continuous exposure to 588 ug/m (0.30 ppm)
of 0,,. Running activity gradually returned to pre-exposure values during the
3
next 5 days of continuous exposure to 588 jag/m (0.30 ppm) of 0.,. If the same
3
mice were subsequently exposed to 1372 ug/m (0.70 ppm) of 0, for an additional
7 days, running activity was depressed to 20 percent of pre-exposure values.
3
Partial recovery was described during the final days of exposure to 1372 yg/m
(0.70 ppm), and complete recovery occurred several days after exposure was
terminated. However, partial tolerance was seen when the air-control mice
3
were subsequently exposed to 392 |jg/m (0.20 ppm) of 03 for 7 days. Konigsberg
and Bachman (1970) used a capacitance-sensing device to record the motor
9-153
-------
TABLE 9-17. CENTRAL NERVOUS SYSTEM ABO BEHAVIORAL EFFECTS OF
M
cn
Ozone
concentration
u§/«3 ppi
110 0.056
98-1960 0.05-1.0
196-3920 0.1-2.0
235-1960 0.12-1.0
392-980 0.2-0.5
588-1372 0.3, 0.7
980-19600 0.5-10.0
980 0.5
Measurement3 * Exposure
method duration and protocol Observed effects(s) Species
d 93 days, continuous No overt behavioral changes. Cholines- Rat
terase activity inhibited at 75 days of
exposure, returning to control levels
12 days after termination of exposure.
HAST 45 min Motor activity progressively decreased Rat
with increasing 03 concentrations up to
0,5 ppa. Slight Increase in frequency
of 3-nin intervals without aotor activity.
CHEH G hr Linear and/or nonotonic decreases in Rat
operant behavior during exposure.
CHEH 6 hr Wheel running activity decreased Rat
iwmotonically with increasing Oa con-
centration. Components of running
were differentially affected at low
vs. high Os concentrations.
NBKI 6 hr Wheel running activity decreased 50%. House
MAST 7 days, continuous Running activity decreased 60% during
first 2 days, returning to control
levels during the next 5 days of expo-
sure; running activity decreased 20X
when 0.3 ppm exposure was followed
immediately by 0.7 ppm 03 exposure.
Adaptation with continued exposure
was apparent.
CHEM 60 s Exposure terminated by nose pokes with Mouse
increasing frequency as Os concentra-
tion increased.
NO 30 rain Elevation of simple and choice reactive Nonhunan
time, priaate
Reference
Eglite, 1968
Konigsberg and
Bachflian, 1970
Weiss et al.,
1981
Tepper et al . ,
1982
Murphy et al . ,
1964
Tepper et al . ,
1983
Reynolds and
Chaffee, 1970
-------
TABLE 9-17. CENTRAL NERVOUS SYSTEM AND BEHAVIORAL EFFECTS OF OZONE (continued)
V£>
H
Ul
Ul
Ozone .
concentration Measurement '
(jg/m-1 ppm method
980-19SO 0.5, 1.0 NBKI
1176- 0.6-8.5 e
16,660
1960 1.0 HAST
NBKI
1960- 1-3 ND
5880
1960 1
1960- 1-3 ND
5880
Exposure
duration and protocol
1 hr
30 min
7 days, continuous
18 months,
8 hr/day
18 months;
8, 6, 24 hr/day
18 months,
8 hr/day
Observed effects(s)c Species
Evoked response to light flashes in the Rat
visual cortex and superior collicus
decreased after exposure.
Avoidance behavior increased with Mouse
increasing 03 concentration.
Reduction in wheel running activity; no Rat
effect of Vitamin E deficiency or
supplementation.
COMT activity decreased at 2 ppm, MAO Dog
activity increased at 1 ppm only.
COMT activity decreased as the daily
exposure increased from 8 to 24 hr.
MAO activity increased at 8 and 16
hr/day and decreased at 24 hr/day.
Alterations in EEG patterns after Dog
9 months, but not after 18 months of
exposure.
Reference
Xintaras et al. ,
1966
Peterson and
Andrews, 1963
Fletcher and
Tappel, 1973
Trains et al . ,
1972
Johnson et al . ,
1976
Measurement method: MAST = Kl-coulometric (Mast meter); CHEM
described.
Calibration method: NBKI = neutral buffered potassium iodide.
^Abbreviations used: COMT = catechol-o-methyltransferase; MAO
Spectrophotometric method with dihydroacridine.
eKI titration with sodium thiosulfate.
= gas phase chemiluminescence; NBKI = neutral buffered potassium iodide; ND = not
= monamine oxidase; EEG = electroencephalogram
-------
activity of rats during a 45-min exposure to 98, 196, 392, 980, and 1960 ug/m
(0.05, 0.10, 0.20, 0.50, and 1.0 ppm) of 03< Compared with control rats,
motor activity following 0, exposure progressively decreased with increasing
3
0, concentrations up to 980 ug/m (0.50 ppm). No greater reduction was obtained
3
at 1960 ug/m (1.0 ppm). In addition, the frequency of 3-min intervals without
measurable motor activity tended to increase slightly (from 1 to 1.25 to
approximately 3) with increasing 03 concentration.
A detailed raicroanalysis of motor activity was undertaken by Tepper
et al. (1982), who exposed rats for 6-hr periods during the nocturnal phase of
their light-dark cycle to 235, 490, 980, and 1960 pg/m3 (0.12, 0.25, 0.50 and
1.0 ppm) of Og. The 3 days preceding an exposure were used for control obser-
vations to measure running activity for each rat in a wheel attached to the
3
home cage. Decreases in wheel running activity occurred at 235 ug/m (0.12 ppm)
and progressively greater decreases in wheel running activity occurred with
increasing 0- concentration. An analysis of the running behavior showed that
the components of running were differentially affected by On. An increase in
the time interval between running bursts primarily accounted for the decreased
motor activity at the low (235 ug/m , 0.12 ppm) 03 concentration. Postexposure
increases in wheel running were seen following this low 0, concentration. At
3
higher 03 concentrations (>490 pg/m, 0.25 ppm), an increase in the time per
wheel revolution, a decrease in the burst length as well as the extended time
interval between bursts contributed to the reduced motor activity. These
higher concentrations also caused a decrease in performance compared to control
for several hours after exposure was terminated.
Effects of 03 on behavior were further investigated by Weiss et al. (1981)
in their studies on the operant behavior of rats during 03 exposure. The term
operant refers to learned behaviors that are controlled by subsequent events
such as food or shock delivery. In this case, rats were trained to perform a
bar-pressing response maintained by a reward with food pellets delivered
according to a 5-min fixed-interval reinforcement schedule. The rats were
3
exposed for 6 hr to 03 concentrations from 196 to 3920 ug/m (0.10 to 2.0 ppm),
with at least 6 days separating successive exposures. Two groups of rats were
tested, one beginning in the morning and the other in the mid afternoon. Ozone-
3
induced decreases were linear from 196 to 2744 ug/m (0.10 to 1.40 ppm) for the
first group; for the second, the decreases were generally monotonic from 196 to
o
3920 ug/m (0.10 to 2.0 ppm). Analysis of the distribution of responses during
9-156
-------
the various 0- exposures indicated concentration-related decreases arose
mainly from the later portions of the sessions and that the onset of the
decline in response occurred earlier at the higher 0- concentrations. In con-
trast to other types of toxicants, 0- did not disrupt the temporal pattern
that characterized response during each fixed-interval presentation. Based on
the sedentary nature of the task, the authors suggested that the inclination
to respond rather than the physiological capacity to respond was impaired.
Kulle and Cooper (1975) studied the effects of 0- on the electrical
3
activity of the nasopalatine nerve in rats. Ozone exposure to 9800 ug/m
(5 ppm) for 1 hr produced an increase in nasopalatine nerve response (action
potential frequency) to amyl alcohol, suggesting that the nerve receptors were
made more sensitive by prior exposure to 03. One-hour air perfusion following
the Og exposure reduced the neural response to amyl alcohol, but not to pre-
exposure levels. The nasopalatine nerve is a branch,of the trigeminal nerve
which responds to airborne chemical irritants. Because most irritants, in-
cluding Qg, also have odorant properties and, therefore, stimulate both
trigeminal and olfactory receptors in the nasal mucosa, it is difficult to
distinguish an irritant response from an odor response as the mechanism for
behavioral effects in laboratory animals.
Effects of 0_ on the CNS have been reported. Trams et al. (1972) measured
biochemical changes in the cerebral cortex of dogs exposed for 18 months to
•3
1960, 3920, or 5880 ug/m (1, 2, or 3 ppm) of 0,. In 8 hr/day exposures,
, *3
reported decreases (35 percent) in the catecholamines norepinephrine and
epinephrine were not statistically significant, although 0- exposure at 3920
3
ug/m (2 ppm) caused a statistically significant decrease in catechol-o-methyl-
transferase (COMT) activity. In contrast, monamine oxidase (MAO) activity was
•3
significantly elevated at 1960 ug/m (1 ppm) of 0.,, but not at 3920 or 5880
3 3
pg/m (2 or 3 ppm) of 03. Increasing daily exposures to 1960 pg/m (1 ppm)
from 8 to 24 hr/day caused a significant decrease in COMT activity, but MAO
activity increased at 8 and 16 hr/day but decreased at 24 hr/day. Concurrently,
Johnson et al. (1976) measured electroencephalographic (EEC) patterns in the
same dogs and noted alterations in EEC patterns after 9 months of exposure to
3
1960 to 5800 ug/m (1 to 3 ppm) of 0-, but not after 18 months of exposure.
The authors noted that it was difficult to correlate the observed EEG changes
with the alterations of metabolic balance described. Furthermore, it was even
more difficult to assess the metabolic and physiologic significance of the
changes without more information abcut chronic 0, exposure.
9-157
-------
9.4.2 Cardiovascular Effects
Very few reports on the cardiovascular effects of 03 and other photo-
chemical oxidants in animals have been published. Brinkman et al. (1964)
studied structural changes in the cell membranes and nuclei of myocardial
muscle fibers in adult mice exposed to 0-. After a 3-week exposure to 392
3
ug/m (0.20 ppm) of 0~ for 5 hr/day, structural changes were noted, but these
effects were reversible about 1 month after exposure. However, because this
study had severe design and methodology limitations, the results should be
considered questionable until independently verified.
Bloch et al. (1971) studied the effects of 03 on pulmonary arterial
pressure in dogs. They exposed 31 dogs to 1.0 ppm of Og daily for various
hours for 17 months. Ten percent (3 dogs) of the animals developed pulmonary
arterial hypertension, and approximately 30 percent (9 dogs) had excessive
systolic pressure, but there was no proportional relationship between pulmonary
arterial hypertension and 0, exposure. Unless sample sizes were too small to
find adequate dose-response effects, the authors attributed the results to
genetic susceptibility.
Revis et al. (1981) studied the effects of 0« and cadmium, singly and
3
combined, in rats. The rats were exposed to 1176 ug/m (0.60 ppm) 0.,,
3
5 hr/day for 3 consecutive days or to 3 mg/m cadmium for 1 hr or to both
pollutants. All exposure treatments resulted in increases in systolic pressure
and heart rate. Neither diastolic pressure or mean pressure was affected. No
additive or antagonistic effects were seen with the pollutant combinations.
Costa et al. (1983) measured heart rate and standard intervals of cardiac
electrical activity from the electrocardiographic (EKG) tracings of rats
exposed to 392, 1568, or 3920 ug/m (0.2, 0.8, or 2 ppm) of Og 6 hr/day, 5
days/week for 62 exposure days as part of a more extensive evaluation of lung
function (Section 9.3.2). Heart rate was not altered by 0» exposure. The
3
predominant effects occurred at the highest 0, concentration (3920 ug/m ,
2 ppm) at which there was evidence of partial A-V blockade and distorted
ventricular activity, often associated with repolarization abnormalities.
Friedman et al. (1983) evaluated the effects of a 4-hr exposure to 588
2
and 1960 ug/m (0.3 and 1.0 ppm) of 03 on the pulmonary gas-exchange region of
dogs ventilated through an endotracheal tube. Pulmonary capillary blood flow
and arterial 0? pressure (PaO-) were decreased 30 min following exposure to
both 0., concentrations, and arterial pH (p.H0) was decreased following exposure
J d
9-158
-------
3
to 1960 |jg/m (1.0 ppm) of 0,. Decreases in pulmonary capillary blood flow
3
persisted 24 hr following exposure to 588 and 1960 jjg/m (0,3 and 1.0 ppm) of
3
DO and as long as 48 hr following exposure to 1960 pg/m (1.0 ppm) of 0~.
Persistent decreases in pH and Pa09 were observed 24 hr following exposure to
3 a L
1960 ug/m (1-0 ppm) of 0~. Pulmonary edema, determined histologically and by
increased lung water content and tissue volume, was observed 24 hr following
3
exposure to 1960 ug/m (1.0 ppm) of 0_. The data indicate that 03 exposure
can cause both acute and delayed changes in cardiopulmonary function.
9.4.3 Hematological andSerum Chemistry Effects
Hematological effects reported in laboratory animals and man after inhala-
3
tion of near-ambient 0., concentrations (£ 1960 ug/m ; £ 1.0 ppm) indicate that
03 or some reaction product of 0~ can cross the blood-gas barrier. In addition
to reports of morphological and biochemical effects of 0, on erythrocytes,
chemical changes have also been detected in serum after iji vitro and in vivo
03 exposure. Hematologieal parameters are frequently used to evaluate 0^
toxicity, because red blood cells (RBCs) are structurally and metabolically
simple and well understood, and because the relatively noninvasive methods
involved in obtaining blood samples from animals and man make blood samples
available for study.
9.4.3.1 Animal Studies - In Vivo Exposures. The effects of jji vivo Q~
exposure in animals, including studies reviewed in the previous 03 criteria
document (U.S. Environmental Protection Agency, 1978), are summarized in Table
9-18. •'-'•••'
Effects of 03 on the blood were first reported by Christiansen and Giese
(1954) after they detected an increased resistance to hemolysis of RBCs from
o
mice exposed to 1960 ug/m (1.0 ppm) for 30 min. Goldstein et al. (1968)
reported a significant decrease in RBC acetylcholinesterase (AChE) activity
3
after exposure of mice to 15,680 ug/m (8 ppm) of 0, for 4 hr. Menzel et al.
(1975a) observed the presence of Heinz bodies in approximately 50 percent of
3
RBCs in the blood of mice exposed to 1666 ug/m (0.85 ppm) of 03 for 4 hr.
About 25 percent of RBCs contained Heinz bodies after continuous exposure of
mice to 0.85 ppm of 0, for 3 days. Heinz bodies are polymers of methemoglobin
formed by oxidant stress; they appear to attach to the inner membrane of the
RBC. However, Chow et al. (1975) detected no significant changes in GSHs,
G-6-PDH, oxidized glutathione reductase, or GSH peroxidase in RBCs of rats or
monkeys exposed to the same 0~ concentration 8 hr/day for 7 days.
9-159
-------
TAfllE 9-18. HEHATOLOGY: ANIMAL ~ IK VIVO EXPOSURE
cn
o
Ozone
concentration
ug/w3
110
118
235
470
941
392
392
392
392-
1960
490
980
1372
588
588
ppm
0.056
0.6
0.12
0.24
0.48
0.2
0.2
0.2
0.2-
1.0
0.25
0.50
0.70
0.3
0.3
Keasurensent3
method
c
UV
ND
UV
UV
UV
UV
UV
UV
Exposure
duration and
protocol
93 days
2.75 hr
4 hr
8 hours/day,
5 days/week,
3 weeks
60 «1n
1-4 hr
2.75 hr
3 hr/day
until death
(2-3 wk)
3 hr
Observed effect(s)
Decreased whole blood cholintsterase,
which returned to normal 12 days after
exposure ceased.
RBC survival decreased at 0.06,
0.12, and 0.48 ppm; no concentra-
tion-response relationship.
Increased osiiotic fragility and
spherocytosis of RBC's.
Increased serum glutamic pyruvic
transaai nase and hepatic ascorbic
acid, No change in blood catalase.
Small decrease in total blood sero-
tinin.
Plasma creatine phosphokinase
activity altered immediately and
15 Bin postexposure; no effect
30 min postexposure. No change
in plasma histamine or plasma
lactic acid dehydrogenase.
RBC survival decreased at 0.25 ppm
only.
Increased mortality in mice
parasitized with Plasmodium
berghei . Increased number of
parasitized red blood cells.
No effect on RBC reduced glutathione,
L-ascorbic acid, hemoglobin, red
blood cell counts. Slight increase
(p = 0.08) in % methemoglobin.
Decreased hematocrit in 03-low
vitamin C group. Generally, vitamin
C deficiency did not increase sensi-
tivity to 03.
Species Reference
Rat Eglite, 1968
Rabbit Calabrese et al . ,
1983a
Rabbit Brinkman et al.,
1964
Mouse Veninga, 1970
Rabbit Veninga, 1967
Mouse Veninga et al.,
1981
Sheep Moore et al . ,
1981a
Mouse Moore et al., 1984
Guinea pig Ballew et al., 1983
-------
TABLE 9-18. HEMATOLOGY: ANIMAL — IN VIVO EXPOSURE (continued)
Ozone Exposure
concentration Measurement duration and
ug/M3 ppi method protocol
627 0.32 UV 6 hr
± dietary
vitamin E
784 0.4 NO 6 hr/day,
5 days/week,
6 months
784 0.4 NO 6 hr/day,
5 days/week,
10 months
784 0.4 NO 10 months
784 0.4 NO 6 hr/day,
fii • 5 days/week,
en 10 months
M
980 0,5 UV 2.75 hr
980 0.5 MAST Continuous,
23 days
980 0.5 NBKI 8 hr/day,
7 days
1254 0.64 UV 8 hr/day »
lyr
1470 0.75 ND 4 hr/day,
4 days
Observed effect(s)
Increased erythrocyte G-6-PD and
decreased AChE (both diets).
Increased plasma vitamin E
(both diets).
No change serum trypsin inhibitor
capacity.
Increase in serum protein esterase.
Increase in serum protein esterase.
Decreased serum albumin concentra-
tion. Increased concentration of
a- and 6-globulins. Not much change
in p-globulin. No change in total
serum proteins.
Decreased erythrocyte GSH.
Increased hemolysis of erythrocytes
of animals depleted of vitamin E.
No such change when rats received
vitamin E supplements.
No change in GSH level or activ-
ities of GSH peroxidase. GSH
reductase, or G-6-PD in erythro-
cytes.
Altered RBC morphology: decreased
number of discocytes, increased
number of knizocytes, stomatocytes,
and spherocytes. No effect on RBC
FA composition.
RBC's: Increased fragility;
decreased GSH, AChE; no effect
on LDH, G-6-PD.
Species
Mouse
Rabbit
Rabbit
Rabbit
Rabbit
Sheep
Rat
Monkey ,
rat
Monkey
Monkey
Reference
Moore et al . , 1980
P'an and Jegier,
1971
Jegier, 1973
P'an and Jegier,
1972
P'an and Jegier,
1976
Moore et al . ,
1981b
Menzel et al., 1972
Chow et al . , 1975
Larkin et al., 1983
Clark et al., 1978
-------
TABLE 9-18. HUWTQLQGY: AHIHAl — IN VIVO EXPOSURE (continued)
H
G\
to
Ozone
concentration
pg/n3 ppa
1568 0.8
1568 0.8
1568 0.8
1666 0.8S
1686 0.86
1960 1.0 .
1960 1.0
1960- 1.0
3920 2
Exposure
Heasureaent duration and
nethod protocol
mm 7 days
NBKI 8 hr/day,
7 days
NBKI Continuous,
29 days
HAST 4 hr
NO 8 hr/day,
§ days/week,
6 months
UV 4 hr ± vitamin E
ND 30 Bin
CHEM 2 or 7 days
Observed effect(s)
Increased activity of OSH pero-
oxidase, pyruvate kinase, and
lactate dehydrogenase; and
decrease in red cell level of
GSH of vitamin E-deficient animals.
Animals in both vitamin E-deficient
and supplemented diet groups exhibited
no change in activities of 6-6-DP,
catalase, and superoxide disnutase
and in levels of thiobarbituric acid
reactants, met hemoglobin, hemoglobin,
and reticulocytes.
No change in total lactate dehydro-
genase activity or isoeniyae pattern
in plasma or erythrocytes.
Increased lysozyne activity by
day 3.
Increased Heinz bodies in RBC's
(decreased with continual exposure).
Increased infestation and mor-
tality after infection with
Plasnodiun berghei. Increased
acid resistance of erythrocytes.
Decreased filterability. No pro-
tection by vitamin E. No lipid
peroxidation.
Increased resistance to erythrocyte
hemolysis.
No changes.
Species Reference
Rat Chow and Kaneko,
1979
Monkey Chow et al . , 1977
Rat Chow et al., 1974
House Menzel et al . , 1975a
House Schlipkoter and
Bruch, 1973
Mouse Dorsey et al., 1983
Mouse Mizoguchi et al.,
1973;
Christiansen and
Giese, 1954
Rat, Cavender et al.,
guinea pig 1977
-------
TABLE 9-18. HEMATOLOGY: ANIMAL — IN VIVO EXPOSURE (continued)
Ozone
concentration
ug/nd ppro
1960 Ja.O
5880 3.0
1960 1
Exposure
Measurement duration and
method protocol
CHEH 4 hr
UV continuously,
2 wk
Observed effect(s)
No effects on oxyhemoglobin affinity,
2,3-DPG concentrations, heme-02
binding.
Increased serum cholesterol , low
density lipoproteins and very
low density lipoproteins. Males
apparently more affected than
females. No effect on trigly
cerides.
Species Reference
Rabbit Ross et al., 1979
Guinea pig Vaughan et al. ,
1984
M
1960
3430
5880
1960
2940
11,760
15,680
1
1.75
3
1
1.5
6.0
8.0
CHEM, UV 5 hr/day,
10 days within
14- day period
CHEM, UV 5 hr/day,
15 days
within 19-day
period
UV 3 days
4 days
4 days
No effect on serum lipids and Rat
lipoproteins at 1 ppm. Concentration
related linear increase in total
lipoprotein-free cholesterol
and high-density lipoprotein
total cholesterol; decrease in
triglycerides.
Increased serum total cholesterol
(p = 0.1) high density lipoprotein-
cholesterol (p = 0.08) and high
density lipoprotein-free cholesterol
(p = 0.006); decrease in trigly-
cerides (p = 0.06).
No effect on SOD, GPx, K* influx Rat
ratios (all levels). Increased Hb,
Hct, echinocytes II & III (6 & 8 ppm);
echinocytes correlated with petechiae
in lungs, indicative of vascular
endothelial damage.
Mole et al., 1985
Larkin et al., 1978
Measurement method: ND = not described; CHEH = gas phase cherailuminescence; UV - UV photometry; NBKI = neutral buffered potassium iodide;
MAST = KI - coulometric (Mast neter); I = iodometric.
Abbreviations used: RBC = red blood cell; G-6-PD = glucose-6-phosphate dehydrogenase; AChE = acetylcholinesterase; GSH = reduced
glutathione; GSH peroxidase = glutathione peroxidase; GSH reductase = glutathione reductase; FA = fatty acid; LDH = lactic dehydrogenase;
'?! E-r 2«3*'»PhosP|wfllycei'ate; SOD = superoxide dismutase; GPx = glutathione peroxidase; K = potassium, Hb = hemoglobin; Hct = henato- •
cm; P6F2a = prostaglandin F2a; PGE2 = prostaglandin E2.
Spectrophotometric method using dihydroacridine.
-------
In more recent studies, Clark et al. (1978) investigated the biochemical
3
changes in RBCs of squirrel monkeys exposed to 1410 yg/m (0.75 ppm) of Q3
4 hr/day for 4 days. They observed an increase in RBC fragility with decreases
in GSH and AChE activities. No changes were detected in G-6-PDH or lactic
dehydrogenase (LDH) activities. After a 4-day recovery period, RBC fragility
was still significantly increased, although to a lesser degree. AChE activity
returned to control levels at 4 days postexposure; however, RBC GSH remained
significantly lowered.
Ross et al. (1979) investigated the effects of 0-, on the oxygen-delivery
3
capacity of erythrocytes. After exposure of rabbits to 1960 or 7880 pg/m (1
or 3 ppm) of 03 for 4 hr, no changes were detected in RBC 2,3-diphosphogly-
cerate concentration, oxyhemoglobin dissociation curve, or heme-oxygen binding
of RBCs. Analysis of blood parameters 24 hr after exposure revealed no delayed
effects of 03.
Alterations in RBC morphology have been previously observed in CK-exposed
laboratory animals and man (Brinkman et al., 1964; Larkin et al., 1978).
o
Similar observations have recently been made in monkeys exposed to 1254 pg/m
(0.64 ppm) of Oo for 8 hr/day over a 1-yr period (Larkin et al., 1983).
Ultrastructural SEM studies of RBC1s following exposure to 0, demonstrated
reduced numbers of normal discocytes and increased numbers of knizoeytes,
stomatocytes, and spherocytes, which were either absent or found in small
numbers in the blood of air-exposed controls. Despite changes in shape, there
were no differences in the fatty acid composition of the erythrocyte total
lipids. Values for hematocrit, hemoglobin, mean corpuscular volume, and red
cell and reticulocyte count were the same in control and 0,,-exposed animals.
3
Moore et al. (1981a) reported reduced RBC survival in sheep exposed to 490 ug/m
(0.25 ppra) of 0,, for 2.75 hr. Similar reductions in RBC survival were reported
3
following 2.75-hr exposures to 0- concentrations as low as 118 and 235 pg/m
(0.06 and 0.12 ppm) in rabbits (Calabrese et al., 1983a).
Vitamin E deficiency has been associated with an increased hemolysis in
rats and other animal species (Scott, 1970; Gross and Melhorn, 1972). Chow
and Kaneko (1979) reported significant increases in RBC GSH peroxidase, pyru-
vate kinase, and LDH activities, and a decrease in RBC GSH after exposure of
3
vitamin E-deficient rats to 1568 ug/m (0.8 ppm) of 03 continuously for 7 days.
These effects were not observed in vitamin E-supplemented rats (45 ppm of
vitamin E for 4 months). The activities of G-6-PD, catalase, superoxide
9-164
-------
dismutase, and levels of TBA reactants, methemoglobin and reticulocytes were
not altered by 0- exposure or by vitamin E status.
Moore et al. (1980) investigated the effects of dietary vitamin E on
3
blood of 9-month-old C57L/J mice exposed to 627 (jg/m (0.32 ppm) of 03 for
6 hr. Animals were maintained on vitamin E-deficient, or supplemented (3.9 mg
tocopherol/100 Ib., twice the minimal daily requirement) diets for 6 weeks
before 0™ exposure. Mice on the vitamin E-deficient diet showed a 24-percent
increase in G-6-PD activity over controls after 03 exposure, and mice fed a
supplemented diet exhibited a 19-percent increase. Decreases in AChE activity
were observed in both vitamin E-deficient (19-percent decrease) and vitamin
E-supplemented (12-percent decrease) groups.
Dorsey et al. (1983) evaluated the effects of 03 on RBC deformability
after exposure of vitamin E-deficient and supplemented (105 mg of tocopherol
3 3
per kg of chow) male CD-I mice to 588 |jg/m (0.3 ppm), 1372 ug/m (0.7 ppm),
or 1960 ug/m (1.0 ppm) of 03 for 4 hr. After incubation of RBCs in buffer
(0.9 percent RBCs) for up to 6 hr at 25°C, the time required for 2.0 ml of RBC
suspension to pass through a 3-[jm pore size filter was determined. Exposure
3 3
of mice to 1960 ug/m (1.0 ppm) or 1372 ug/m (0.7 ppm) of 03 and incubation
of RBCs for 6 hr resulted in a significant increase in filtration time of RBCs
from 0~-exposed mice, and a lack of protection by dietary vitamin E. The
3
hematocrit of vitamin E-deficient mice exposed to 1960 (jg/m (1.0 ppm) of 0~
was significantly greater than that of nonexposed vitamin E-supplemented mice.
The increased hematocrit was attributed to a loss of RBC deformability, and
sphering resulting in decreased packing of cells during centrifugation for
hematocrit determination. No TBA reactants were detected in the blood of
exposed animals, with or without vitamin E.
The influence of vitamin C deficiency on erythrocytes of guinea pigs
3 ' ' '
exposed to 588 ug/m (0.3 ppm) for 3 hr and examined 0.5 or 3 hr post-exposure
was studied by Ballew et al. (1983). Ozone caused no effect on reduced gluta-
thione levels in erythrocytes. There was a slight increase (p = 0.08) in
percent methemoglobin in 0.~-exposed animals. Vitamin C levels did not signifi-
cantly influence these results. Plasma L-ascorbic acid levels were not affec-
ted by 0, exposure. Hematocrits were decreased in animals on the low vitamin C
diet that were exposed to 03- Hemoglobin and red blood cell counts were
unaffected by 03.
Moore et al. (1984) infected mice with Plasmodium berghei (a blood-borne
3
malarial parasite) 1 day prior to exposure to 588 ug/m (0.3 ppm) Q3. The
9-165
-------
exposure lasted for 3 hr/day until death, or approximately 2 to 3 wk. Mice
exposed to 0-, did not live as long as the controls. Ozone-exposed mice also
had an increase in the number of parasitized red blood cells. The authors
hypothesize that there are 2 potential mechanisms responsible: 03 may have
altered the erythrocyte membrane, making it more permeable to IP. berghei, or
Og increased the reticulocyte count, reticulocytes possibly being more sensitive
^•° £• berghei infestation. These results are consistent with those of Schlipkoter
and Bruch (1973), who reported, without statistical analysis, an increase in
infestation with £. berghei and higher mortality in mice exposed for 6 mo
(8 hr/day, 5 days/wk) to 1686 Mfl/m (0.86 ppm) O.
9.4.3.2 In Vitro Studies. The effects of in vitro 0, exposure of animal
........... — » ^
blood( have been studied by a number of investigators, and these reports are
summarized in Table 9-19.
The effects of i_n vitro 03 exposure on human RBCs have been evaluated by
using a number of different end points, such as increases in complement-mediated
cell damage (Goldstein et al., 1974a), formation of Heinz bodies (Menzel
et al., 1975b), decreases in RBC native protein fluorescence (Goldstein and
McDonagh, 1975), and decreases in concanavalin A agglutinability (Hamburger
et al . , 1979). Exposure of RBCs or their membranes to 0- has also been shown
to inhibit (Na+ - K-*-) ATPase (Kindya and Chan, 1976; Chan et al . , 1977; Koontz
and Heath, 1979; Freeman et al., 1979; Freeman and Mudd, 1981). Kindya and
Chan (1976) proposed that inhibition of ATPase by 0,, caused spherocytosis and
increased fragility of RBCs after 03 exposure. (See Tab-le 9-20 for a summary
of the human in vitro studies.)
Kesner et al. (1979) demonstrated that Og-treated phospholipids inhibited
RBC membrane ATPase. Addition of semicarbazide to Og-exposed phospholipids
before mixing with RBC membranes substantially reduced the inhibitory effect,
suggesting that the inhibitors may be carbonyl compounds. In addition, a
slower- forming semicarbazide-insensitive inhibitor was formed.
Verweij and Steveninck (1980, 1981) reported that semicarbazide and also
p-aminobenzoic acid (PABA) might protect by acting as 03 scavengers. Spectrin
(a major glycoprotein component of the RBC membrane) solution was treated by
bubbling 0»-containing 02 through the solution at 4 ml/min (2.5 uM/min of 03)
for 1 or 9 min. Semicarbazide (40 pM) or PABA (40 pM) inhibited the cross-
linking of 03-exposed spectrin. The inhibition of AChE and hexokinase activi-
ties of RBC ghosts exposed to 0~ was also partially prevented by these two
+
agents, as was K influx into whole RBCs. The authors attributed the inhibition
9-166
-------
TABLE 9-19. HEMATOLOGY: ANIMAL — IN VITRO EXPOSURE
Exposure
Ozone Measurement duration and
concentration method protocol
W3
M
cr\
-o
980-
3920
1960-
13,132
2156
4508
0.5 CHEM 2 hr
2.0
1.0 NBKI 90 min-
6.7 4 hr
1.1 UV 16 hr
2.3
Observed effect(s) Species
Decrease in agglutination of erythro- Rat
cytes by concanavalin A.
Decreased erythrocyte catalase levels Rat,
at > 5 ppm when animals were pretreated mouse
with atninotriazole.
No effect on hemoglobin. No change Mouse
in organic free radicals as measured
by EPR spectra*. No statistics.
Reference
Hamburger and
Goldstein, 1979
Goldstein, 1973
Case et al., 1979
Measurement method: CHEM = gas phase chemiluminescence; UV = UV photometry; NBKI = neutral buffered potassium iodide.
-------
TABLE 9-20. HEHATOLOGY: HUMAN - IN VITRO EXPOSURE
H
a\
co
Ozona3
concentration
980 pg/ra3 (0.5 ppm)
1960 jjg/ra3 (1.0 ppm)
Oa-treated phospholipids
4 uM/ndn
Methyl ozonide
10-4-2xlQ-3 M
750 nH/nin
106 nM/min
300 nM/min
0-9.8 uM/g of Hb
0.84 |iM/nin
78400 ug/m3 (40 ppm)
1,960 ug/m3 (1.0 ppn)
Measurement
method
CHEM
NO
I
NO
NBKI
NBKI
NBKI
NBKI
NBKI
NBKI
Exposure
duration and
protocol
0.5-2 hr
5, 10, 15, and
20 Bin
1 min
30 rain
14.3 or 43,0
nMol of 03 per 10s
cell equivalent
5, 10, 20, 30,
40 and 50 Bin
NO
0-2 hr
2 hr
20 and 60 min
Observed effect(s)
Decreased agglutination of RBCs by
concanavalin A.
Decreased ATPast activity.
Decreased ATPase activity.
Heinz body formation. Prevented
by dietary vitamin E.
RBC — No effect on ATPase.
Decreased cation transport.
RBC ghosts ~ decreased ATPase
activity.
Decreased activity of purified
Oj-proteinase inhibitor.
Decreased glyceraldehyde-3-PD.
Decreased ATPase.
No statistics.
Decreased 6SH. No effect on
Hb or on glucose uptake.
Increased complement-mediated cell
damage.
Decreased native protein fluore-
Species
Hunan
Human
(RBC ghosts)
Hunan
(RBC ghosts)
Human
Human
Human
Human
(RBC ghosts)
Hunan
(RiCs,
RBC ghosts)
Human
Human
Reference
Hamburger et al
Kesner et a1_, ,
Kindya and Chan
Henzel et al . ,
. , 1979
1979
, 1976
1975b
Koontz and Heath,
1979
Johnson, 1980
Freeman et al , ,
1979
Freeman and Hudd,
1981
Goldstein et al
1974a
Goldstein et al
* »
. ,
scence. No statistics.
(RBC ghosts) 1975
-------
TABLE 9-20. HEMATOLOGY: HUMAN - IN VITRO EXPOSURE (continued)
(£>
Ozone8 Measurement
concentration method
40 nM/min I
2.5 (jM/nrin I
2.5 jjM/min I
?Not ranked by concentration; listed by
Measurement method: NO = not described
Exposure
duration and
protocol
4 min
20, 40, and 60
min
20, 40, and
60 min
reported values.
; CHEM = gas phase
Observed effect(s)
Decreased ATPase activity; lost 40%
membrane sulfhydryls. Lipid per-
oxidation and protein crossl inking
detected.
Pretreatment with semicarbazide
prevented crosslinking.
Cross-linking of membrane proteins
inactivation of glyceraldehyde-3-
phosphate dehydrogenase.
Crosslinking of spectrin. Decreased
ACHase activity. Increased K+ leak-
age from RBCs. Semicarbazide and
p-amino benzole acid prevented
these 03 effects.
cheroi luminescence; NBKI = neutral buffered
Species
Human
(RBC ghosts)
Human
Human
(RBC ghosts)
potassium iodide;
Reference
Chan et al.
Verweij and
Steveninck,
Verweij and
Steveninck,
, 1977
1980
Van
1981
I = iodometric.
-------
of ATPase to oxidation of phospholipids with subsequent cross-linking of
membrane protein by lipid peroxidation products. Because the reaction of
ozonolysis products with semicarbazide and PABA during 0, treatment of RBCs
was not directly measured in these studies, the protective mechanism remains
unclear.
In a recent study, Freeman and Mudd (1981) investigated the iji vitro
reaction of 0,, with sulfhydryl groups of human RBC membrane, proteins, and
cytoplasmic contents. After exposure of RBCs to 0, in 02 at 20 ml/min (0.84
p:Mol/min of 03) for up to 2 hr, oxidation of intracellular GSH was observed.
Ozone exposure produced membrane disulfide cross-links in RBC ghosts but not
in intact RBCs. Neither oxyhemoglobin content nor glucose uptake was affected
by 03 exposure of RBCs. These data support earlier studies of Menzel et al.
(1972) that reported decreased RBC GSH levels following exposure of rats to
3
980 ug/m (0.5 ppm) of 03 continuously for 23 days.
Although jm vitro studies using animal and human RBCs have provided
information on the possible mechanism by which 0, may react with cell membranes
and RBCs, extrapolation of these data to in vivo 0- toxicity in man is diffi-
""""" O
cult. In most jm vitro studies, RBCs were exposed by bubbling high 03 concen-
trations (> 1 ppm) through cell suspensions. Not only were the 03 concentra-
tions unrealistic and the method of exposure nonphysiological, but the toxic
species causing RBC injury may be different during in vitro and in vivo 0,,
™"rm~ v ::::::'"::n •—— -—.—,—„. ^
exposures. Because of its reactivity, it is uncertain that 03 per se reaches
the RBCs after inhalation but may instead appear in blood in the form of less
reactive products (e.g., lipid, peroxides). However, during in vitro exposure
of RBC suspensions, 03 or highly reactive free-radical products (e.g., hydroxyl
radical, superoxide anion, singlet oxygen) may be the cause of injury.
9,4.3.3 Changes In Serum. In addition to O3's effects on RBCs, changes have
been detected in the serum of animals exposed to 0,. P'an and Jegier (1971)
3
investigated the effects of 784 ug/m (0.4 ppm) of 03 6 hr/day, 5 days/week
for 6 months on the serum trypsin inhibitor capacity (TIC) of rabbits. With
the exception of a sharp rise after the first day of exposure, TIC values-
remained within normal limits. However, after exposure for 10 months, the TIC
had progressively increased to about three times the normal level (P'an and
Jegier, 1972). Microscopic evaluation suggested that the rise in TIC may have
been due to the thickening of small pulmonary arteries. The results from this
study'are questionable, however, because the rabbits may have had intercurrent
infectious disease, which was more severe in the exposed animals (Section 9.3.1).
9-170
-------
P'an and Jegier (1976) also reported changes in serum proteins after
3 3
exposure of rabbits to 784 [jg/m (0.4 ppm) and 1960 [jg/m (1.0 ppm) of 0~.
3
Following exposure to 784 [jg/m (0.4 ppm) of 03 for 105 days, the albumin
concentrations began to decrease, and a- and 6-globulin concentrations began
to increase. At the end of 210 days of exposure, the mean albumin level fell
16 percent, the crglobulin level rose 78 percent, and the 6-globulin levels
fell 46 percent. No significant changes were observed in total protein concen-
tration.
Chow et al. (1974) observed that the serum lysozyme activity of rats
increased significantly during continuous (24 hr/day) but not during intermit-
2
tent (8 hr/day) exposure to 1568 |jg/m (0.8 ppm) of 0~ for 7 days. The in-
creased release of lysozyme into the plasma was suggested to be a result of 0-
damage to alveolar macrophages.
Veninga et al. (1981) reported that short-term exposures of mice to low
03 concentrations induced changes in serum creatine phosphokinase (CPK)
activity. Ozone doses were expressed as the product of concentration and
3
time; the maximum 03 concentration was 1600 pg/m (0.8 ppm), and the maximum
exposure time was 4 hr. Alterations in CPK were detected immediately and
15 min after termination of the exposure. By 30 min postexposure, the CPK
activities had returned to control levels. Neither plasma histamine nor plasma
LDH was altered by the range of 03 doses employed. The authors concluded that
these responses may represent adaptation of the animals to 03 toxicity by
enhanced metabolic processes.
Serum lipids and lipoproteins of rats exposed continuously for 2 wk to
3
1960 |jg/m (1 ppm) were determined (Vaughan et al., 1984). Serum from each
guinea pig was sampled before and' immediately after the 2-wk exposure and
30 days .after exposure ceased. Thus, each animal served as its own control
and there was no air-exposure group. Immediately after exposure, cholesterol,
low density lipoproteins-, and ve-ry low density lipoproteins were elevated in
males. Generally, females had similar effects, but no changes ,in very low
density lipoproteins. Triglycerides were not affected in either sex immediately
after exposure. Although statistical comparisons of sex susceptibility were
not performed, it appears that males were more affected than females. Statis-
tical tests of the post-exposure group were unclear, but it appears that
levels of cholesterol, low density lipoproteins, and very low density lipopro-
teins had returned to pre-exposure values.
9-171
-------
Serum lipids and lipoproteins have also been evaluated in male rats after
repeated 0- exposure (Mole et al., 1985). A concentration response study
o
involved exposure to air; 1960, 3430, and 5880 ug/m (1, 1.75, and 3 ppm) 03
for 5 hr/day for 10 exposure days within a 14-day period. For a given rat,
serum samples taken 2 days prior to the first exposure were compared to samples
taken 20 hr after the last exposure;, each animal served as its own control.
Another group of animals was exposed to air and sampled pre- and postexposure.
Shifts in the 0--exposed rats (pre- vs. postexposure) were compared statisti-
cally to shifts in the air-exposed animals. Ozone caused a concentration-related
linear increase in total lipoprotein-free cholesterol and high density lipo-
protein total cholesterol (both the free and esterified components) and a
decrease in total lipoprotein triglycerides. There was no effect on high
density lipoprotein-tryglycerides or on total lipoprotein-free fatty acids.
2
At the 1960 ug/m (1 ppm) 03 level, none of the values was elevated signifi-
cantly over controls. In a sampling time study (Mole et al., 1985), rats were
2
exposed to 1960 ug/m (1 ppm) 03 for 5 hr/day for 15 days in 5-day segments
within a 19-day period. Serum samples were taken from each rat 4 days prior
to the first exposure and at 7 times (0 to 44 hr) after the last exposure.
Ozone increased serum total chlolesterol (p = 0.1), high density lipoprotein-
cholesterol (p = 0.08), and high density lipoprotein-free cholesterol (p =
0.006), and decreased triglycerides (p = 0.06). The changes appeared to be
maintained over the 44-hr post-exposure period and were greater than those
2
observed at the 1960 ug/m (1 ppm) 03 level of the concentration-response
study described above that used fewer days of exposure. Thus, 03 caused a
mild hypercholesterolemia and hypotriglyceridemia.
Both the Vaughan et al. (1984) and Mole et al. (1985) studies report
/
increases in serum cholesterol. There is some disparity between results for
other lipoproteins. The Vaughan et al. study did not account for changes that
can be produced by exposure stress alone, as indicated by Mole._et_al. (19.85).
The Mole et al. (1985) studies showed only marginally significant effects.
Thus, a possible conclusion from the rat and guinea pig studies is that short-
term exposure to 1960 ug/m (1 ppm) has the potential of elevating cholesterol
in animals. Elevation in human serum cholesterol is a risk factor in human
coronary heart disease (Gottov 1979; Dawber, 1980).
9.4.3.4 Interspecies Variations. The use of animal models to investigate
the effects of 03 on the blood is complicated, because few species respond
9-172
-------
like humans. The rodent model has been most commonly used to predict the
effects of 0- on human RBCs (Calabrese et al., 1979). However, the reliability
of this model was challenged by Calabrese and Moore (1980).on the following
grounds: (1) ascorbic acid synthesis was significantly increased in mice
following 03 exposure (Veninga and Lemstra, 1975), (2) ascorbic acid protected
human G-6-PD-deficient RBCs jji vitro from the oxidant stress of acetylphenyl-
hydrazine (Winterbourn, 1979), and (3) humans lack the ability to synthesize
ascorbic acid. Although Calabrese and Moore (1980) stressed that this hypo-
thesis is based on a very limited data base, they,point out the importance of
developing animal models that can accurately predict the response of human
G-6-PD-deficient humans to oxidant stressor agents. In another report, Moore
et al. (1980) suggested that C57L/J mice may present an acceptable animal
3
model, because these mice responded to 0, exposure (627 ug/m , 0.32 ppm for 6
«3
hr) in a manner similar to that of humans, with increases in serum vitamin E
and G-6-PD activity. Unlike many other mouse strains, the C57L/J strain has
low G-6-PD activity, which is similar to that found in human RBCs. Moore
et al. (1981b) also followed up on the proposed use of Dorset sheep as an
animal model for RBC G-6-PD deficiency in humans (National Research Council,
1977). However, Dorset sheep were found to be no more sensitive than normal
humans with respect to 03-induced changes in GSH and also differed from humans
in the formation of methemoglobin. Further studies (Calabrese et al., 1982,
1983b,d; Williams et al., 1983a,b,c) demonstrated that the responses of sheep
and normal human erythrocytes were very similar when separately incubated with
potentially toxic Q-> intermediates, but G-6-.PD-deficient human erythrocytes
were considerably more susceptible. Consequently, the authors also questioned
the value of the sheep erythrocyte as a quantitatively accurate predictive
model.
9.4..4 .Reproductive and .Teratpge.m'c Effects ,
Pregnant animals and developing fetuses may be at greater risk to effects
from photochemical oxidants, because the volume of air inspired by females
generally increases from 15 to 50 percent during pregnancy (Altman and Dittmer,
1971). Before 1978, experiments designed to investigate the reproductive
effects of photochemical oxidants often used complex mixtures of gases, such
as irradiated auto exhaust (see Section 9.5), or they used oxidant concentra-
tions greater than those typically found in ambient air. Brinkman et al.
9-173
-------
(1964) exposed pregnant mice to lower concentrations of 03, but the results of
their experiments are difficult to interpret, because the time of 0, exposure
during gestation and postparturition was not specified. They reported that
3
mice exposed to 196 or 392 ug/m (0.1 or 0.2 ppm) of 03 for 7 hr/day and 5
days/week over 3 weeks had normal litter sizes, compared with air-exposed
controls. However, there was greater neonatal mortality in the litters of
3
0--exposed mice, even at the exposure level of 196 [jg/m (0.1 ppm) of 03
(Table 9-21). Unfortunately, without more details on the period of exposure,
it is impossible to ascertain whether the decreased infant survival rate was
due to development interference j_n utero, to a direct effect on the pups, or
to a nutritional deficiency caused by parental anorexia or reduced lactation,
or a combination of these effects. When using a similar experimental protocol,
o
Veninga (1967) found that mice exposed to 392 ng/m (0.2 ppm) of 03 for 7 hr/day,
5 days/week during embryo!ogical development and the 3 weeks after birth
(total exposure time not reported) had an increased incidence of excessive
tooth growth, although no statistical evaluation was provided.
In more recent experiments, Kavlock et al. (1979) exposed pregnant rats
to 0, for precise periods during organogenesis. No significant teratogenic
effects were found in rats exposed 8 hr/day to concentrations of Oq varying
from 863 to 3861 ug/m3 (0.44 to 1.97 ppm) during early (days 6 to 9), mid
(days 9 to 12), or late (days 17 to 20) gestation, or the entire period of
organogenesis (Days 6 to 15). Continuous exposure of pregnant rats to 2920 ug/
m (1.49 ppm) of 0, in midgestation resulted in increased resorption of embryos.
q
A single dose of 150 mg/kg sodium salicylate followed by 1960 ug/m (1.0 ppm)
of 0~ during midterm produced a significant synergistic increase in the resorp-
tion rate, a decrease in maternal weight change, and a decrease in average fetal
3
weight. Exposure of pregnant rats 8 hr/day to 862 pg/m (0.44 ppm) of 03
throughout the period of organogenesis also resulted in a significant decrease
in average maternal weight gain.-
In a follow-up study, Kavlock et al. (1980) investigated whether i_n utero
exposure to 0, can affect postnatal growth or behavioral development. In con-
trast to the results of Brinkman et al. (1964), neonatal mortality of rats was
3
not increased by exposure to 2940 [jg/m (1.5 ppm) of 0, for periods of 4 days
3
during gestation. Pups from litters of females exposed to 1960 yg/m (1-0
ppm) of 03 during mid- (days 9 to 12) or late (days 17 to 20) gestation exhibi-
ted significant dose-related reductions in weight 6 days after birth. Pups
9-174
-------
TABLE 9-21. REPRODUCTIVE AND TERATOGENIC EFFECTS OF OZONE
vo
1
l_l
~J
in
Ozone
concentration
ng/B3 ppm
196 0, 1
392 0.2
862 0.44
2920 1.49
3
Measurement Exposure
method duration and protocol
ND 7 hr/day, 5 days/week
for 3 weeks
ND 7 hr/day, 5 days/week
for 3 weeks
I" 8 hr/day over entire
period of organogenesis
(days 6 to 15)
Continuous during mid-
gestation
Observed effect(s) Species
Increased neonatal mortality £4.9 to 6.8% House
vs. 1.6 to 1.9% for controls) .
Unlimited growth of incisors (5.43. incidence Mouse
vs. 0.9% in controls) .
Decreased average maternal weight gain. Rat
Increased fetal resorption rate (50% vs. 9%
for controls).
Reference
Brinkman et al. , 1964
Veninga, 1967
Kavlock et al. , 1979
1960 1.0
1960 1.0
2940 1.5
Continuous during late
gestation
Continuous during mid-
(day 9 to 12) or late
(days 17 to 20) gestation
Continuous during late
gestation (days 17 to 20)
Slower development of righting, eye opening,
and horizontal movement; delayed grooming
and rearing behavior.
Average weight reduced 6 days after birth.
3 males (14.3%) were permanently runted.
Rat
Kavlock et al., 1980
Measurement method: ND = not described, I = iodoroetric (Saltzntan and Gilbert, 1959).
No statistical evaluation.
-------
from the late gestation exposure group were affected to a greater extent and
for a longer period of time after parturition. In fact, several males exposed
3
to 2940 |jg/m (1,5 ppm) of 03 during late gestation were also significantly
slower in the development of early movement reflexes and in the onset of
grooming and rearing behaviors. The authors pointed out that it is impossible
to distinguish between prenatal and postnatal contributions to the behavioral
effects, because foster parent procedures were not used to raise the pups.
9.4.5 Chromosomal and Mutational Effects
9.4.5.1 Chromosomal Effects of Ozone. A large portion of the data available
on the chromosomal and mutational effects of 0, was derived from investigations
3
conducted above 1,960 ug/m (1 ppm) of 03, and their relevance to human health
is questionable. However, for completeness of the review of the literature,
and for possible insight into the mechanisms by which 0™ may produce genotoxi-
city, this discussion will not be limited to data derived from research conducted
3
at or below 1,960 ug/m (1 ppm) of 0-,- Data derived predominantly from In
3
vitro experiments conducted at CL concentrations in excess of 1,960 (jg/m (1
ppm) of 0- will be discussed .first (Table 9-22), followed by a discussion of
the genotoxicity data from both i_n vitro and In vivo research conducted at or
below 1 ppm of 03 (Table 9-23).
The potential for genotoxic effects relating to 0- exposure was predicted
from the radiomimetic properties of 03. The decomposition of 03 in water
produces OH and HOp radicals, the same species that are generally considered
to be the biologically active products of ionizing radiation. Fetner (1962)
reported that chromatid deletions were induced in a time-dependent manner in
3
human KB cells exposed to 15,680 M9/m (8 ppm) of 03 for 5 to 25 min. The
chromatid breaks were apparently identical to those produced by x-rays. A
10-min exposure to 8 ppm of 0., was slightly more efficient in the production
of chromatid breaks than 50 rad of x-rays'. Significant mitotic delay was
measured in neuroblasts from grasshoppers (Chortophaga viridifaciata) exposed
to 3500 to 4500 ug/L of Og in a closed system (Fetner, 1963).
Scott and Lesher (1963) measured a sharp loss of viability with Escherichia
coli as the 0™ concentration was increased. Viability was reduced to zero
111 *5
when cells were exposed to 1 pg/ml of Q~. Damage 'to cell membranes was evident
by the leakage of nucleic acids and other cellular components from cells
exposed to 0.18 ug/ml of 0-
9-176
-------
TABLE 9-22. CHROMOSOMAL EFFECTS FROM IN VITRO EXPOSURE TO HIGH OZONE CONCENTRATIONS
a
concentration
Measurement
method
Exposure
duration and protocol
Observed effect(s)
Species
Reference
15,680 MO/M8 (8ppm)
UKI
5-25 min
Chroinatid deletions.
Humans KB
cells
Fetner, 1962
98,000 ug/m3 (50 ppro)
MAST"
30 min
Tex mutants deficient in
repair of x-ray-induced
DMA strand breaks were more
sensitive to lethal effects
of Oa than were the wild-type
repair-proficient parental strains
Escherichia
coTi
Hamelin and
Chung, 1974
98,000 M9/B3 (50 ppm)
MASF
30 Bin
DNA Polymerase I mutant strains
(KMBL 1787, 1789, 1791) were more
sensitive to the cytotbxic effects
of 03, and OKA was degraded to a
greater extent in the first 3 hr
after 03 exposure than strain KMBL
1788, which contains a normal DNA
Polymerase I.
E. coli
Hamelin et al.,
1977a
98,000 pg/m3 (50 ppa)
MAST"
30 min
Hucoid mutant strains (MQ 100 &
105) obtained by treating MQ 259
with 03 yet having full complement
of DNA repair enzymes were shown
to be more sensitive to Oa and
degraded DNA to a greater extent
than the Ion + (MQ259) strain.
E. coll
Hamelin et al.,
1977b
98,000 pg/m3 (50 ppra)
MAST"
up to 3 tirs 15 different DNA repair-deficient
strains were tested for sensitivity
to the cytotoxic effects of 03; DNA
Polytnerase I was involved in DNA
repair but Polymerases I and II
and DNA synthetic genes dna A, B,
and C were not; recombinational
repair pathways, assayed with rec
A and rec B strains, were only
partially involved in the repair
of 03-induced DNA damage.
E. coli
Hamelin and Chung,
1978
-------
TABLE 9-22. CMROHQS0HM. EFFECTS FROM IN VITRO EXPOSURE TO HIGH COHCINTRATIONS (continued)
M
~J
oo
Oa Measurement
concentration nethod
0.1 ug/»l [51 pp«] UV
0.5 ug/ml [255 ppin] UV
0.18 fig/ml [92 ppn] UKI
1.0 |ig/ml [510 ppm]
0.5-6 ug/ml [255- NBKI
3061 ppn]
1-10 ug/nl [510- NBKI
5100 ppm]
5X [SO, 000 ppm] NBKI
3.5-4.5 Mi/«1 [1786- UKI
2296 ppm]
2% [20,000 ppm] e
8% [80,000 ppm] 'GPT
Exposure
duration and protocol Observed effect(s)
60 nln (70 8l/«1n) Preferential degradation of yeast
RNA at the N-glycosyl linkage;
sugar-phosphate linkage was
Oa stable.
30 rain (330 ml/rnin) 5-ribonucleotide guanosine
monophosphate was degraded
most rapidly.
NO Release of nucleic acids;
cell lethality.
0-5 nin Os reacts with pyrinndine
bases-froin nucleic acids
(thyinidine > cytosine >
uraci 1 ) .
30 nin Cell death and nonspecific
chromosomal aberrations:
shrunken and fragmented nuclei,
clumped metaphase chromosomes
and chromosome bridges.
0-40 min Rapid loss of glycolytic and
respiratory capacity; loss of
tumorigenicity after 20 min.
exposure.
ND Hitotic delay.
3 min Abnormal nuclei; fragmentation.
5, 15, 60 s Rapid degradation of nucleic
acid bases, nucleosides or
nucleotides in 0.05M phosphate
buffer, pH 7.2.
Species
Yeast
E. coli
E. coli
Chick
embryo
fibroblasts
Mouse
as cites
cells
Chortophaga
viridifaciata
Am. oyster
ND
Reference
Shiniki et al.,
1983
Scott and Lesher,
1963
Prat et al., 1968
Sachsenmaier
et al,, 1965
Fetner, 1963
Maclean et al . ,
Ii73
Christensen and
Giese, 1954
Not ranked by air concentration; listed by reported exposure values and [approximate ppm conversions].
Measurement method: MAST = Kl-coulometric (Mast meter); NBKI = neutral buffered potassium iodide; UV = UV photometry; GPT = gas phase titration;
UKI = unbuffered potassium iodide.
C03 flov* rate given in (ml/min):, when available. ND = not described.
Concentrations of 03 were not measured in the cell suspensions.
eOa analyzer (Fisher and Porter, Warminster, PA).
-------
TABLE 9-23. CHROMOSOMAL EFFECTS FROM OZONE CONCENTRATIONS AT OR BELOW 1960 ug/m3 (1 Ppm)
H
»-J
VD
03
Concentration
M8/"i'i
294
412
1940
451
2548-
14,700
3234-
27,832
3920
392
470-
588
ppm
0.15
0.21
0.99
0.23
1.3-
7.5
1.65-
14.2
2.0
0.2
0.24-
0.3
Measurement3'
method
NBKI
NBKI
NBKI
NBKI
NBKI
MAST
UKI
MAST
UKI
Exposure0
duration and protocol
5 hr
5 hr
2 hr
5 hr
(in vitro)
ND
(in vitro)
ND
(in vitro)
5-90 rain
(in vitro)
5 hr
(in vivo)
5 hr
CLE vivo)
Observed effect(s) Species Reference
No effect induced by 03 treatment Mouse Gooch et a!,,
on the frequency of chromosome or 1976
chromatid aberrations in Chinese
hamster or mouse peripheral Hamster
blood lymphocytes stimulated with
PHA; no effect on spermatocytes
in mice 8 wk following exposure.
Peripheral blood lymphocytes exposed Human
to 03 in culture 12 hr after stlnr lymphocytes
ulation with PHA showed no increase
in chromosome or chromatid aberrations.
Peripheral blood lymphocytes exposed Human
to Oa in culture, 36 hr after PHA lymphocytes
stimulation, showed no change in the
frequency of chromosome or chromatid
aberrations at any concentration
except 7.23 ppm of Og.
No apparent increase in the fre- Human
quency of chromosome or chromatid lymphocytes
aberrations 12 or 36 hr after
PHA stimulation.
Combined exposure to 03 and radi- Hamster Zelac et al.,
ation (227-233 rad) produced an 1971b
additive effect on the number
of chromosome breaks measured in
peripheral blood lymphocytes.
A significant increase in chro- Hamster Zelac et al.,
mosorae aberrations (deletions, 1971a
ring dicentrics) in the peri-
pheral blood lymphocytes; in-
creased break frequency was
still apparent at 6 and 16 days
following exposure.
-------
TABLE 9-23. CBRQMOSOHAL EFFECTS FROM OZONE CONCENTRATIONS AT OR BELOW 1860 (jg/ra3 (1 ppo) (continued)
f
(-•
CO
o
03
Concentration
ug/P ppi
490- 0.2S-
1960 1.0
588- 0.3-
1568 0.8
843 0.43
3920 2.0
1960- 1.0-
9800 5.0
Beasurenent3' Exposure
method duration and protocol
UV 1 hr
(in vitro)
UV 8 days,
continuous
Cifi vitro)
UV, 5 hr
HBKI (in vivo)
6 hr
ijn v|yo)
CHEM 24 hr
NBKI (in vivo)
Observed effect(s) Species Reference
Dose- related increase in SCE fre- Hunan Guerrero et
quency in WI-38 diploid fibroblasts fibroblasts al., 1979
exposed in culture.
Growth of cells from lung, breast, Hunan Sweet et al.,
and uterine tumors were inhibited tumor 1980
to a greater degree than IHR-90, cells
a nontunor diploid fibroblast.
Increase in chromatid-type Hamster Tice et al.,
aberrations in peripheral blood 1978
lymphocytes of 03-exposed hamsters;
Increase in deletions at 7 days
and increase in achromatic lesions
at 14 days after exposure; ehronosoae-
type lesions were not significantly
different; no chromosomal aberrations
in bone Marrow lymphocytes; no change
in SCE frequency in peripheral blood
lymphocytes.
No change in SCE frequency in peri- Mouse
pheral blood lymphocytes.
Variable decrease in the molecular Mouse Chaney, 1981
weight of DNA from peritoneal exu-
date cells of 03 exposed mice
becoming significant at 5 ppm;
significant induction of single-
strand breaks at 5 ppm.
Measurement method: HAST = Kl-coulometric (Mast meter); NBKI = neutral buffered potassium iodide; UV = UV photometry
Calibration method: UKI = unbuffered potassium iodide; NBKI = neutral buffered potassiua iodide.
CND = not described.
Abbreviations used: PHA = phytohemagglutinin; SCE = sister chronatid exchange.
-------
The molecular mechanism for the clastogenic and lethal effects resulting
from Qg exposure are not precisely known. Bubbling 8 percent CL through a
phosphate buffer solution (Q.05M, pH 7.2) containing DNA caused an immediate
loss in absorption at 260 nm and an increase in the absorption of the solution
at wavelengths shorter than 240 nm (Christensen and Giese, 1954). A similar
rate of degradation was observed with RNA and the individual purine and pyrinr
idine bases, nucleosides, and nucleotides. In a more recent report, Shiniki
et al. (1983) examined the degradation of a mixture of 5' nucleotides, yeast
t-RNA or tobacco mosaic virus RNA with 03 (0.1 to 0.5 mg/L). The guanine
moiety was found to be the most 0,.-labile among the four nucleotides, whether
the guanine was present as free guanosine monophosphate or incorporated into
RNA. The sensitivity to degradation by 03 among the four nucleotides was
found to be, in decreasing order, GMP > UMP > CMP > AMP (GMP = guanosine
monophosphate, U = uridine, C = cytidine, A = adenosine). Even after exten-
sive ozonolysis of yeast t-RNA (0.5 |jg/ml,. 30 min) and substantial degradation
of the guanine moieties, the RNA migrated as a single band on polyacrylamide
gels. The band exhibited the same-mobility as the intact t-RNA, indicating
that although the glycosidic bond between the sugar and the base is 03-labile,
the sugar-phosphate backbone was intact and extremely stable against 0-. Prat
et al. (1968) investigated the reactivity of the pyrimidines in E._ coll DNA
with 0^ (0.5 to 6 mg/L, 0 to 5 min) and radiation. Ozone preferentially
reacted with thymidine, then with cytosine and uracil, in decreasing order of
reactivity. The results are slightly different from those reported by Shiniki
et al. (1983) in that the reactivity with uracil and cytidine" are in reversed
order.
There is evidence that single-strand breaks in DNA may contribute to the
genotoxic effects of 0-j. Radiosensitive lex mutants of E. coli, which were
O .~ '~—
known to be defective in the repair of x-ray-induced single-strand breaks in
DNA, were-found to be significantly more sensitive to the cytotoxic effects of
Qv than the repair-proficient parental strain (Hamelin and Chung, 1974).
In an effort to investigate the nature of the 0.,-induced lesion in DNA,
Hamelin and co-workers investigated the survival of bacterial strains with
known defects in DNA repair. Closely related strains of £_._ coli K-12 with
mutations in DNA polymerase I were shown to be more sensitive to the cytotoxic
effects of 0~ than the DNA polymerase proficient (pol +) strain (Hamelin et
al., 1977a). Polymerase I-deficient strains also exhibited an extensive
9-181
-------
degradation of DNA in response to 03 or x-ray treatment. The authors concluded
that DNA polymerase I plays a key role in the repair of lesions produced in E.
coli DNA by 0_ and that the unrepaired damage was responsible for the enhanced
™nmmr::r~--n £
degradation of DNA and the enhanced cell killing observed in the pol- mutants.
This interpretation of the data may not be entirely correct, because an enhanced
degradation of DNA and an increased sensitivity to cell killing were also
observed in a Ion mutant strain of £._ coli K-12 (Hamelin et al., 1977b). The
Ion mutant appears to have a full complement of DNA repair enzymes. With
these mutants, there may be an enhanced DNA repair activity (evidenced by the
extensive degradation of DNA), and the enhanced activity of the Ion gene
products was thought to be responsible for the increased cell killing observed
with these strains when they were exposed to 03.
Although DNA polymerase I was shown to be involved in the repair of
0,-induced DNA damage (Hamelin et al., 1977a), JL_ coli cell strains with
mutations in DNA polymerase II or III were not found to be more sensitive to
0, than the wild-type, suggesting that these enzymes are not involved in the
repair of DNA damaged by 0, (Hamelin and Chung, 1978). Mutant strains of g._
coli with defects in DNA synthesis (DNA A, B, C, D, and G) showed no enhanced
sensitivity to CU. Therefore, the DNA gene products are probably not involved
in the repair of (U damage. Recombinational repair mutants, rec A and rec B,
only showed a slightly increased sensitivity to 0~ than the wild-type, suggest-
ing that the rec gene products are only partially involved in the repair of
Og-induced DNA lesions (Hamelin and Chung, 1978).
Other effects have been observed than those described above on bacteria.
In the commercial American oyster exposed to CU-treated sea water (Maclean et
al., 1973), fertilization occurred less readily and abnormal nuclei (degenera-
tion, fragmentation) were observed approximately twice as frequently. Sachsen-
maier et al. (1965) observed a rapid loss of glycolytic and respiratory capacity
and subsequent loss of tumorigenicity in mouse ascites cells treated with CL.
These authors also reported that chicken embryo fibroblasts exposed to CU (1
to 10 ul/ml) for 30 min exhibited nonspecific alterations in cells resembling
those seen after x-ray damage, including shrunken nuclei, clumped metaphase
chromosomes, arrested mitosis, chromosome bridges and fragmented nuclei.
In the studies described up to this point, the investigators have predomi-
nantly examined the in vitro effects of extremely high Og concentrations on
biological systems or biologically important cellular components. Although
9-182
-------
these investigations may be important for the elucidation of the types of
damage or responses that might be expected to occur at lower CL concentrations,
the most relevant data on the genotoxicity of CL should be obtained from
3
investigations where the 03 concentration did not exceed 1960 (jg/m (1 ppm).
Research, conducted at or below 1 ppm of 0, will be presented below (See Table
9-24).
Several investigators have examined the j_n vivo cytogenetic effects of 03
in rodents and human subjects. Until the reports of Zelac et al. (1971a,b),
the toxic effects of Q» were generally assumed to be confined to the tissues
directly in contact with the gas, such as the respiratory epithelium. Due to
the highly reactive nature of CU, little systemic absorption was predicted.
Zelac, however, reported a significant increase in chromosome aberrations in
3
peripheral blood lymphocytes from Chinese hamsters exposed to 392 pg/m (0.2
ppm) for 5 hr. Chromosome breaks, defined as the sum of the number of deletions,
rings and dicentrics, were scored in lymphocytes collected immediately after
0» exposure and at 6 and 15 days postexposure. At all sampling times, there
was an increase in the break frequency (breaks/ cell) in the_03-exposed animals
when compared with nonexposed control animals. Zelac et al. (1971b) reported
that Q3 was additive with radiation in the production of chromosome breaks.
Both 0» and radiation produced chromosome breaks independently of each other;
Simultaneous exposure to 0.2 ppm of 03 for 5 hr and 230 rad of radiation
resulted in the production of 40 percent more breaks than were expected, from
either agent alone and 70 percent of the total number of.breaks expected from
the combined effects of the two agents, if it was assumed that the effects
were .additive. .
Chaney (1981) investigated the effects of 03 exposure on mouse peritoneal
exudate cells (peritoneal macrophages) stimulated by an i.p. injection of
3
glycogen. Mice were subsequently exposed by inhalation to 1960 or 9800 ug/m
(1 or 5 ppm) of 0., for 24 hr. A significant reduction in the average molecular
weight of the DNA was observed in the peritoneal exudate cells from mice
3 ' 3
exposed to 9800 ug/m (5 ppm) of 03 but not in animals exposed to 1960 ug/m
(1 ppm) of OT for 24 hr. The reduction in the average molecular weight of DNA
in 03~exposed animals indicated the induction of single-strand breaks in the
DNA. It should be noted, however, that the alkaline sucrose gradient method
of determining the average molecular weight of the DNA does not discriminate
between the frank strand-breaks in DNA, produced as a direct effect of the 03
treatment, and the induction of alkaline-labile lesions in DNA by 03, which
9-183
-------
TABLE 9-24. MUTAT10NAL EFFECTS OF OZOHE
concentration Measurement
ug/i3 pjw
method
Exposure .
duration and protocol
Observed effect(s)
Species
Reference
t->
00
196 0.1 HAST
58,800 30 NBKI
98,000 50 UV
60 rain
(2.1 nl/min)
3 hr
30 rain
(2.1 ml/min)
Various mutated, growth factor auto-
trophic states, were recovered; mutant
strains differed from parental strains
in sensitivity to UV light and excessive
production of capsular polysaccharide.
Induction of a dominant lethal muta-
tion during stages of spermatogenesis;
spern were found to be twice as sen-
sitive as earlier stages.
Radiation-sensitive mutant strains
defective in repair of single strand
Escherichia coli
(HQ 259)
Drosophila
virilis
Saccharomyces
cerevisae
Hamelin and
Chung, 1975a
Erdman and
Hernandez, 1982
Oubeau and
Chung, 1979
(rad 51) and double strand (rad 52)
DMA breaks were more sensitive to
03 cell killing than either the
wild-type or the UV light repair
deficient strain (rad 3); recom-
binational repair-deficient strain
(rad 6) was moderately sensitive
to 03.
98,000 50
UV
30 Bin
Induction of forward mutations at 2
loci of adenine biosynthesis (strain
C16-11C); induction of reversion
nutation at 6 genetic loci (strain
XV 185-14C); induction of intra-
genic and intergenic recombinational
mutants (strain 07); 03 was 2Q-20Gx
less mutagenic than equitoxic expo-
sures to UV light, x-rays, or MNNG.
Saccharomyces
cerevisae
Dubeau and
Chung, 1982
Measurement method:
QS flow rates given in (ml/min), where available.
HAST = Kl-coulometric (Mast meter); NBKI = neutral - buffered potassium iodide; UV = UV photometry
-------
would be converted to strand breaks under the alkaline condition of the assay.
Although these experiments do not prove that 0~ exposure can cause strand
3
breaks in DNA, they do indicate an 03 effect on DNA at 9800 ug/m (5 ppm) of
03 for 24 hr.
Because of the importance of the reports by Zelac et al. (1971a,b) that
indicated that significant levels of chromosome aberrations in Chinese hamster
peripheral blood lymphocytes collected as late as 15 days after 0~ exposure by
inhalation, Tice et al. (1978) tried to repeat the experiments of Zelac as
3
closely as possible. Chinese hamsters were exposed to 843 ug/m (0.43 ppm) of
0- by inhalation for 5 hr. The authors investigated chfomatid and chromosome
aberrations in peripheral blood lymphocytes and bone marrow of control and
0~-exposed animals immediately after exposure and at 7 and 14 days after 0-,
o ' - • • t *5
exposure. They also investigated the sister chromatid exchange (SCE) frequency
in peripheral blood lymphocytes of Chinese hamsters. In separate experiments,
SCE frequencies in C57/B1 mice exposed to 2 ppm of Q3 for 6 hr were examined
in peripheral blood cells collected from the animals immediately after 0-,
exposure and at 7 and 14 days after 03 exposure.
The authors reported no significant increase in the SCE frequency of the
Q3-exposed hamsters or mice at any sampling time, nor did they observe a
significant increase in the number of chromosome aberrations of phytohemagglu-
tinin (PHA)-stimulated peripheral blood or bone marrow cells. The only report-
ed statistically significant differences were observed in peripheral blood
lymphocytes, in which there was an increase in the number of chromatid deletions
and achromatic lesions in the 7- and 14-day samples, respectively. Both types
of chromatid aberration were observed at consistently higher frequencies in
the blood samples of the 03-exposed animals, frequently in the range of 50 to
100 percent increases over the control values. Statistically significant dif-
ferences were assigned at the 1 percent level of significance. It is not
clear how a slightly more rigorous evaluation of significance (e.g., p < 0.05)
would have influenced the interpretation of the data.
Although both Zelac et al. (1971a,b) and Tice et al. (1978) reported sig-
nificant increases in chromosome aberrations in peripheral blood lymphocytes
3
following 5-hr exposures to 392 to 843 ug/m (0.2 to 0.4 ppm) of 03, the types
of lesions observed in the two studies were clearly different. Tice et al.
observed chromatid-type lesions and no increase in the chromosome aberrations,
whereas Zelac et al. reported a significant increase in the number of chromosome
9-185
-------
aberrations. There were a number of differences in the experimental protocols
that may have produced the seemingly different results:
1. The animals were exposed to different concentrations of CL.
Zelac et al. (1971a) administered 470 to 590 ug/m3 (0.24 to 0.3
ppm) of Go to Chinese hamsters for 5 hr, whereas Tice et al.
o ,,
(1978) exposed animals to an atmosphere of 840 \ig/m (0.43 ppm)
of Do for 5 hr.
2. Zelac stimulated peripheral blood lymphocytes into DNA synthesis
with pokeweed mitogen, which is mainly a B-lymphocyte mitogen.
In the experiments of Tice et al. (1978), lymphocytes were
stimulated with PHA, which is a T-lymphocyte mitogen (Ling and
Kay, 1975).
3. Zelac cultured lymphocytes with the mitogen jn vitro for 3 days
(72 hr), whereas Tice et al. cultured lymphocytes with mitogen
for 52 hours.
Because of the longer incubation time with the mitogen in the experiments of
Zelac et al. (1971a), lymphocytes may have converted chromatid type aberra-
tions, like those reported by Tice et al. (1978), into chromosome aberrations
with another round of DNA synthesis. Because the experiments of Zelac et al.
and Tice et al. were conducted with peripheral blood lymphocytes stimulated by
*
two different mitogens (pokeweed vs. PHA), the cytogenetic consequences of 03
exposure were examined in different populations of lymphocytes. If one of the
populations of lymphocytes was more sensitive to 03 than the other, different
cytogenetic responses could be expected when PHA was used as a mitogen, com-
pared with the results with the use of polkweed mitogen.
There is evidence that the B-lymphocyte may be more sensitive to 0~ than
the T-lymphocyte. Savino et al. (1978) measured the effects of 03 on human
cellular and humoral immunity by measuring rosette formation with human lympho-
cytes (See Chapter 10). Rosette formation measures the reaction of antigenie
red cells with surface membrane sites on lymphocytes. Different antigenie
RBCs are used to distinguish T-lymphocytes from B-lymphocytes. Rosette forma-
tion with B-lymphocytes was significantly depressed in eight human subjects
3
exposed to 784 yg/m (0.4 ppm) of 03 by inhalation for 4 hr, A similar inhibi-
tion of rosette formation was not observed with T-lymphocytes from the same
9-186
-------
subjects. The depressed B-cell responses persisted for 2 weeks after 0^
exposure, although partial recovery to the pre-exposure level was evident.
It cannot be stated with any certainty how the differences in the CU
exposure, the choice of mitogen, and the length of the mitogen exposure may
have contributed to the differences in the results reported by Zelac et al.
(1971a,b) and by Tice et al. (1978). There are sufficient differences in the
experimental protocols of the two reports so that the results need not be con-
sidered directly contradictory.
An assumption that is made in all of the reports in which lymphocytes are
stimulated with mitogens is that the lymphocytes from the CL-exposed animals
and the control animals ara equally sensitive to the mitogenic stimulus. This
assumption is probably not correct, because in investigations by Peterson et
al. (1978a,b) the proliferation of human lymphocytes exposed to PHA was signi-
ficantly suppressed in blood samples taken immediately after the subjects were
3
exposed to 784 ug/m (0.4 ppm) of 0, for 4 hr (See 10.7). Other reports have
suggested that 0^ might inhibit or inactivate the PHA receptor on lymphocytes
(see Gooch et al., 1976). Because the ability to measure chromosome aber-
rations in mitotically arrested cells is absolutely dependent on the induction
of Gg or G-^ cells into the cycling state, cells exposed to sufficient concen-
trations of Og would not be stimulated to divide, and hence no O^-induced cy-
togenic effects would be observed in activated cells. In their report, Tice
et al. (1978) stated that the lymphocytes of the Q~-exposed animals in their
experiments "did tend to be worse than those from controls." Only a small
difference in the number of responding lymphocytes could make large differences
in the results of the experiments if Q~~damaged lymphocytes were selected
against in the cytogenetic investigations.
In other investigations with rodents, Gooch et al. (1976) analyzed bone
3
marrow samples from Chinese hamsters exposed to 451 ug/m (0.23 ppm) of 0^ for
5 hr. Marrow samples were taken at 2, 6, and 12 hr following 0, exposures.
3
In separate experiments, male C3H mice were exposed to 294 or 412 ug/m (0.15
or 0.21 ppm) of 03 for 5 hr, or to 1940 ug/m3 (0.99 ppm) of 03 for 2 hr.
Blood samples were drawn from these animals at various times for up to 2 weeks
following 0, exposure. The mice were killed 8 weeks following 0~ exposure, and
spermatocyte preparations were made and analyzed for reciprocal translocations.
Data from the Chinese hamster bone marrow samples and the mouse leukocytes in-
dicated that there was no effect induced by 0~ treatment on the frequency of
9-187
-------
chromatid or chromosome aberrations, nor were there any recognizable reciprocal
trans! ocations in the primary spermatocytes.
Several investigators have examined the effects of 0., on human cells in
C3 —
vitro. Fetner (1962) observed the induction of chromatid deletions in human
KB cells exposed to 15,680 pg/m3 (8 ppm) of Q3 for 5 to 25 min. Sweet et al.
(1980) reported that the growth of human cells from breast, lung, and uterine
a
tumors was inhibited by exposure to 588 to 1568 (jg/m (0.3 to 0.8 ppm) of 0,,
«3
for 8 days in culture.
Guerrero et al. (1979) performed SCE analysis on diploid human fetal lung
cells (WI-38) exposed to 0, 490, 980, 1470, or 1960 yg/m3 (0, 0.25, 0.5, 0.75,
or 1.0 ppm) Oo for 1 hour in vitro. A dose-related increase in the SCE fre-
quency was observed in the WI-38 human fibroblasts exposed to Og. In the same
report, the authors stated that no significant increase in the SCE frequency
over control values was observed in peripheral blood lymphocytes from subjects
exposed to 03 by inhalation (Chapter 10). Unless the lymphocyte is intrinsi-
cally less sensitive to the induction of SCE by 03 than the WI-38 human fetal
lung fibroblast, the results indicate that exposure of human subjects to 980
2
ug/rn (0.5 ppm) of 03 for 2 hr did not result in a sufficiently high con-
centration of 03 or Oo reaction products in the circulation to induce an in-
crease in the SCE frequency in the lymphocytes. From the authors' data on the
induction of SCE in WI-38 cells, the concentration of 0« required to induce
3
SCE in human cells is approximately 490 (jg/m (0.25 ppm) for 1 hour.
Gooch et al. (1976) also investigated the effects of 0- exposure on human
cells IB yjtrjo. In these experiments, lymphocytes were stimulated with PHA
for 12 or 36 hr before the 03 exposure to obviate the potential problems of
CL inactivation of the PHA receptor. Human leukocyte cultures v/ere exposed to
3
3920 ug/m (2 ppm) of 03 for various lengths of time to accumulate total 0~
exposure doses of 3234 to 27,832 HS/m per hour (1.65 to 14.2 ppm/hr). The
results showed no increase in the chromatid and chromosome aberrations at any
total dose, with the possible exception of an apparent spike in chromatid
2
aberrations at a total exposure of 14,170 \ig/m (7.23 ppm/hr). The significance
of this observation is unclear because the data showed no dose-response increase
in the number of chromatid aberrations at concentrations near 7.25 ppm/hr, and
the authors did not report how, or indeed if, the data were statistically
evaluated for differences in chromatid aberrations.
In summary, in vitro 0» exposure has been shown to produce toxic effects
_"•» — — — $
on cells and cellular components including the genetic material. Cytogenetic
9-188
-------
toxicity has been reported in cells in culture and in cells isolated from
animals if 0- exposure has occurred .at sufficiently high levels and for suffi-
ciently long periods,
9.4.5.2 Mutational Effects of Ozone. The mutagenic effects of 0,, have been
investigated in surprisingly few instances (Table 9-24). No publication to
date has investigated the mutagenic effects of 03 in mammalian cells.
Sparrow and Schairer (1974) measured an increase in the frequency, over
the background level, in the induction of pink or colorless mutant cells or
groups of cells in petal and/or stamen hairs of mature flowers of various
blue-flowered Tradescantia. No 03 concentration was reported in this publica-
tion.
LL coli, strain MQ 259, were mutated to various growth factor auxotrophic
states, including requirements for most common amino acids5 vitamins, and
3
purines and pyrimidines (Hamelin and Chung, 1975a). Ozonated air (196 |jg/m ,
0.1 ppm) was passed through the bacterial suspensions at a rate of 2.1 L/min
for 30 min. Many of the Q~-induced mutant strains were either more or less
sensitive to UV light than the parental strain. Other mutant strains, called
mucoid mutants, had apparent defects in DNA repair pathways and were charac-
terized (Hamelin and Chung, 1975b) as producing excessive amounts of capsular
polysaccharide.
Erdman and Hernandez (1982) investigated the induction of dominant lethal
2
mutations in Drosophila virilis exposed to 58,800 |jg/m (30 ppm) of 0, for 3
hr. 0™ induced dominant lethal mutations at various stages of spermatogenesis.
The sperm-sperm bundle stage was the most sensitive to 0-,, and the meiotic
cells were the least sensitive,
Dubeau and Chung (1979, 1982) have investigated the mutagenic and cyto-
toxic effects of 0- on Saccharomycescerevisae. Several different strains
were utilized to investigate forward, reverse, and recombinational mutations.
3
ozone (98,000 yg/m , 50 ppm; 30 to 90 min) induced a variety of forward and
reverse mutations as well as gene conversion and mitotic crossing-over. Both
base-substitution and frame-shift mutations were induced by 0,. Ozone was
shown to be more recombinogenic than mutagenic in yeast, probably as a result
of the induction of strand breaks in DNA, either directly or indirectly.
In the investigation of Dubeau and Chung (1982), the mutagenic potency of
3
0~ (98,000 M9/m . 50 PP^; 30 to 90 min) was compared with other known mutagens.
2
The positive controls were UV light (1.54 J/m per second, 1-min exposure),
N-methyl-N'-nitrosoguanidine (MNNG) (50 yg/ml, 15 min), or x-rays (2 kR/min,
9-189
-------
40 min). By comparing the induced mutation frequency at similar cellular
survival levels for Q3, MNNG, UV light, and x-rays, it was shown that 0- was a
very weak mutagen. Induced mutation frequencies were generally 20 to 200
times lower for 03 than for the other three mutagens.
In summary, the mutagenic properties of 0~ have been demonstrated in
procaryotic and eucaryotic cells. Only one study, however, (Hamelin and
Chung, 1975a, with Ju_ coll) investigated the mutagenic effect of 0-, at concen-
trations of less than 1 ppm. The results clearly indicate that if cells in
culture are exposed to sufficiently high concentrations of 0_ for sufficiently
long periods, mutations will result. The relevance of the presently described
investigations to human or even other mammalian mutagenicity is not apparent.
Additional studies with human and other mammalian cells will be required
before the mutagenic potency of 03 toward these species can be determined.
9.4.6 Qther Extrapulmonary' Effects
9.4.6.1 Liver. A series of studies reviewed by Graham et al. (1983a) have
shown that 0, increases drug-induced sleeping time in animals (Table 9-25).
The animal was injected with the drug (typically pentobarbital), and the time
to the loss of the righting reflex and the sleeping time (time between loss
and regaining of the righting reflex) were measured. Because the time to the
loss of the righting reflex was very rarely altered in the experiments described
below, it will not be discussed further. Animals awake from pentobarbital-
induced sleep, because liver xenobiotic metabolism transforms the drug into an
inactive form. Therefore, this response is interpreted as an extrapulmonary
effect.
Gardner et al. (1974) were the first to observe that 03 increases pentobar-
bital-induced sleeping time. Female CD-I mice were exposed for 3 hr/day for
3
up to 7 days to 1960 yg/m (1.0 ppm) of 03 and the increase was found on
days 2 and 3 of exposure, with the greatest response occurring on day 2. Com-
o
plete tolerance did not occur; when mice were pre-exposed (1960 (jg/m , 1.0 ppm;
3 hr/day for 7 days) and then challenged with a 3-hr exposure to 9800 pg/m
(5.0 ppm) on the eighth day, pentobarbital-induced sleeping time increased
greatly.
A series of follow-up studies was conducted to characterize the effect
further. To evaluate female mouse strain sensitivity, one outbred (CD-I) and
two inbred (C57BL/6N and DBA/2N) strains were compared (Graham et al., 1981).
9-190
-------
TABLE 9-25, EFFECTS OF OZONE ON THE LIVER
Ozone
concentration
ug/n3 ppm
196- 0.1-
9800 5
Measurement3' Exposure
method duration and protocol
CHEH 3 hr/day,
GPT 1-17/days
Observed effects(s)
Increase In pentobarbital- induced
sleeping time with following expo-
sure regimens; 0.1 ppm, IS or 16 days;
0,25 ppm, 6 or 7 days; 0.5 ppn,
2 or 3 days; 1 ppm, 1, 2, or 3 days;
S ppm, 1 day. No effects at days
before or after days given above.
Species Reference
House Graham et al . ,
(female) 1981
H
588
1470
5880
19,600
1600
(max)
1960
1960
0.3
0.75
3
10
0.82
(max)
1
1
UV
NBKI
UV
ND
CHEM
GPT
3 hr
3 hr
4 hr
(max)
3 hr/day
5 hr
No effect on liver reduced ascorbic
acid levels.
No effect on hepatic benzo(a)pyrene
hydroxylase activity.
Decrease in hepatic reduced ascorbic
acid content. Actual exposure regi-
mens not reported, only maximal
levels given.
Increase in pentobarbital -induced
sleeping time after 2 or 3 days,
but not other days (up to 7 days).
No tolerance to a challenge of 03
(9800 MS/1"3. 5 ppm x 3 hr) to mice
pre-exposed to 1 ppn for 7 days.
Increase in pentobarbital-induced
sleeping time in 3 strains of female
Rat
Hamster
Mouse
House
(female)
Mouse, rat,
hamster
Calabrese et al. ,
1983c
Palmer et al. ,
1971
Veninga et al. ,
1981
Gardner et al . ,
1974
Graham et al . ,
1981
mice (CD-I, C57BL, and D1A), female
rats, and male and female hamsters.
Male mice and rats not affected,
even when male mice received 3 days
(5 hr/day) of exposure. The increase
in male hamsters was less than the
increase in female hamsters..
(male and
female)
-------
TABLE 9-25, EFFECTS OF OZONE ON THE LIVER (continued)
Ozone
concentration
jjgTS8 ppi
1960 1
I960 1
Measurement3'
method
CHEH
GPT
CHEH
GPT
Exposure
duration and protocol
5 hr/day,
1,2,3 or
4 days
5 hr
Observed effects(s)
Increase in pentobarbital- induced
sleeping time at 1,2, and 3 days,
decreased with increasing days of
exposure. 24-hr postexposure for
each group, no effects occurred.
Increase in hexobarbital- and thiopen-
tal-induced sleeping tine and zoxazo-
Species
House
(female)
House
(female)
Reference
Graham et al . ,
1981
Graham et al . ,
1982a
&
to
lamine-induced paralysis time. Pre-
treatment with mixed function oxidase
inducers (phenobarbital, pregneolone-
I6a-carbon1trile, and p-naphthofla-
vone, but not pentobarbital} decreased
phenobarbital-induced sleeping time in
CD-I mice, and 03 increased the sleeping
tine in all groups. Pretreatntent with
inhibitors (SF525A, piperonyl butoxide)
reduced the sleeping time, but 03 increa-
sed the sleeping time, with the magnitude
of the increase becoming larger as the
dose of inhibitor was increased.
1960 1 CHEH 5 hr
9800 5 GPT 3 hr
No effect on hepatic cytochrome Mouse Graham et al . ,
p-450 concentration, aminopyrine (female) 1982b
N-demethylase, or p-nitroanisole
0-demethylase activities. Aniline
hydroxylase activity increased at
5 ppm (3 hr) and at 1 ppm (5 hr/day
x 2 days). No change in liver
to body weight ratios.
-------
TABLE 9-25. EFFECTS OF OZONE ON THE LIVER (continued)
UD
H
VO
U>
Ozone
concentration
ug/n3 ppm
1960 1
9800 5
1960 1
3920 2
Measurement*' Exposure
method duration and protocol Observed effects(s) Species
CHEH 5 hr At 1 ppm: 71% increase in plasma House
GPT half-life of pentobarbital , decrease (female)
(p = 0.06) in slope of clearance curve.
At 5 ppm: 106% increase in plasma
half-life of pentobarbital; decrease
in slope of clearance curve; no
effect on concentrations of pen-
tobarbital in brain at time of
awakening; no change in type of
pentobarbital metabolites in
serum/or brain.
ND 90 rain No effect on hepatic cytochrome Rabbit
P-450 concentration.
UV 8 hr/day Supplementing or depriving rats of Rat
vitamin E or selenium altered the
03 effect. 03 caused changes in
several |n vitro enzyme activities
in the iTver and kidney (see text).
Reference
Graham 1979;
Graham et al . ,
1983, 1985
Goldstein
et al., 1975
Reddy et al , ,
1983
aMeasureraent method: CHEM = gas-phase cheniluminescence; NBKI - neutral buffered potassium iodide; UV = UV photometry; ND = not described
Calibration nethod: GPT = gas phase titration
-------
3
Ozone (I960 jjg/m , 1.0 ppm for 5 hr) increased pentobarbital-induced sleeping
time in all strains. To determine whether this effect was sex- or species-
specific, male and female CD-I mice, rats, and hamsters were exposed for 5 hr
to 1960 |jg/m3 (1.0 ppm) of Og (Graham et al., 1981). The females of all
species exhibited an increased pentobarbital-induced sleeping time. Male mice
and rats were not affected. Male hamsters had an increase in sleeping time,
but this increase was less (p = 0.075) than the increase observed in the
females. Thus, the effect is not specific to strain of mouse or to three
»
species of animals, but it is sex-specific, with females being more susceptible.
Female CD-I mice were exposed to 196 to 9800 yg/m (0.1 to 5.0 ppm) of 0Q for
3
3 hr/day for a varying number of days (Graham et a!., 1981). At 1960 |jg/m
(1.0 ppm), effects were observed after 1, 2, or 3 days of exposure, with the
3
largest change occurring on day 2. At 980 (jg/m (0.5 ppm), the greatest
increase in pentobarbital-induced sleeping time was observed on day 3, but at
o
490 |jg/m (0.25 ppm), 6 days of exposure were required to cause an increase.
At the lowest concentration evaluated (196 |jg/m3, 0.1 ppm), the increase was
only observed at days 15 and 16 of exposure. Thus, as the concentration of Q~
was decreased, increasing numbers of daily 3-hr exposures were required to
significantly increase pentobarbital-induced sleeping time. Once the maximal
effect occurred, increasing the number of exposures resulted in a diminution
of the effect. Generally, effects were observed around an approximate C X T
(concentration, ppm x time, hr) value of 5. Also, CD-I female mice were ex-
2
posed to four different concentrations of 03 (1960 to 196 ug/m , 1.0 to 0.1 ppm)
for 5 to 20 hr continuously in a fashion yielding a C x T value of 5 (Graham
et al., 1981). All regimens increased pentobarbital-induced sleeping time.
3
The time to recovery was examined in mice exposed to 1960 ug/m (1.0 ppm) of
0,~ for 5 hr/day. Recovery was complete within 24 hr after exposure, whether
exposure was for 1, 2, or 3 days.
To determine whether the previous responses were specific to pentobar-
bital, other drugs with known and different mechanisms for termination of
3
action were used in CD-I female mice exposed to 1960 ug/m (1.0 ppm) of Og for
5 hr (Graham et al., 1982a). Ozone increased sleeping time induced by hexo-
barbital and thiopental and paralysis time induced by zoxazolamine. Within
the liver, pentobarbital and hexobarbital metabolism is more related to cyto-
chrome P-450 than to cytochrome P-448-dependent activities. Zoxazolamine
metabolism is more related to cytochrome P-448 than to cytochrome P-450.
9-194
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Other major differences in the nature of the metabolism (i.e., aliphatic vs.
aromatic) also exist between these three drugs. In contrast to the above,
sedation from thiopental is terminated because of drug redistribution. Thus,
it would appear that 03 might affect some aspects of both drug redistribution
and metabolism. Although there are different mechanisms involved in hexobarbital,
zoxazolamine, and pentobarbital metabolism, it is possible that some common
component(s) of metabolism may have been altered.
CD-I female mice were pretreated with mixed-function oxidase inducers and
inhibitors with partially characterized mechanisms of action to relate any
potential differences in the effect of 0, to differences in the actions of the
agents. Mice were exposed to 1960 ug/m (1.0 ppm) of 03 or air for 5 hr
before measurement of pentobarbital-induced sleeping time (Graham et al.,
1982a). Again, the effect of 0- was observed, but mechanisms were not eluci-
dated.
The effect of 03 on hepatic mixed-function oxidases in CD-I female mice
was evaluated in an attempt to relate to the sleeping time studies (Graham et
al. , 1982b). A 3-hr exposure to concentrations of 03 as high as 9800 [jg/m
(5.0 ppm) did not change the concentration of cytochrome P-450 or the activi-
ties of related enzymes (aminopyrine N-demethylase or p-nitroanisole 0-demethy-
o
lase). However, this exposure regimen and another (1960 [jg/m , 1.0 ppm, 5
hr/day for 2 days) increased slightly the activity of another mixed-function
oxidase, aniline hydroxylase. Goldstein et al. (1975) also found no effect of
a 90-min exposure to 1960 ug/m (1.0 ppm) of 0- on liver cytochrome P-450
levels in rabbits. Hepatic benzo(a)pyrene hydroxylase (another mixed-function
oxidase) activity of hamsters was unchanged by a 3-hr exposure to up to
19,600 ug/m3 (10 ppm) of QS (Palmer et al., 1971).
Pentobarbital pharmacokinetics in female CD-I mice were also examined. A
3-hr exposure to 9800 ug/m (5.0 ppm) of 03 did not affect brain concentra-
tions of pentobarbital at-time of awakening, even though sleeping time was
increased (Graham et al. , 1985). Therefore, it appears that 03 did not alter
the sensitivity of brain receptors to pentobarbital. A similar exposure
regimen also did not alter the pattern of brain or plasma metabolites of
pentobarbital at various times up to 90 min postexposure (Graham et al.,
1985). Following this exposure, first-order clearance kinetics of pentobarbital
were observed in both the air and 0, groups, and 0, increased the plasma
half-life by 106 percent (Graham et al. , 1985). Mice exposed to 1960 ug/m3
(1.0 ppm) of 0-, for 5 hr had a 71 percent increase in the plasma half-life of
9-195
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pentobarbital. This ozone exposure resulted in a decrease (p = 0.06) in the
slope of the clearance curve. Clearance followed first-order kinetics with a
one-compartment model in this experiment also.
In summary, the mechanism(s) for the effect of 03 on pentobarbital-induced
sleeping time are not known definitively. However, it is hypothesized (Graham
et al., 1983) that some common aspect(s) of, drug metabolism is quantitatively
reduced, whether it is direct (e.g., enzymatic) or indirect (e.g., liver blood
flow) or a combination of both. In addition, drug redistribution is apparently
slowed. It is unlikely that ozone itself caused these effects at target sites
distant from the lung (see Section 9.2). Because of the free-radical nature
of oxidation initiated by 0~, a myriad of oxygenated products, several of
which have toxic potential, may be formed in the lung (Section 9.3.3).
However, the stability of such products in the blood and their reaction with
organs such as the liver are speculative at present.
Reddy et al. (1983) studied the effects of a 7-day (8 hr/day) exposure of
rats to 3920 ug/m3 (2.0 ppm) of 03 on liver xenobiotic metabolism by perform-
ing jm vitro enzyme assays. Although lower 03 levels were not tested, this
study is presented because it indicates the potential of 0-, to cause hepatic
and kidney effects. The rats used were either supplemented or deficient in
both vitamin E and selenium. Ozone exposure caused a decrease in microsomal
cytoehrome P-450 hydroperoxidase activity in livers of rats deficient in both
substances, whereas an increase resulted in the supplemented animals. Rats
deficient in vitamin E and selenium experienced a decrease in liver microsomal
epoxide hydrolase activity after 03 exposure; no effect was observed in supple-
mented rats. Glutathione S-transferase activity was increased in the liver
and kidney in both the supplemented and deficient groups. Selenium-independent
glutathione peroxidase activity was not significantly affected in the livers
of the supplemented or deficient rats. However, 0~ decreased selenium-dependent
glutathione peroxidase activityin the-livers of supplemented rats and-caused
an increase in deficient rats. In the kidney,, both these groups of animals
had an increase in this enzyme activity. Other groups of rats (deficient in
vitamin E, supplemented with selenium; supplemented with vitamin E, deficient
in selenium) were examined also and in some cases, different results were
observed. The authors interpreted these results (along with pulmonary effects)
as a compensatory mechanism to protect cells from oxidants. A more extensive
interpretation of the effects depends on the nutritional status and the presence
9-196
-------
of other compounds metabolized by the affected enzymes. For example, epoxide
hydrolase, which was decreased in the vitamin E- and selenium-deficient rats,
metabolizes reactive epoxides to dihydrodiols. The metabolism of a substance
such as benzo(a)pyrene would be expected to be affected by such a change.
However, because of the complexity of the metabolism of a given chemical, such
as benzo(a)pyrene, a precise interpretation is not possible at this time.
Veninga et al. (1981) exposed mice to 03 and evaluated hepatic reduced
ascorbic acid content. The authors expressed the exposure regimen in the form
of a C x T value from about 0.2 to 3.2. Actual exposure regimes cannot be
3
determined. They stated that the maximal 03 level was 1600 ug/m (0.82 ppm)
and the maximal exposure time was 4 hr, which would have resulted in a C x T
of about 3.2. Animals were studied at 0, 30, and 120 min postexposure. It
appeared that immediately after exposure, a C x T value < 0.4 caused a decrease
in the reduced ascorbic acid content of the liver. At a C x T value of 0.4
and 0.8, there appeared to be an increase that was not observed at higher
values. For the 30-min postexposure groups, the increase in reduced ascorbic
acid shifted, with the greatest increase being at about a C x T value of 1.2
and no change occurring at a C x T value of 2.0. The 120-min postexposure
group was roughly similar to the immediate post-exposure group. No effects
occurred 24 hr postexposure.
Hepatic reduced ascorbic acid levels were also studied by Calabrese
3
et al. (1983c) in rats exposed for 3 hr to 588 ug/m (0.3 ppm) of 03 and
examined at 5 postexposure periods up to 24 hr. Rats had significantly increased
ascorbic acid levels in both the 03 and air .groups, with the greater change
taking place in the air group. Thus, there were no changes due to 03. Likewise,
there was no 0 effect on reduced ascorbic acid content in the serum.
9.4.6.2 The Endocrine System. A summary of the effects of 03 on the endo-
crine system, gastrointestinal tract, and urine is given in Table 9-26.
Fairchild and co-workers were the first to observe the involvement of the
3
endocrine system in 0,, toxicology. Miceiexposed to 11,368 ug/m (5.8 ppm) for
4 hr were protected against mortality by a-naphthylthiourea (ANTU) (Fairchild
et al., 1959). Because ANTU has antithyroidal activity and can alter adrenal
cortical function, Fairchild and Graham (1963) hypothesized a possible inter-
action of 03 with the pituitary-thyroid-adrenal axis. In exploring the hypo-
thesis, they exposed mice and rats for 3 to 4 hr to unspecified lethal con-
centrations of 03. Thyroid-blocking agents and thyroidectomy increased the
9-197
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TABLE 9-26. EFFECTS OF OZONE ON THE ENDOCRINE SYSTEH, GASTROINTESTINAL TRACT, AND URINE
Ozone
concentration Measurement
yg/i* ppi aethod
5.4
21
no
490
2940
980
1960
0.003 c
0.01
O.OSi
0.25 d
1.5
0.5 I
1
Exposure
duration and
protocpl
93 days,
continuous
2 hr
30 nin
5 hr/day,
4 days
Observed effect(s) Species
From 6th wk to end of exposure, 0.056 pp« Rat
increased the urine concentration
of 17-ketosteroids. After 93 days of
exposure to 0.056 ppn, the ascorbic and
level of the adrenal glands was decreased.
No data were presented for these effects.
1.5 pp« of 03 (30 Bin) inhibited gastric Rat
mortality; recovery was rapid. The
lower level caused no effects.
No effects on thyroid release of 131I, Rat
96-384 hr post 131I injection.
Reference
Eglite, 1968
Roth and Tansy, 1972
Fairchild et al., 1964
1470
H
vo
CO
0.7S
m
4-8 hr
LM and TEM changes in parathyroid glands.
Loss of "clusterlike" arrangement of
parenchyma. Dilated capillaries.
Vacuolated chief cells. Increased RER,
prominent Golgi, abundant secretory
granules.
Rabbit
Atwal and Wilson, 1974
1470 0.75 NO
1470 0. 75 ND
1470 0.75 ND
1568- 0.8- NBKI
2940 1.5
(range) (range)
48 hr
postexposure
1-20 days
48 hr
postexposure
12-18 days
48 hr,
3, 10-13, 18
days post-
exposure
6 hr/day,
4 days/wk
about 19 wk
Early postexposure parathyroid glands * Rabbit
enlarged and congested with focal
vasculitis. After 7 days postexposure,
parenchyma! atrophy, leukocyte infiltra-
tion and capillary proliferation. Authors
suggest lesions may be due to autoimmune
reactions.
Mi crovascular changes in the parathyroid Dog
f glands, including hemorrhage, endothelial
"proliferation, platelet aggregation, and
lynphocyte infiltration.
Ciliated cysts found in parathyroid gland. Dog
Hallory body-like inclusions found in chief
cells of parathyroid with highest incidence
being 10-13 days postexposure.
Lower titratable acidity of urine, Rat
with no changes in levels of creatine,
uric acid/creatinine, ami no acid
nitrogen/creatinine, or excretion of
12 ami no acids.
Atwal et al., 1975
Atwal and Pens ingh, 1981
Perns ingh and Atwal , 1983
Atwal and Pensingh, 1984
Hathaway and Terrill, 1962
-------
TABLE 9-26. EFFECTS OF 010NE ON THE ENDOCRINE SYSTEM, GASTROINTESTINAL TRACT, AND URINE (continued)
Ozone
concentration
Hg/na
1960
3920
7840
1960
ppm
1
2
4
1
Measurement8
method
I
ND
Exposure
duration and
protocol
5 hr •
24 hr
:• Observed effect(s)b
Decreased release of 131I from thyroid,
48-384 hr post mlnjection to all 03
levels above 1 ppm. -
Decreased serum level of thyroid-
stimulating hormone from anterior
Species
Rat
Rat
Reference
Fairchild et al. ,
demons and
Garcia, 1980a,b.
1964
f
pituitary, thyroid hormones (Ta, T4,
and free T4), and protein-bound iodine;
no change in unsaturated binding capacity
of thyroid-binding globulin in serum; in-
crease in prolactin levels; no change in
levels of corticotropin, growth hormone,
luteinizing hormone, follicle stimulating
hormone from pituitary or insulin. Thy-
roidectomy prevented the effect on TSH
levels. There was no effect 'on the
circulating half-life of 131I-TSH, The
anterior pituitaries had fewer cells, but
more TSH/cell. The thyroid gland was also
altered. Exposures to between 0,2 to 2 ppm
for unspecified lengths of tine up to a
potential maximum of 500 hr also caused
a decrease in TSH levels.
1960 1
ND
24 hr
9800
CHEM
3 hr
Decreased serum levels of T3, T4, and TSH.
In thyroidectomized and" hypophy sectoral'zed
rats, the decrease in T4 was greater when
rats were supplemented with T4 in the
drinking water.
Rat
demons and Wei, 1984
79,800 > 5.0 I
> 3 hr
< 8 hr
Anti-thyroid agents, thyroidectony,
hypophysectomy, and adrenal ectomy
protected against 03- induced mor-
tality. Injection of thyroid hormones
decreased survival after Q3 exposure.
Mouse,
rat
Fairchild et al. ,
1959; Fairchild and
Graham, 1963;
Fairchild, 1963.
Increased levels of 5-hydroxytryptamine
in lung; decreases in brain; no change
in kidney.
Rat
Suzuki, 1976.
Measurement method: CHEM = gas phase chemiluminescence; NBKI = neutral buffered potassium iodide; I = iodometric
(Byers and Saltzman, 1959); ND = not described.
Abbreviations used: LM = light microscopy; TEM = transmission electron microscopy; RER = rough endoplasmic reticulum;
T3 = triiodothyronine; T4 = thyroxine.
Spectrophotometric technique (dihydroacridine).
Flow rates from ozonator.
-------
survival of mice and rats acutely exposed to 0~, and injections of the thyroid
hormones, thyroxine (T*), or triiodothyronine (I,) decreased their survival.
This response in animals with altered thyroid function was not specific to a
concomitant altered metabolic rate, because another drug (dinitrophenol),
which increases metabolism, had no effect on the CL response.
Hypophysectomy and adrenalectomy also protected against Ov-induced mor-
tality, presumably in rodents (Fairchild, 1963). Hypophysectomy would prevent
the release of thyroid-stimulating hormone, thereby causing a hypothyroid con-
dition as well as preventing the release of adrenocorticotropic hormone (ACTH),
which would cause a decreased stimulation of the adrenal cortex to release
hormones. This confirms the above-mentioned finding of thyroid involvement in
0, toxicity and suggests that a decrease in adrenocorticosteroids reduces 0.,
toxicity. Rats that have been adrenalectomized and treated with adrenergic
blocking agents are more resistant to 03 than rats that have only been adrena-
lectomized, indicating that decreases in catecholamines reduce CL toxicity.
Potential tolerance to the effect of 03 on thyroid activity was also
investigated by Fairchild et al. (1964). -A variety of exposure regimens were
used for the rats, and the release of I was used as an index of thyroid
•3
function. A 5-hr exposure to 1960, 3920, or 7840 pg/m (1, 2, or 4 ppm) of 0~
131 131
inhibited the release of I at several time periods post injection of I
(48, 96, 192, and 384 hr). The rats were injected before the 5-hr exposure,
presumably shortly before exposure. Twenty-four hr post-injection, only the
highest 0, concentration showed an effect. Rats were also exposed for 5
hr/day for 4 days to either 980 or 1960 ug/m3 (0.5 or 1.0 ppm) of Ov At 96,
131
192, and 384 hr post-injection of I, no effects were observed. Thus,
tolerance appeared to have occurred, a finding consistent with lethality
studies with 0- (Matzen, 1957a). In another study, rats were exposed to 3920
3
(jg/m (2.0 ppm) of 0- 5 hr/day for 2 days and challenged with a 5-hr exposure
3
to 7840 yg/m (4.0 ppm) on the third day. These animals exhibited a greater
effect than rats that received only the 7840-ug/m (4.0 ppm) challenge. This
difference persisted for 48, 96, and 192 hr post-injection. Thus, although it
appears that tolerance occurs, it results in a condition that leads to stimula-
tion of thyroid activity after a subsequent exposure to an 03 challenge.
demons and Garcia (1980a,b) extended this area of research by investi-
gating the effects of 0_ on the hypothalamo-pituitary-thyroid axis of rats.
Generally, these three endocrine organs regulate the function of each other
9-200
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through complex feedback mechanisms. Either stimulation or inhibition of the
hypothalamus regulates the release of thyrotropin-releasing hormone (TRH).
The thyroid hormones (1~3 and T.) can stimulate TRH, Thyrotropin-releasing
hormone and circulating thyroid hormones (T.3 and T.) regulate secretion of
thyroid-stimulating hormone (TSH) from the anterior pituitary. Stimulation of
3
the thyroid by TSH releases T~ and T,. A 24-hr exposure to 1960 ug/m (1.0
ppm) of CL caused decreases in the serum concentrations of TSH, T3, T», free
T., and protein-bound iodine. There was no change in the uptake of T3, and
thus no change in the unsaturated binding capacity of thyroid-binding globulin
in the serum. Prolactin levels were increased also, but no alterations were
observed in the concentrations of other hormones (corticotropin, growth hormone,
luteinizing hormone, follicle-stimulating hormone, and insulin). Plasma TSH
3
was also evaluated after continuous exposures to between 392 and 3920 ug/m
(0.2 and 2.0 ppm) for unspecified lengths of time in a fashion to result in a
concentration x time relationship between about 2 and 100. Plasma TSH was
decreased after a C x T exposure of about 6. These data cannot be independently
interpreted, because the specific exposure regimens were not given. The
authors state, without any supporting data, that the decrease in TSH levels
persisted "beyond two weeks" when exposure was continued. Therefore, it
appears that tolerance may not have occurred. Thyroidectomized rats exposed
to 1960, 3920, 5880 or 7840 ug/m3 (1.0, 2.0, 3.0, or 4.0 ppm) of DS for 24 hr
did not exhibit a decrease in the levels of TSH. Exposure to (presumably)
3
1960 ug/m (1.0 ppm) for 24 hr did not alter the circulating half-life of
125
I-labeled TSH injected into the rats, and therefore, there apparently is no
effect on TSH once it is released from the pituitary.
3
To evaluate pituitary function further, rats exposed 24 hr to 1960 ug/m
(1.0 ppm) of ozone were immediately subjected to a 45-min exposure to the cold
(5°C) (demons and Garcia, 1980a,b). The anterior pituitary released an
increased level^pf TSH, indicating., that the hypothalamus was still able to
respond (via increased TRH) after 0, exposure. The increase was greater in
the 0- group, which might indicate increased production of TRH or increased
sensitivity to TRH. In addition, the anterior pituitaries of the 0, group had
fewer cells than the air group. The cells from the 0,,-exposed rats had more
TSH and prolactin per 1000 cells, irrespective of whether the cells had received
a TRH treatment. The cells from the 0- group also released a greater amount
of TSH, but not prolactin, into the tissue culture medium.
9-201
-------
The thyroid gland itself was altered by the 00 exposure (apparently 1960
3
, 1.0 ppm, for 24 hr). Ozone increased thyroid weight without changing
protein content (e.g., edema) and decreased the release of T. per milligram of
tissue. There was no change in T. release per gland, demons and Garcia
(1980a,b) interpreted these findings as an 0~-induced lowering of the hypo-
thalamic set point for the pituitary-thyroid axis and a simultaneous reduction
of prolactin-inhibiting-factor activity in the hypothalamus,
3
The effect of a 24-hr exposure to 1960 ug/m (1 ppm) 03 on exogenous T4
levels was examined in rats by demons and Wei (1984) to increase understanding
of their previous studies. Normal, thyroidectomized, and hypophysectomized
rats were exposed to Qg and serum T. levels were measured. Ozone reduced
serum T^ levels in normal rats, but not the other groups of rats which had
reduced levels of 1. prior to exposure. However, when the thyroidectomized
and hypophysectomized rats received supplemental T. in their drinking water
that increased their serum T. level, 03 caused a decrease in serum T*.
Generally, the higher the pre-exposure T* levels, the greater the 03~induced
reduction in 1, levels. Similar observations were made for thyroidectomized
rats when serum T3 levels were measured. Exposure to CU also decreased plasma
TSH levels in normal rats and in thyroidectomized rats supplemented with T* in
the drinking water. These observations led the authors to hypothesize that
the results are not due to a reduction in the hypothalamus-pituitary-thyroid
axis as previously suggested (demons and Garcia, 1980a,b), but that 03 causes
decreases in serum T,, T*, and TSH by peripheral changess possibly changes in
serum binding.
The susceptibility of the parathyroid gland to 0~ exposure was inves-
tigated by Atwal and co-workers. In the initial study (Atwal and Wilson,
3
1974), rabbits were exposed to 1470 ug/m (0.75 ppm) of 03 for 4 to 8 hr, and
the parathyroid gland was examined with light and electron microscopy at 6,
18, 22, and 66 hr after exposure. The parathyroid gland exhibited increased
activity after 03 exposure. Changes included hyperplasia of chief cells;
hypertrophy and proliferation of the rough endoplasmic reticulum, free ribo-
somes, mitochondria, Gqlgi complex, and lipid bodies; and an increase of
secretion granules within the vascular endothelium and capillary lumen. Such
changes suggested an increased synthesis and release of parathormone, but
actual hormone levels were not measured.
9-202
-------
Atwal et al. (1975) also investigated possible autoimmune involvement in
3
parathyroiditis of rabbits following a 48-hr exposure to 1470 (jg/m (0.75 ppm)
of Oq- The authors stated that there was both a "continuous" and an "inter-
mittent" 48-hr exposure, without specifying which results were due to which
exposure regimen. Animals were examined between 1 and 20 days postexposure.
Hyperplastic parathyroiditis was observed to be followed by capillary proli-
feration and leukocytic infiltration. Cytologic changes included the presence
of eosinophilic leukocytes, reticuloendothelial and lymphocytic infiltration,
disaggregation of the parenchyma, and interstitial edema. A variety of alter-
ations were observed by electron microscopy, including atrophy of the endo-
plasmic reticulum of the chief cells, atrophy of mitochondria, degeneration of
nuclei, and proliferation of the venous limb of the capillary bed. The alter-
ations to the parathyroid gland were progressive during postexposure periods.
Parathyroid-specific autoantibodies were detected in the serum of 0.,-exposed
rabbits, suggesting that the parathyroiditis might be due to inflammatory
injury with an autoimmune causation. Microvascular changes in the parathyroid
were further studied by Atwal and Pemsingh (1981) in dogs exposed to 1470 ug/m3
(0.75 ppm) of 03 for 48 hr. They reported focal hemorrhages, vascular endothe-
lial proliferation, intravascular platelet aggregation, and lymphocytic infiltra-
tion. A potential autoimmunity after 0, exposure was also observed by Scheel
et al. (1959), who showed the presence of circulating antibodies against lung
tissue.
In another study, Pemsingh and Atwal (1983) studied cells (APUD-type)
within ciliated cysts of the parathyroid gland of dogs exposed for 48 hr to
3
1470 ug/m (0.75 ppm) 0~. Examination was made at several times post-exposure
(3, 10-13, and 28 days). Ozone caused ciliated cysts in 5 out of 12 exposed
dogs and 1 out of 4 control, dogs. Cysts in the 03-exposed dogs had different
cellular content, namely the presence of APUD-type cells on the cyst wall.
The interpretation of these findings is not- clear. The ultrastructure .of
chief cells, which are the hormone-producing cells, was also examined in dogs
exposed identically to those described above (Atwal and Pemsingh, 1984).
Mallory body-like inclusions (i.e., accumulations of filaments in the perinuclear
area) were found within the chief cells of the parathyroid of 0.,-exposed dogs.
Additional alterations were observed. The highest incidence of this change
was at 10 to 13 days post-exposure.
9-203
-------
Also classed as a hormone is 5-hydroxytryptamine (5-HT), and it too
interacts with 0- toxicity (Suzuki, 1976). It has a variety of activities,
including bronchoconstriction and increased capillary permeability; it can be
a neurotransmitter. Although rats were exposed to a high concentration (9800
3
|jg/m , 5,0 ppm) of 0_ for 3 hr, this study is discussed because extrapulmonary
effects were observed. This exposure caused an increase in the 5-HT content
of the lung and spleen, a decrease in 5-HT in the brain, and no change in the
levels of 5-HT in the liver or kidney.
Adrenal cortex function after a 93-day (continuous) exposure to 0, was
3
investigated in rats (Egl.ite, 1968). Exposure to 110 ug/m (0.056 ppm), but
not lower levels, increased the urine concentration of 17-ketosteroids from
the 6th week of exposure to the end of exposure. There was also a decrease in
ascorbic acid in the adrenals. The generation and monitoring methods for the
0, exposures were not sufficiently described. The authors described the
effects as statistically significant but did not specify the statistical
methods. No data were provided. Therefore, these results need to be con-
firmed before accurate interpretation is possible.
9.4.6.3 Other Effects. Rats were exposed for 6 hr/day, 4 days/week, for
about 19 weeks, and analyses were performed on urine collected for the 16 hr
following the exposure week (Hathaway and Terrill, 1962). Ozone exposures
were uncontrolled, ranging from 1568 to 2940 (0.8 to 1.5 ppm). All parameters
were not measured for each week of'exposure. On days 91 and 112 after initial
exposure, there was a lower titratable acidity and higher pH in the urine of
Qg-exposed animals. Titratable acidity was also lower on day 98. Ozone did
not alter the levels of creatinine, creatine, uric acid/ creatinine, ami no
acid nitrogen/creatinine excretions, or excretion of 12 ami no acids. The
lungs and kidneys were examined histologically, and no consistent differences
were observed. The authors interpreted the results as a reflection of respira-
tory alkalosis, assuming no kidney• toxic-i-ty.
Gastric secreto-motor activities of the rat were investigated by Roth and
3
Tansy (1972). A 2-hr exposure to 490 ug/m (0.25 ppm) caused no effects.
•a
Thirty minutes of exposure to 2940 ug/m (1.5 ppm)'inhibited gastric motility,
but activity tended to return towards normal for the remaining 90 min of
exposure. Recovery had occurred by 20 min postexposure. However, these
results are questionable because ozone was monitored only by ozonator flow
rates.
9-204
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9.5 EFFECTS OF OTHER PHOTOCHEMICAL OXIDANTS
9.5.1 Pe rpxyacety1 N i1rate
Very little information on the toxicity of peroxyacetyl nitrate (PAN) has
appeared in the literature since the previous criteria document on photochemi-
cal oxidants (U.S. Environmental Protection Agency, 1978). The document
reviewed the results of inhalation experiments with mice that tested PAN's
lethal concentration (LCgp) (Campbell et al. , 1967); its effects on lung
structure (Dungworth et al. , 1969); and its influence on susceptibility to
pulmonary bacterial infections (Thomas et al., 1979). The concentration of
3 3
PAN used in these studies ranged from 22.3 mg/m (4.5 ppm) to 750 mg/m (150
3
ppm). They are considerably higher than the 0.232 mg/m (0.047 ppm) daily
maximum concentration of PAN reported in recent years for ambient air samples
in areas having relatively high oxidant levels (Chapter 5) and are of question-
able relevance to the assessment of effects on human health.
Campbell et al. (1967) estimated that the LC5Q for mice ranged from 500
to 750 mg/m3 (100 to 150 ppm) for a 2-hr exposure to PAN at 80°F (25°C). Mice
in the 60- to 70-day-old age group were more susceptible to PAN lethality than
mice ranging from 98 to 115 days in age. Temperature also influenced the
lethal toxicity of PAN; a higher LC5Q (less susceptibility) was seen at 70°F
(125 ppm) than at 90°F (85 ppm). In a follow-up study, Campbell et al. (1970)
characterized the behavioral effects of PAN by determining the depression of
voluntary wheel-running activity in mice.1 Exposures to 13.9, 18.3, 27.2,
31.7, and 42.5 mg/m3 (2.8, 3.7, 5.5, 6.4, and 8.6 ppm) of PAN for 6 hr de-
pressed both the 6-hr and 24-hr activity when compared to similar pre-exposure
periods. Depression was more complete and occurred more rapidly with higher
exposure levels. However, the authors indicated that PAN was less toxic than
0,, when compared to similar behavioral data reported by Murphy et al. (1964)
(Section 9.4.1).
3
Dungworth et al, (1969) reported that daily exposures of mice to 75 mg/m
(15 ppm) of PAN 6 hr/day for 130 days caused a 30-percent weight loss compared
to sham controls, 18 percent mortality, and pulmonary lesions. The most
prevalent lesions were chronic hyperplastic bronchitis and proliferative
peribronchiolitis.
Thomas et al. (1979) found that mice exposed to 22.3 mg/m3 of PAN (4.5
ppm) for 2 hr and subsequently challenged with a Streptococcus sp. aerosol for
1 hr showed a significant increase in mortality and a reduction in mean survival
9-205
-------
rate, compared to mice exposed to air. No effect on the incidence of fatal
pulmonary infection or survival time was observed in mice challenged with
•3
Streptococcus sp. 1 hr before the pollutant exposure (27.2 mg/m of PAN for '3
hr), Thomas at al. (1981a) published additional data that extend observations
of reduced resistance of mice to streptococcal pneumonia over a range of
o
exposures to PAN. A single 2- or 3-hr exposure to PAN at 14.8 to 28.4 mg/m
(3.0 to 5.7 ppm) caused a significant increase in the susceptibility of mice
to streptococcal pneumonia. The mean excess mortality rate ranged from 8 to
3
39 percent. Mice exposed to 0~ at 0.98 mg/m (0.5 ppm) and challenged with
the Streptoccus sp. aerosol resulted in a mean excess mortality (38 percent) that
was almost equivalent to the excess mortality for the group'exposed to 28.4
o
mg/m of PAN. The results agreed with earlier reports that PAN is less toxic
2
than Og to mice exposed under ambient conditions. Exposure to 7.4 mg/m (1.5
ppm) of PAN 3 hr/day, 5 days/week for 2 weeks had no appreciable effect,
although no statistics were provided. Neither exposure routine altered the
morphology, viability, or phagocytic activity of isolated macrophages, although
there was a decrease in ATP levels. The other noticeable effect was that
macrophages isolated from the animals that were repeatedly exposed failed to
attach themselves to a glass substrate. These investigators also studied
whether a chronic infection initiated with an exposure to Mycobacteriurn tubercu-
losis (RIRv) was influenced by subsequent exposure to PAN. The exposure to
this oxidant (25 mg/m for i
growth in the lungs of mice.
3
this oxidant (25 mg/m for 6 days) did not alter the pattern of bacterial
9.5.2 Hydrogen Peroxide
Hydrogen peroxide (H«0?) has been reported to occur in-trace amounts in
urban air samples (Chapter 5), but very little is known about the effects of
HpOn from inhalation exposure. Most of the early work on H202 toxicity in-
volved exposure to very high concentrations. Oberst et al. Q954) investigated
the inhalation toxicity of 90 percent H909, vapor in rats, dogs, and rabbits at
3
concentrations ranging from 10 mg/m (7 ppm) daily for six months to an 8-hr
o
exposure to 338 mg/m (243 ppm). After autopsy, all animals showed abnormali-
ties of the lung. In a recent experiment, Last et al. (1982) exposed rats for
3
7 days to > 95-percent H^O- gas with a concentration of 0.71 mg/m (0.5 ppm)
in the presence of respirable ammonium sulfate particles. No significant
effects were observed in body weight, lung lobe weights, and protein or DNA
9-206
-------
content of lung homogenates. The authors suggested that because HpQp is highly
soluble, it is not expected to penetrate to the deep lung, which may account
for the absence of observed effects.
The majority of studies on H?0p explore possible mechanisms for the
effects of HpOp. These include direct cellular effects associated with i_n
vitro exposure to H000 and biochemical reactions to H000 generated in vivo.
::::::::::::::::: £ £» £- £ —
Hydrogen peroxide may affect lung function by the alteration of pulmonary
surfactant. Wilkins and Fettissoff (1981) found that lo"2 to lO'^N of H202
increased the surface tension of saline extracts of dog lung homogenates. The
3
authors estimated that a dog breathing 1.4 mg/m (1 ppm) of H9Q~ for 30 hr
-2
would build up a pollutant concentration of 10 M in the surfactant, assuming
that all the HpOp was retained by the lungs. However, as stated above, this
estimate of tissue dose is not realistic, because most of the HpOp would be
absorbed in the upper airway.
Another mechanism by which H909 may affect ventilation is by changing the
^ *• -A
tone of airway smooth muscle. Stewart et al. (1981) reported that 10 M of
HpQp caused significant constriction of strips of subpleural canine lung
parenchyma and of bovine trachealis muscle. In the distal airway preparation
(canine), this contraction was reversed by catalase. Pretreatment of both
proximal (bovine) and distal muscle strips with meclofenamate or iodomethacin
markedly reduced the response to HpOp. This suggested that the increase in
airway smooth muscle tone produced by HpOp involves prostaglandin-like sub-
stances.
The potential genotoxic effects from in vitro HpOp exposure have been
evaluated in isolated cell systems. Bradley et al. (1979) reported that HpOp
produced both toxicity and single-strand DMA breaks but was not mutagenic at
concentrations up to 530 pM. They observed a significant increase in the
frequency of reciprocal sister chromatid exchanges in V-79 Chinese hamster
cells at a concentration of 353 |jM of HpQp. However, the authors pointed out
that sister chromatid exchange frequency was not necessarily equivalent to
increased mutant frequency. In subsequent experiments, Bradley and Erickson
(1981) confirmed these observations in V-79 Chinese hamster lung cells and
were unable to detect any DNA-protein or DNA-DNA crosslinks with 353 uM of
HpOp for 3 hr at 37°C. Increases in the frequency of sister chromatid exchanges
in Chinese hamster cells have also been found by MacRae and Stich (1979),
Spelt and Vogel (1982), and Speit et al. (1982). Wilmer and Natarajan (1981)
9-207
-------
reported only a slight enhancement in the frequency of sister chromatid ex-
changes in Chinese hamster ovary cells following treatment with up to 10 M of
HgOg. In comparison, cells were killed with a concentration of 10 M of H2Q2.
Similarly, HpQp (10 mg/ml) was negative in the Ames mutagenicity assay
(Ishidate and Yoshikawa, 1980), and several other investigators have confirmed
the lack of mutagenicity for H202 (Stich et a!,, 1978; Kawachi et a!., 1980).
Johnson et al. (1981) reported that the intrapulmonary instillation of
glucose oxidase, a generator of HpO,,, increases lung permeability in rats. A
greater increase in lung permeability was achieved by the addition of a com-
bination of glucose oxidase and lactoperoxidase than by the glucose oxidase-
HoCU-generating system alone. Horseradish peroxidase did not effectively
substitute for lactoperoxidase in the potentiation of damage. Injury was
blocked by catalase but not by superoxide dismutase (SOD), suggesting that
HjjQ, or its metabolites, rather than superoxide, were involved. Because
horseradish peroxidase did not potentiate the glucose oxidase damage, the
authors speculated that the mechanism of injury occurs through the action of a
hall de-dependent pathway described for cell injury produced by H202 and lac-
toperoxidase/myeloperoxidase (MPO) (Klebanoff and Clark, 1975). Any source of
H^O* plus MPO and a halide cofactor is capable of catalyzing many oxidation
and halogenation reactions (Clark and Klebanoff, 1975), but other possible
mechanisms of oxygen radical production have been proposed (Halliwell, 1982).
Carp and Janoff (1980) have shown that a H202 generating system with MPO
and Cl will suppress the elastase inhibitory capacity of the protease inhibi-
tor (BMPL) present in bronchial mucus. This antiprotease is capable of inhibit-
ing the potentially dangerous proteases found in human polymorphonuclear
leukocytes (PMNs), including elastase and cathepsin-G. Inactivation of BMPL
could make the respiratory mucosa more susceptible to attack by inflammatory
cell proteases. Human PMN have not been shown to contain MPO but macrophages
may contain analogous forms of peroxidase.
In other i_n vitro experiments, Suttorp and Simon (1982) demonstrated that
H202 generated by glucose oxidase was cytotoxic to cultured lung epithelial
cells (L9 cells) in a concentration-dependent fashion. Cytotoxicity was
51
measured by determining Cr release from target cells. Cytotoxicity was
prevented by the addition of catalase. It was stressed that there is no
established identity between the L? cell line and the jji situ type 2 pneumo-
cytes from which they were derived.
9-208
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9.5.3 Formic Acid
lexicological interest in formic acid has focused primarily on its role
as the metabolic end product of methanol and on any similarity the effects
from inhalation exposure to formic acid vapor may have with methanol toxicity.
Unfortunately, the concentrations (20 ppm) used in the two studies discussed
below are over three orders of magnitude greater than the trace concentrations
(<0,015 ppm) reported in the highest oxidant areas of Southern California (see
Chapter 5).
Zitting and Savolainen (1980) exposed rats to 20 ppm (0.8 |jmol/L) formic
acid vapor, 6 hr/day for 3 and 8 days. Tissue samples were analyzed for
neurochemical effects as well as effects on drug-metabolizing enzymes in the
liver and kidneys. Enzyme profiles were variable, depending on the sampling
period during exposure. However, a general pattern developed. Cerebral
glutathione concentration and acid proteinase activity increased after 3 and
8 days of exposure, respectively, indicating possible lipid peroxidation
associated with cerebral hypoxia. Decreased kidney ethoxycoumarin deethylase
activity, cytochrome P-450 content, and glutathione concentrations were also
consistent with changes due to lipid peroxidation. The liver, which is less
sensitive to tissue hypoxia, showed only small increases in deethylase activity
although associated with a reduction in glutathione. Since prolonged low-level
cerebral hypoxia can potentially lead to demyelination and subsequent nerve
degeneration, the authors repeated the study to look specifically at effects
on glial cells (Savolainen and Zitting, 1980). Rats were exposed to 20 ppm
formic acid vapor, 6 hr/day, 5 days/week for 2 or 3 weeks. Again, enzyme
profiles were indicative of metabolic responses to tissue hypoxia, providing
evidence for potential central nervous system toxicity at high formic acid
vapor concentrations.
9.5.4 Complex Pollutant Mixtures • ' ' •
Additional toxicological studies have been conducted on the potential
action of complex mixtures of oxidants and other pollutants. Animals have
been exposed under laboratory conditions to ambient air from high oxidant
areas, to UV-irradiated and nonirradiated reaction mixtures of automobile
exhaust and air, and to other combinations of interactive pollutants. Although
these mixtures attempt to simulate the photochemical reactions produced under
actual atmospheric conditions, they are extremely difficult to analyze because
9-209
-------
of their chemical complexity. Variable concentrations of total oxidants,
carbon monoxide, hydrocarbons, nitrogen oxides, sulfur oxides, and other
unidentified complex pollutants have been reported. For this reason, the
studies presented in this section differ from the more simplified combinations
of 0~ and one or two other nonreactive pollutants discussed under previous
sections of the chapter. The effects described in animals exposed to UV-
irradiated exhaust mixtures are not necessarily uniquely characteristic of CU,
but most of them could have been produced by 0,,. In most cases, however, the
biological effects presented would be difficult to associate with any one
pollutant.
Research on ambient air and UV-irradiated or nonirradiated exhaust mix-
tures is summarized in Table 9-27. Long-term exposure of various species of
animals to ambient California atmospheres have produced changes in the pul-
monary function of guinea pigs (Swann and Balchum, 1966; Wayne and Chambers,
1968) and have produced a number of biochemical, pathological, and behavioral
effects in mice, rats, and rabbits (Wayne and Chambers, 1968; Emik and Plata,
1969; Emik et al., 1971). Exposure to UV-irradiated automobile exhaust con-
taining oxidant levels of 0.2,to 1.0 ppm produced histopathologic changes
(Nakajima et al., 1972) and increased susceptibility to infection (Hueter et
al., 1966) in mice. Both UV-irradiated and nonirradiated mixtures produced
decreased spontaneous running activity (Hueter et al., 1966; Boche and Quilligan,
1960) and decreased infant survival rate and fertility (Kotin and Thomas,
1957; Hueter et al., 1966; Lewis, et al., 1967) in a number of experimental
animals. Pulmonary changes were demonstrated in guinea pigs after short-term
exposure to irradiated automobile exhaust (Murphy et al., 1963; Murphy, 1964)
and in dogs after long-term exposure to both irradiated and nonirradiated
automobile exhaust (Lewis et al., 1974; Orthoefer et al., 1976). Irradiation
of the air-exhaust mixtures led to the formation of photochemical reaction
products that were biologically more active than nonirradiated mixtures. The
concentration of total oxidant as expressed by CU ranged from 588 to 1568
3
|jg/m (0.30 to 0.80 ppm) in the irradiated exhaust mixtures, compared to only
a trace or no oxidant detected in the nonirradiated mixtures.
The description of effects following exposure of dogs for 68 months to
automobile exhaust, simulated smog, oxides of nitrogen, oxides of sulfur, and
their combination has been expanded in a monograph by Stara et al. (1980).
The dogs were examined after 18 months (Vaughan et al., 1969), 36 months
9-210
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TABLE 9-27. EFFECTS OF COMPLEX POLLUTANT MIXTURES
ro
H
Concentration,
(ppra)
Pollutant
Exposure
duration and
protocol
Observed effect(s)c
Species
Reference
A. Ambient air
0.032 - 0.050
9.1 - 13.5
0.044 - 0.077
0.019 - 0.144
0.057
1.7
2.4
0.019
0.015
0.004
0.062 - 0.239
0.03 - 0.07
2.7 - 4.4
0.27 - 0.31
13 - 38
2-9
0.09 - 0.70
0.4 (max)
°x
cfi
N02
NO
0,,
c8
HC
N02
NO
PAN
ox
NU2
HC
°x
c8
HC
NOX
°x
Lifetime
study,
continuous
2.5 years,
continuous
13 months,
continuous
1 year,
continuous
19 weeks,
continuous
No clear chronic effects; "suggestive"
changes in pulmonary function, morphology,
and incidence of pulmonary adenomas in
aged animals. Increased 17-ketosteroid
excretion in guinea pigs. Decreased
glutamic oxalacetic transaminase in blood
serum of rabbits.
Reduced pulmonary alkaline phosphatase
(rats); reduced serum glutamic oxaloacetic
transaminase (rabbits); increased pneu-
monitis (mice); increased mortality (male
mice); reduced body weights (mice); de-
creased running activity (male mice); no
significant induction of lung adenomas
(mice).
Decreased spontaneous running activity.
Expiratory flow resistance increased on
days when 0 reached > 0.30 ppn or at com-
bined concentrations of > 40 ppm of CO,
16 ppm of HC, and 1.2 ppm of NO . Indi-
dividual sensitivity demonstrated. Tem-
perature was an important variable.
No consistent effects on conception rate,
litter rate, or newborn survival.
Mouse,
rat,
hamster,
guinea pig,
rabbit
Mouse,
rat,
rabbit
Mouse
Guinea pig
Mouse
Wayne and Chambers,
1968
Emik et a!., 1971
Enrik and Plata, 1969.
Swann and Balchum, 1966
Kotin and Thomas, 1957
-------
TABLE 9-27, EFFECTS OF COMPLEX POLLUTANT MIXTURES (continued)
V
NJ
Concentration,3 .
(pom) Pollutant0
Exposure
duration and
protocol
Observed effect(s)c
' Species
Reference
B. Automobile exhaust
0.012-
3.0
0.04 -
0.06 -
0.06 -
0.04 -
0.1S -
0.4 -
6
20 -
0.1 -
0.2 -
5
40 -
0.2 -
100
24 -
0.1 -
0.1 -
0.42 -
0.02 -
0.65
1.10
5.00
1.20
0.2
0.5
l.B
36
100
0.5
0.6
8
60
0.4
30
1.0
2.0
0.49
0.03
03 (max)
HC (propylene)
N02
NO
S02
03
N02
NO
HC (CH4)
CO
N0*
HC*
CO
Os
CO
HC (CH4)
N02
NO
S02
H2S04
0.5-6 hr
(diesel)
1.5-23 10
4 weeks,
5 days/week,
2-3 hr/day
18-68 months,
7 days/week,
16 hr/day
2-3 years
recovery In
ambient air.
UV-irradiatlon of propylene, S02, NO, and
N02 produced 03 and a mutagenic moiety
when collected particles were tested by
the pi ate- Incorporation test. Irradiation
did not alter and 03 tended to reduce
the mutagenic response.
Increased pulmonary infection. Decreased
fertility and infant survival. No signi-
ficant changes in pulmonary function. De-
creased spontaneous running activity during
the first few weeks of exposure.
Histopathologic changes resembling
tracheitis and bronchial pneumonia at
the higher concentration range of oxidants.
No cardiovascular effects
No significant differences In collagen:
protein ratios; prolyl hydroxylase in-
creased with high concentrations of all
mixes.
Salmonella
typhJBur1u«
House,
rat,
hamster,
guinea pig
Mouse
Dog
• Dog
Claxton and Barnes, 1981
Hueter et al . , 1966
Lewis et al., 1967
Nakajima et al . , 1972
Bloch et al., 1972, 1973
Gillespie, 1980
Orthoefer et al . , 1976
Pulmonary function for groups receiving
oxidants [irradiated exhaust (I) + SOX]
18 months: no effects
36 months: no effects
Dog
Vaughan et al., 1969
Lewis et al., 1974
-------
TABLE 9-27. EFFECTS OF COMPLEX POLLUTANT MIXTURES (continued)
K>
M
U!
Concentration,
(ppm)
Pollutant"
Exposure
duration and
protocol
Observed effect(s)
Species
Reference
61 months: N2 washout increased (I);
R. increased (I, I+SO ).
2 years recovery: P-.C02 increased (I+SQ );
Vp increased (I, I+SOX); Dico/TLC decreased
and V increased in all groups; lung com-
partment volumes increased (I+SOX).
Morphology (32-36 months recovery): air
space enlargement; nonciHated bronchiolar
hyperplasia; foci of ciliary loss with and
without squamous metaplasia in trachea
and bronchi.
Dog
Dog
Dog
Lewis et al., 1974
Gillespie, 1980
Hyde et al,, 1978
0.33 - 0.82
0.16 - 5.50
0.16 - 4.27
0.12 - 2.42
0.02 - 0.20
°x
NOg
NO
Formaldehyde
Acrolein
4-6 hr
Increased pulmonary flow resistance, in- Guinea pig Murphy et al., 1963;
creased tidal volume, decreased breathing Murphy, 1964
frequency due to formaldehyde and acrolein
at low 0 : aldehyde ratio. Decreased
tidal volume, increased frequency, in-
creased pulmonary resistance due to 0
and NO at high '0 : aldehyde ratio.
C. Other complex mixtures
0.08
0.7i
2.05
1.71
0.3
1.0
2.0
Os
S02
T-2 Butene
acetal dehyde
03
N02
S02
4 weeks
7 days/week
23 hr/day
2 weeks
7 days/week
23 hr/day
Alteration in distribution of ventilation Hamster Raub et al., 1983b
(AN2) and increased diffusing capacity.
Voluntary activity (wheel running) Mouse Stinson and Loosli, 1979
decreased 75% after 1-3 days, returning
to 853) of pre-exposure levels by the end
of 14 days.
-------
TABLE 9-27. EFFECTS OF COMPLEX POLLUTANT MIXTURES (continued)
Concentration,9
(ppm)
0.40 - 0.52
1.0 - 2.15
1.25
Pollutant6
03
Ox (§as vapor)
Ox (gas vapor)
Exposure
duration and
protocol Observed effect(s)
24 hr Decreased spontaneous wheel running
activity.
19 weeks, Decreased conception rate, litter rate,
continuous and newborn survival.
Species
Mouse
Mouse
Reference
Boche and
1960
Kotin and
Quilligan,
Thomas, 1957
.Ranked by nonspecific oxidant concentration (Os or 0 ).
Abbreviations used: 0$ = ozone; 0 = oxidant; CO = carbon
83 = ozone; 0 = oxidant; CO = carbon monoxide; NO = nitrogen oxide, K02 = nitrogen dioxide;
NO = nitrogen oxides; S02 = sulfur dioxide; HC = hydrocarbon; CH4 = methane; H2S04 = sulfuric acid; PAN - peroxyacetyl nitrate.
c
See Glossary for the identification of pulmonary symbols.
-------
(Lewis et. al., 1974), 48 to 61 months (Bloch et al. , 1972, 1973; Lewis et
al., 1974), and 68 months (Orthoefer et al., 1976) of exposure; the dogs were
examined again 24 months (Gillespie, 1980) or 32 to 36 months (Orthoefer et
al., 1976; Hyde et al., 1978) after exposure ceased. Only those results
pertaining to oxidant exposure are described in this section, which limits the
discussion to groups exposed to irradiated automobile exhaust (I) and irradiated
exhaust supplemented with sulfur oxides (I+SO ). See Table 9-27 for exposure
s\
concentrations.
No specific cardiovascular effects were reported during the course of
exposures (Bloch et al., 1971, 1972, 1973) or 3 years after exposure (Gillespie,
1980). Similarly, Orthoefer et al.-(1976) reported no significant biochemical
differences in the collagen to protein ratio in tissues of dogs exposed for 68
months or after 2.5 to 3 years of recovery in ambient air. However, prolyl
hydroxylase levels were reported to have increased in the lungs of dogs exposed
to I and I+SO , when compared to control air and the nonirradiated exhaust
J\
alone or in combination with SO .
/v
No significant impairment of pulmonary function was found after 18 months
(Vaughan et al., 1969) or 36 months (Lewis et al., (1974) of exposure. However,
by 61 months of exposure, Lewis et al. (1974) reported increases in the nitrogen
washout of dogs exposed to I, and higher total expiratory resistance in dogs
exposed to both I and I+SO , when compared to their respective controls receiv-
/\
ing clean air and SO alone. Two years after exposure ceased, pulmonary
/\
function was remeasured by Gillespie (1980). These measurements were made in
a different laboratory than the one used during exposure, but consistency
among measurements of the control group and another set of dogs of similar age
at the new laboratory indicated that this difference did not have a major
impact on the findings. Arterial partial pressure of C0? increased in the
group exposed to I+SO and total deadspace increased in the the I and I+SO
)\ />
groups. The diffusing capacity for carbon monoxide (D, ) was similar in all
exposure groups, but when normalized for total lung capacity (TLC), the D|CO/TLC
ratio was smaller in exposed groups than in the air control group. Mean
capillary blood volumes also increased in all exposed groups. No changes in
lung volumes were reported at the end of exposure (Lewis et al., 1974).
However, when lung volumes from these animals were measured 2 years later,
increases were reported in the I+SO group. Unfortunately, the sample size of
1 /\
dogs exposed to I was too small (n = 5) to permit meaningful comparisons. In
9-215
-------
general, pulmonary function changes were found to be similar in all groups
exposed to automobile exhaust alone or supplemented with SO . Exposure to
Pv
these mixtures with or without UV-irradiation produced lung alterations normal-
ly associated with injury to the airway and parenchyma.
The functional abnormalities mentioned above showed relatively good
correlation with structural changes reported by Hyde et al. (1978). After 32
to 36 months of recovery in clean air, morphologic examination of the lungs by
light microscopy, scanning electron microscopy, and transmission electron
microscopy revealed a number of exposure-related effects. The displaced
volume of the fixed right lung was larger in the I-exposed group. Both the I
and I+SQ groups showed random enlargement of alveolar airspaces centered in
f\
respiratory bronchioles and alveolar ducts. Small hyperplastic lesions were
observed at the junction of the terminal bronchiole and the first-order respi-
ratory bronchiole. Foci of ciliary loss associated with squamous metaplasia
were also observed in the intrapulmonary bronchi of the I+SO group. However,
A
because these was no significant difference in the magnitude of these lesions,
oxidant gases and SO did not appear to act in an additive or synergistic
X
manner.
Additional work on irradiated and nonirradiated automobile exhaust has
been presented by Claxton and Barnes (1981). The mutagenicity of diesel
exhaust particle extracts collected under smog-chamber conditions was evaluated
by the Salmonella typhimurium pi ate-incorporation test (Ames et al., 1975).
The authors demonstrated that the irradiation of propylene, S02, NO, and NQ"2
produced 0, and a mutagenic moiety. In baseline studies on diesel exhaust, in
which Qg was neither added nor produced, the mutagenicity of each sample was
similar under dark or UV-light conditions. When 0, was introduced into the
smog chamber, the mutagenicity of the organic compounds was reduced.
The behavioral effect of a nonirradiated reaction mixture was examined by
Stinson and Loosli (1979). Voluntary wheel-running activity was recorded
3
during a continuous 2-week exposure to synthetic smog containing 588 pg/m
(0.3 ppm) of 0,, 1 ppm of NOp, and 2 ppm of S0?, or to each component separate-
ly. An immediate decrease in spontaneous activity occurred after 1 to 3 days
of exposure, returning to 85 percent of the original activity by the end of
exposure. Activity returned to basal levels 5 days after breathing filtered
air. Ozone alone produced a response that was similar to that of the synthetic
smog mixture. Since N02 and SOp alone had only moderate effects, the authors
concluded that 0~ had the major influence on depression of activity.
9-216
-------
More 'recently, Raub et al. (1983b) reported pulmonary function changes in
hamsters exposed 23 hr/day for 4 weeks to a nonirradiated reaction mixture of
trans-2-butene, CL, and SCL. Decreases in the nitrogen washout slope and
o £
increases in the diffusing capacity indicated a significant compensatory
change in distribution of ventilation in the lungs of exposed animals. Animals
compromised by the presence of elastase-induced emphysema were unable to
respond to this pulmonary insult in the same manner as animals without impaired
lung function.
9.6 SUMMARY
9.6.1 Introduction
The biological effects of 0., have been studied extensively in animals and
a wide array of toxic effects have been ascribed to CL inhalation. Although
much has been accomplished to improve the existing data base, refine the con-
centration-response relationships and interpret better the mechanisms of CL
effects, many of the present data were not accumulated with the idea that
quantitative comparisons to man would be drawn. In many cases, only qualita-
tive comparisons can be made. To maximize the extent that animal toxicological
data can be used to estimate the human health ris*k of exposure to CL, the
qualitative as well as quantitative similarities between the toxicity of 0, to
animals and man must be considered more carefully in the future. Significant
advances have been made in understanding the, toxicity of 0, through appropriate
animal models. This summary highlights the significant results of selected
studies that will provide useful data for better predicting and assessing, in
a scientifically sound manner, the possible human responses to 0-,.
Summary figures and tables are presented in the following sections. The
practical purpose of this presentation of the data is to help the reader focus
on what types of effects or responses have been reported, what concentrations
have been tested (1.0 ppm and lower), and as a convenient list of references
with each of the biological parameters measured. Studies were selected for
inclusion in these figures and tables on the basis of specific criteria presen-
ted below:
1. Studies have been cited when the reported effects are clearly due to 0^
exposure. Effects due to mixtures of CL with other pollutants have been
summarized in a separate figure and table. Studies involving exercise,
9-217
-------
diet deficiencies, or other possible modifiers of response to 03 have not
been included.
2. Cited studies report the effects of CL exposure over a broad range of
animal species and strains and for varying lengths of time. Specific
details on animal species, exposure duration, and observed biological
effects can be obtained from the tables within the body of this chapter,
3. Each closed symbol on the figures represents one or more studies conducted
at that particular concentration that caused effects. Specific references
can be found in the accompanying tables.
4. Each open circle represents one or more studies that used the given
concentration, but reported no significant effects. No-effect levels are
also indicated by brackets in the accompanying tables.
5. Only pulmonary function effects were divided by short-term (<14 days) and
long-term exposures to follow the discussion in the text.
In order to keep this section brief, and concise, it was necessary to be somewhat
selective in determining what and how this information would be presented. A
number of important factors, such as the specific length of exposure, were not
included. Also, the parameter selected to illustrate a specific response was
usually broad and very general. For example, the category "decreases in
macrophage function" includes such diverse endpoints as measurements of lyso-
somal and phagocytic activity, macrophage mobility, or chemotactic response.
These responses may or may not be related to one another. Thus, care must be
taken in how these data are used and interpreted. The only appropriate use is
to gain an overview of the broad array of the effects of ozone and the concen-
trations which did and did not cause these effects.
9.6.2 RegionalDosimetry in the Respiratory Tract
The amount of 0» acting at a given site in the lung is related to the
airway luminal concentration at that level. As a result, 0, does not immediately
interact with cellular components of the respiratory tract. Instead, it first
comes into contact with the mucous or surfactant layer lining the airway. It
should be noted that CL is quite reactive chemically. Reactions with components
9-218
-------
of this layer cause an increase in total absorption of 03 in the upper airways
and in a reduction of the amount of CL reaching sensitive tissues. The site
at which uptake and subsequent interaction occur and the local dose (quantity
of CL absorbed per unit area per time), along with cellular sensitivity, will
determine the type and extent of the injury. Also, the capacity for responding
to a specific dose may vary between animals and humans because of dissimilarities
in detoxification systems, pharmacokinetics, metabolic rates, genetic makeup,
or other factors. Thus, along with the above, a knowledge of the complex
process of gas transport and absorption is crucial to understanding the effects
of On and other oxidants in humans.
The animal studies that have been conducted on ozone absorption are
beginning to indicate the quantity and site of 03 uptake in the respiratory
tract. Experiments on the nasopharyngeal removal of CL in animals suggest
that the fraction of 0, uptake depends inversely on flow rate, that uptake is
greater for nose than for mouth breathing, and that trachea! and chamber
concentrations are positively correlated. Only one experiment measured 0~
uptake in the lower respiratory tract, finding 80 to 87 percent uptake by the
lower respiratory tract of dogs (Yokoyama and Frank, 1972). At present,
however, there are no reported results for human nasopharyngeal or lower
respiratory tract absorption. Caution must be used in estimating nasopharyngeal
uptake for normal respiration based upon experiments employing unidirectional
flows.
To further an understanding of 0,, absorption, mathematical models have
been developed to simulate the processes involved and to predict 03 uptake by
various regions and sites within the respiratory tract. The model of Aharonson
et al. (1974) has been used to analyze nasopharyngeal uptake data. Applied to
Oo data, the model indicates that the average mass transfer coefficient in the
nasopharyngeal region increases with increasing air flow, but the actual
percent uptake decreases.
Three models have been developed to simulate lower respiratory uptake
(McJilton et al. , 1972; Miller et al., 1978b, 1985). These models are very
similar in their treatment of 0» in the airways (taking into account convection,
diffusion, wall losses, and ventilatory patterns) and in their use of morpho-
logical data to define the dimensions of the airways and liquid lining. The
models differ in their treatment of the mechanism of absorption. Both of the
models of Miller and co-workers take into account chemical reactions of 0,
with constituents of the liquid lining, whereas the model of McJilton et al.
9-219
-------
does not. The models of Miller et al. differ in their treatment of chemical
reactions, as well as in the fact that the newer model includes chemical reac-
tions of 03 in additional compartments, such as tissue and blood.
Tissue dose is predicted by the models of Miller et al. to be relatively
low in the trachea, to increase to a maximum between the junction of the
conducting airways and the gas-exchange region, and then to decrease distally.
This is not only true for animal simulations (guinea pig and rabbit) but it
is also characteristic of the human simulations (Miller et al., 1978b; 1985).
A comparison of the results of Miller and co-workers with morphological
data (that shows the centriacinar region to be most affected by 03) indicates
qualitative agreement between predicted tissue doses and observed effects in
the pulmonary region. However, comparisons in the tracheobronchial region
indicate that dose-effect correlations may be improved by considering other
expressions of dose such as total absorption by an airway and by further
partitioning of the mucous layer compartment in mathematical models. Further
research is needed to define toxic mechanisms, as well as to refine our know-
ledge of important chemical, physical, and morphological parameters.
At present, there are few experimental results that are useful in judging
the validity of the modeling efforts. Such results are needed, not only to
understand better the absorption of 03 and its role in toxicity, but also to
support and to lend confidence to the modeling efforts. With experimental
confirmation, models which further our understanding of the role of 0~ in the
respiratory tract will become practical tools.
The consistency and similarity of the human and animal lower respiratory
tract dose curves obtained thus far lend strong support to the feasibility of
extrapolating to man the results obtained on animals exposed to 03. In the
past, extrapolations have usually been qualitative in nature. With additional
research in areas which are basic to the formulation of dosimetry models,
quantitative dosimetric differences among species can be determined. If in
addition, more information is obtained on species sensitivity to a given dose,
significant advances can be made in quantitative extrapolations and in making
inferences about the likelihood of effects of 03 in man. Since animal studies
are the only available approach for investigating the full array of potential
disease states induced by exposure to 03, quantitative use of animal data is
in the interest of better establishing CL levels to which man can safely be
exposed.
9-220
-------
9.6.3 Effects of Ozone onthe Respiratory Tract
9.6.3.1 Morphological Effects. The morphological changes which follow
exposure to less than 1960 \jg/m (1.0 ppm) 0, are very similar in all species
of laboratory mammals studied. Of the many specific cell types found in the
respiratory system, two types, ciliated cells and type 1 alveolar epithelial
cells, are the cells most damaged morphologically following 0-, inhalation.
Ciliated cells are found in the conducting airways, e.g., trachea, bronchi,
and nonrespiratory bronchioles. Ciliated cells function in the normal clearance
of the airways and the removal of inhaled foreign material. Following 0-
exposure of experimental animals, damaged ciliated cells have been reported in
all of these conducting airways (Schwartz et a!., 1976; Castleman et a!,,
1977). In rats, damage to ciliated cells appears most severe at the junction
of the conducting airways with the gas exchange area (Stephens et al., 1974a;
Schwartz et al., 1976). Damage to type 1 alveolar epithelial cells is limited
to those cells located near this junction, i.e., the centriacinar or proximal
alveolar region of the pulmonary acinus (Stephens et al., 1974b; Schwartz et
al., 1976; Castleman et al., 1980; Barry et al., 1983; Crapo et al., 1984).
Type 1 alveolar cells form most of the blood-air barrier where gas exchange
occurs. Severely damaged ciliated and type 1 alveolar epithelial cells are
shed (sloughed) from the tissue surface and are replaced by multiplication of
other cell types less damaged by 0, (Evans et al., 1985). This process has
been most extensively studied in the centriacinar region where nonciliated
bronchiolar cells and type 2 alveolar epithelial cells become more numerous
(Evans et al., 1976a,b,c; Lum et al., 1978). Some of these nonciliated bron-
chiolar and type 2 cells differentiate into ciliated and type 1 cells, respec-
tively. Cell multiplication in bronchioles may be more than that required for
replacement of damaged ciliated cells, and nonciliated bronchiolar cells may
become hyperplastic (Castleman et al., 1977; Ibrahim et al., 1980; Eustis et
al, , 1981) and sometimes appear as nodules (Zitm'k et al., 1978; Moore and
Schwartz, 1981; Fujinaka et al., 1985). Inflammatory changes characterized by
a variety of leukocytes with alveolar macrophages predominating, intramural
edema, and fibrin are also seen in the centriacinar region (Stephens et al.,
1974a; Schwartz et al. , 1976; Castleman et al., 1977; Fujinaka et al., 1985).
The damage to ciliated and centriacinar type 1 alveolar epithelial cells
and the inflammatory changes tend to occur soon after exposure to concentrations
9-221
-------
3
of 0, as low as 392 pg/m (0.2 ppm). Damage to centriacinar type 1 alveolar
epithelium in rats has been well documented as early as 2 hours after exposure
3
to Og concentrations of 980 \ig/m (0.5 ppm) (Stephens et al.s 1974a). In the
same publication the authors report damage to centriacinar type 1 alveolar
o
epithelial cells after 2 hours exposure to 392 ug/m (0.2 ppm) 03, but this
portion of their report is not documented by published micrographs (Stephens
et al., 1974a). Loss of cilia from cells in the rat terminal bronchiole
3
occurs following exposure to 980 |jg/m (0.5 ppm) 03 for 2 hours (Stephens et
al., 1974a). Damage to ciliated cells has been seen following exposure of
3
both rats and monkeys to 392 ug/m (0.2 ppm) 03, 8 hr/day for 7 days (Schwartz
et al., 1976; Castleman et al., 1977). Centriacinar inflammation has been
3
reported as early as 6 hours after exposure to 980 ng/m (0.5 ppm) 0- (Stephens
3
et al., 1974b) and 4 hours after exposure to 1568 ug/m (0.8 ppm) 03 (Castleman
et al., 1980).
During long-term exposures, the damage to ciliated cells and to centriacinar
type 1 cells and centriacinar inflammation continue, though at a reduced rate.
Damage to cilia has been reported in monkeys following 90-day exposure to 980
3
ug/m (0.5 ppm) 0,,, 8 hr/day (Eustis et al., 1981) and in rats exposed to 980
q 3
ug/ra (0.5 ppm) 03, 24 hr/day for 180 days (Moore and Schwartz, 1981). Damage
to centriacinar type 1 cells was reported following exposure of young rats to
490 ug/m (0.25 ppm) Q~, 12 hrs/day for 42 days (Barry et al., 1983; Crapo et
•s ,,
al., 1984). Changes in type 1 cells were not detectable after 392 ug/nr (0.2
3
ppm) 03, 8 hr/day for 90 days but were seen in rats exposed to 980 ug/m (0.5
ppm) for the same period (Boorman et al., 1980). Centriacinar inflammatory
3
changes persist during 180-day exposures of rats to 980 pg/m (0.5 ppm) 03, 24
hr/day (Moore and Schwartz, 1981) and one-year exposures of monkeys to 1254
ug/ii (0.64 ppm) 03, 8 hr/day (Fujinaka et al., 1985),
Remodeling of distal airways and centriacinar regions occurs following
long-term exposures to 03. Rats develop respiratory bronchioles between the
terminal bronchiole to alveolar duct junction seen in control rats (Boorman et
al., 1980; Moore and Schwartz, 1981). In monkeys, distal airway remodeling
results in increased volumes of respiratory bronchioles which have thicker
walls and a smaller internal diameter (Fujinaka et al., 1985). The walls of
centriacinar alveoli are also thickened (Schwartz et al., 1976; Boorman et
al.» 1980; Barry et al., 1983; Crapo et al., 1984; Last et al., 1984a). Studies
of the nature of these thickened interalveolar septa and bronchiolar walls
9-222
-------
revealed increases in inflammatory cells, fibroblasts, and amorphous extracel-
lular matrix (Last et al., 1984a; Fujinaka et al., 1985). Three studies
provide morphological evidence of mild fibrosis (i.e., local increase of
collagen) in centriacinar interalveolar septa following exposure to < 1960
3
pg/m (< I ppm) of 03 (Last et al.5 1979; Boorman et al., 1980; Moore and
Schwartz, 1981). Changes in collagen location or amounts, or both, which occur
with the remodeling of the distal airways, were reported in two of those studies
(Boorman et al., 1980; Moore and Schwartz, 1981).
While morphometry of small pulmonary arteries is not commonly studied in
0,,-exposed animals, pulmonary artery walls thickened by muscular hyperplasia
3
and edema were reported in rabbits exposed to 784 pg/rn (0.4 ppm) 03, 6 hr/day,
5 days/week for 10 months (P'an'et al., 1972). Thickened intima and media in
3'
pulmonary arterioles were reported in monkeys exposed to 1254 ug/m (0.64 ppm)
03, 8 hr/day for 1 year (Fujinaka et al., 1985).
Several of the effects of 0,, inhalation persisted after the 0~ inhalation
ended and the animals breathed only filtered air several days or weeks. Lungs
3
from rats exposed to 1568 (jg/m (0.8 ppm) 0, for 72 hours appeared normal 6
days after the end of the exposure (Plopper et al., 1978). However, incomplete
resolution of the nonciliated bronchiolar epithelial hyperplasia was reported
in monkeys 7 days after 50 hours exposure to 1568 |jg/m (0.8 ppm) 0, (Castleman
......... , 3
et al.5 1980) and in mice 10 days after a 20-day exposure to 1568 jjg/m (0.8
ppm) 03, 24 hr/day (Ibrahim et al., 1980). Centriacinar inflammation and
distal airway remodeling were still apparent 62 days after a 180-day exposure
to 980 |jg/m3 (0.5 ppm) Og, 24 hr/day (Moore and Schwartz, 1981).
While not all species of laboratory mammals have been studied following a
single 0, exposure regimen or using the same morphological techniques because
investigators have asked different biological questions, there is a striking
similarity of morphological effects in the respiratory system of all species
studied. The cell types most damaged are the same. One of these cells, the
type 1 alveolar epithelial cell, has a wide distribution in the pulmonary
acinus and yet is damaged only in one specific location in all species studied.
The other, the ciliated cell, appears damaged wherever it is located in the
conducting airways. Damage to these cells is seen within hours after exposure
to concentrations of 0- much lower than 1 ppm and continues during exposures
of weeks or months. Hyperplasia of other cell types is reported to start
early in the exposure period, to continue throughout a long-term exposure, and
9-223
-------
when studied, to persist following postexposure periods of days or weeks.
Centriacinar inflammation is also seen early and is reported throughout long
exposure periods. Duration of centriacinar inflammation during postexposure
periods has been studied less often and appears dependent upon length of the
exposure period.
Other effects which have been reported in fewer studies or in a more
limited number of species include distal airway remodeling and thickened pulmo-
nary arteriolar walls. Remodeling of distal airways has only been reported
in rats and monkeys after long-term exposures. In rats, remodeling of distal
airways has been reported to persist for several weeks after the 03 exposure
has ended. Thickened pulmonary arteriolar walls have been reported only
twice, once after long-term exposure of rabbits and once after long-term
exposure of monkeys.
Studies on the morphologic effects of 0~ exposures of experimental animals
are summarized in Figure 9-4 and Table 9-28 (see Section 9.6.1 for criteria
used to summarize the studies).
9.6.3.2 Pulmonary Function. One of the limitations of animal studies is
that many pulmonary function tests comparable to those conducted after acute
exposure of human subjects are difficult to interpret. Methods exist, however,
for obtaining similar measurements of many variables pertinent to understand-
ing the effects of ozone on the respiratory tract, particularly after longer
exposure periods. A number of newer studies reported here reflect recent
advances in studying the effects of 03 on pulmonary function in small animals.
Changes in lung function following ozone exposure have been studied in
mice, rats, guinea pigs, rabbits, cats, dogs, sheep, and monkeys. Short-term
3
exposure for 2 hr to concentrations of 431 to 980 ug/m (0.22 to 0.5 ppm)
produces rapid, shallow breathing and increased pulmonary resistance during
exposure (Murphy et al., 1964; Yokoyama, 1969; Watanabe et al., 1973; Amdur
et al., 1978). The onset of these effects is rapid and the abnormal breathing
pattern usually disappears within 30 min after cessation of exposure. Other
changes in lung function measured following short-term ozone exposures lasting
3 hr to 14 days are usually greatest 1 day following exposure and disappear by
7 to 14 days following exposure. These effects are associated with premature
closure of the small, peripheral airways and include increased residual volume,
closing volume, and closing capacity (Inoue et al., 1979).
Studies of airway reactivity following short-term ozone exposure of 1 to
2 hr duration in experimental animals show that 03 increases the reactivity of
9-224
-------
**.aSSP A
6.1-
0.2-
g 0.3-
Q.
Q.
| 0.4-
* 4
I'-'
i 4
1 4
4
4
4
4
4
'
r 4
1 1
1
1 (
1 4
» 4
> 4
1
1
1 4
1 4
i i
1 <
t
1
l 4
l
1 1
l 1
»
1
1
t
1
1 •
1
>
Figure 9-4. Summary of morphological effects in experimental animals
exposed to ozone. See Table 9-28 for reference citations of studies ~
summarized here.
$-225
-------
TABLE 9-28. SUMMARY TABLE: MORPHOLOGICAL EFFECTS OF OZONE
IN EXPERIMENTAL ANIMALS
Effect/response
03 concentration, ppm
References
Damaged ciliated
and type 1 cells
Proliferation of non-
ciliated bronchiolar
and type 2 cells
Centriacinar
inflammation
[0.2], 0.5, 0.8
0.2, 0.5, 0.8
0.2, 0.35
0.25
0.25
0.26, 0.50, 1.0
0.5
0.5
0.5
0.5, 0.8
0.5, 0.8
0.54, 0.88
0.8
0.8
0.85
0.2, 0.35
0.35, 0.50, 0.70,
0.75, 1.0
0.5
0.5
0.5
0.5, 0.8
0.54, 0.88
0.64
0.7
0.8
0.8
0.8
1.0
[0.2], 0.5, 0.8
0.2
0.2, 0.5, 0.8
0.25
0.25
0.35
0.5
0
0.5, 0.8
0.5, 0.8
0.5, 0.8
0.54, 0.88
0.54, 0.88
0.64
0.8
1.0
Boorman et al. (1980)
Schwartz et al. (1976)
Castleman et al. (1977)
Barry et al. (1983)
Crapo et al. (1984)
Boatman et al. (1974)
Stephens et al. (1974b)
Moore and Schwartz (1981)
Evans et al. (1985)
Eustis et al. (1981)
Mel lick et al. (1975, 1977)
Stephens et al. (1974a)
Castleman et al. (1980)
Plopper et al. (1978)
Stephens et al. (1978)
Castleman et al. (1977)
Evans et al. (1976b)
Evans et al. (1985)
Zitnik et al. (1978)
Moore and Schwartz (1981)
Eustis et al. (1981)
Freeman et al. (1974)
Fujinaka et al. (1985)
Evans et al. (1976a)
Castleman et al. (1980)
Lum et al. (1978)
Ibrahim et al. (1980)
Cavender et al. (1977)
Boorman et al. (1980)
Plopper et al. (1979)
Schwartz et al. (1976)
Barry et al. (1983)
Crapo et al. (1984)
Castleman et al. (1977)
Stephens et al. (1974b)
Moore and Schwartz (1981)
Mellick et al. (1975, 1977)
Brummer et al. (1977)
Last et al. (1979)
Stephens et al, (1974a)
Freeman et al. (1974)
Fujinaka et al. (1985)
Castleman et al. (1980)
Freeman et al. (1973)
9-226
-------
TABLE 9-28. SUMMARY TABLE: MORPHOLOGICAL EFFECTS OF OZONE
IN EXPERIMENTAL ANIMALS (continued)
Effect/response
Distal airway
remodel ing
Thickened pulmonary
arteriolar walls
03 concentration, ppm
[0.2], 0.5, 0.8
0.2, 0.5, 0.8
0.5
0.64, 0.96
0.64
1.0
0.4
0.64
References
Boorman et al. (1980)
Schwartz et al. (1976)
Moore and Schwartz (1981)
Last et al. (1984a)
Fujinaka et al. (1985)
Freeman et al . (1973)
P'an et al. (1972)
Fujinaka et al. (1985)
the lungs to a number of stimuli. Mild exercise, histamine aerosol inhalation,
and breathing air with reduced oxygen or elevated carbon dioxide concentrations
caused rapid, shallow breathing in conscious dogs immediately following 2-hr
exposures to 1100 to 1666 ng/m3 (0.56 to 0.85 ppm) of 03 (Lee et al., 1979,
1980). Aerosolized ovalbumin caused an increased incidence of anaphylaxis in
mice preexposed to 980 or 1568 pg/m (0.5 or 0.8 ppm) of 03 continuously for 3
to 5 days (Osebold et al. , 1980). In addition, increased airway sensitivity
to histamine or cholinomimetic drugs administered by aerosol or injection has
been noted in several species after exposure to 980 to 5880 [jg/m (0.5 to 3.0
ppm) of 03 (Easton and Murphy, 1967; Lee et al., 1977; Abraham et al., 1980,
1984a,b; Gordon and Amdur, 1980; Gordon et al., 1981, 1984; Roum and Murlas,
1984). The mechanism responsible for 0.,-induced bronchial reactivity is still
uncertain but may involve more than one specific factor. Ozone has been shown
to cause increased sensitivity of vagal sensory endings in the dog airway (Lee
et al., 1977, 1979, 1980), Ozone exposure may also enhance the airway respon-
siveness to bronchoconstrictors by altering sensitivity of the airway smooth
muscle.-directly or through re-leased cellular mediators (Gordon et al. , 1981,
1984; Abraham et al., 1984a,b). In some species, increased airway hyperreac-
tivity may be explained by increased transepithelial permeability or decreased
thickness of the airway mucosa (Osebold et al., 1980; Abraham et al., 1984b).
Ozone exposure may also decrease airway hyperreactivity by causing mucous
hypersectetion, thereby limiting the airway penetration of inhaled bronchocon-
strictors (Abraham et al., 1984a).
9-227
-------
The time course of airway hyperreactivity after exposure to 980 to 5880
pg/m (0.5 to 3.0 ppm) of 0~ suggests a possible association with inflammatory
cells and pulmonary inflammation (Holtzman et a!., 1983a,b; Sielczak et al.,
1983; Fabbri et al., 1984; O'Byrne et al., 1984a,b; Murlas and Roum, 1985).
However, the time course of responsiveness is variable in different species
and the relationships between airway inflammation and reactivity at different
concentrations of 03 are not well understood. Additional studies that demon-
strate increased collateral resistance following 30 min local exposure of 03
or histamine in sublobar bronchi of dogs (Gertner et al., 1983a,b,c,1984)
suggest that other mechanisms, along with amplification of reflex pathways,
may contribute to changes in airway reactivity depending not only on the
concentration of 03 in the airways but also on the extent of penetration of
ozone into the lung periphery.
The effects of short-term exposures to 03 on pulmonary function and
airway reactivity in experimental animals are summarized in Figure 9-5 and
Table 9-29 (see Section 9.6.1 for criteria used in developing this summary).
3
Exposures of 4 to 6 weeks to ozone concentrations of 392 to 490 pg/m
(0.2 to 0,25 ppm) increased lung distensibility at high lung volumes in young
rats (Bartlett et al., 1974; Raub et al., 1983a). Similar increases in lung
3
distensibility were found in older rats exposed to 784 to 1568 pg/m (0.4 to
0.8 ppm) for up to 180 days (Moore and Schwartz, 1981; Costa et al., 1983;
3
Martin et al., 1983). Exposure to 03 concentrations of 980 to 1568 ug/m (0.5
to 0.8 ppm) increased pulmonary resistance and caused impaired stability of
the small peripheral airways in both rats and monkeys ( Wegner, 1982; Costa
et al., 1983; Yokoyama et al., 1984; Kotlikoff et al., 1984). The effects in
monkeys were not completely reversed by 3 months following exposure; lung
distensibility had also decreased in the postexposure period, suggesting the
development of lung fibrosis which has also been suggested morphologically and
biochemically.
The effects of long-term exposures to ozone on pulmonary function and
airway reactivity in experimental animals are summarized in Figure 9-6 and
Table 9-30 (see Section 9.6.1 for criteria used in developing this summary).
9.6.3.3 Biochemical Effects The lung is metabolically active, and- several
key steps in metabolism have been studied after 03 exposure. Since the proce-
dures for such studies are invasive, this research has been conducted only in
animals. Effects, to be summarized below, have been observed on antioxidant
metabolism, oxygen consumption, proteins, lipids, and xenobiotic metabolism.
9-228
-------
^
jp
<*&
0°'
X*
(rf
\«
u.u —
0.1-
0.2-
E 0.3-
a
a
i 0.4-
o
1
S 0.5-
0)
u
8 0.6-
o>
<3 0.7-
0.8-
0.9-
1.0-
'
«
i
<
<
<
<
i
i . (
i
i
t " <
•
t a
C
(
1
(
(
1
(
I
r
•
i
>
i 1
>
I
<
C
i <
l (
1
«J
)
1
i (
(
(
i 1
)
i
i
i
i
Figure 9-5. Summary of effects of short-term ozone exposures on
pulmonary function in experimental animals. See Table 9-29 for
reference citations of studies summarized here.
-------
TABLE 9-29. SUMMARY TABLE: EFFECTS ON PULMONARY FUNCTION
OF SHORT-TERM EXPOSURES TO OZONE IN EXPERIMENTAL ANIMALS
Effect/response 03 concentration, ppm
References
Increased breathing
frequency
0.22, 0.41, 0.8
0.34, 0.68, 1.0
0.5
Amdur et al. (1978)
Murphy et al. (1964)
Yokoyama (1969)
Decreased tidal volume 0.34, 0.68, 1.0 Murphy et al. (1964)
Decreased lung
compliance
Increased residual
volume (RV),
closing capacity
(CC), and closing
volume (CV)
Decreased diffusion
capacity
[0.22], 0.41, 0.8
0.26, 0.5, 1.0
1.0
0.24 - 1.0
Amdur et al. (1978)
Watanabe et al. (1973)
Yokoyama (1974)
Inoue et al. (1979)
0.26, 0.5, 1.0
Watanabe et al. (1973)
Increased pulmonary
resistance
Increased airway
reactivity
:
[0.22]
0.26, 0.5, 1.0
0.5
1.0
[0.1]-0.8
[0.1]-0.8, 1.0
0.5, 1.0
0.7
1.0
Amdur et al. (1978) :
Watanabe et al . (1973)
Yokoyama (1969)
Yokoyama (1974)
Gordon and Amdur (1980)
Gordon et al. (1981, 1984)
Abraham et al. (1980, 1984a,b)
Lee et al. (1977)
Holtzman et al. (1983a,b)
The lung contains several compounds (e.g., vitamin E, sulfhydryls, gluta-
thione) and enzymes (e.g., glutathione peroxidase, glutathione reductase,
glucose-6-phosphate dehydrogenase, and superpxide dismutase) that function as
antioxidants, thereby defending the lung against oxidant toxicity from the
oxygen in air, from oxidants produced during metabolic processes, and from
oxidizing air pollutants such as ozone. Obviously, this protection is only
partial for 0- since exposure to ozone causes numerous effects on lung struc-
ture, function, and biochemistry. Acute exposure to high ozone levels (2920
ug/m , 2 ppm) typically decreases antioxidant metabolism, whereas repeated
o
exposures to lower levels (between 272 and 1568 ug/m , 0.2 and 0.8 ppm) in-
creases this metabolism (DeLucia et al., 1975b). In rats maintained on normal
9-230
-------
VD
M
CO
"" >x V*
:yp \ , • • i.
0*~ o6"
>
i
4
1
• . . <
<
1
»
1
Figure 9-6. Summary of effects of long-term ozone exposures on
pulmonary function in experimental animals. See Table 9-30 for
reference citations of studies summarized here.
-------
TABLE 9-30. SUMMARY TABLE: EFFECTS ON PULMONARY FUNCTION
OF LONG-TERM EXPOSURES TO OZONE IN EXPERIMENTAL ANIMALS
Effect/response 0'3 concentration, ppm
References
Increased lung volume
Increased pulmonary
resistance
Decreased lung
compliance
Decreased inspiratory
flow
Decreased forced
expiratory volume
(FEV,) and flow
(FEFjr
[0.08], [0.12], 0.25
0.2
[0.2], 0.8
0.4
0.2, 0.8
0.5, 1.0
0.64
0.64
0.5, 0.8
0.64
0.2, 0.8
0.64
Raub et al. (1983a)
Bartlett et al. (1974)
Costa et al. (1983)
Martin et al. (1983)
Costa et al. (1983)
Yokoyama et al. , 1984
Wegner (1982)
Kotlikoff et al.5 1984
Eustis et al. (1981)
Wegner (1982)
[0.08], 0.12, 0.25 Raub et al. (1983)
Costa et al. (1983)
Wegner (1982)
diets, this response has been observed after a week of continuous or intermit-
tent exposure to 392 ug/m3 (0.2 ppm) 03 (Mustafa, 1975; Mustafa and Lee, 1976;
Plopper et al., 1979). Similar responses are seen in monkeys and mice, but at
3
higher concentrations (980 [jg/m , 0.5 ppm) (Fukase et al., 1978; Mustafa and
Lee, 1976).
The effects of 03 on oxygen consumption have been studied since oxygen
consumption is a fundamental parameter of cellular metabolism, reflecting
energy production by cells. As with antioxidant metabolism, acute exposure to
3
high ozone levels Q> 3920 |jg/m ; > 2 ppm) decreases metabolism (and thus,
3
oxygen consumption); repeated exposure to lower levels (> 1568 [jg/m , 0.8 ppm)
increases oxygen consumption (Mustafa et al.5 1973; Schwartz et al., 1976;
Mustafa and Lee, 1976)% Effects in rats on normal diets have been observed
3
after a short-term exposure to ozone levels as low as 392 Mg/m (0.2 ppm)
(Schwartz et al., 1976; Mustafa et al., 1973; Mustafa and Lee, 1976). Monkeys
3
are affected at a higher level of ozone (980 yg/m , 0.5 ppm).
9-232
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Similar patterns of response for both antioxidant metabolism and oxygen
consumption are observed after exposure to ozone. A 7-day exposure to ozone
produces linear concentration-related increases in activities of glutathione
peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and
succinate oxidase (Mustafa and Lee, 1976; Chow et al., 1974; Schwartz et al. ,
1976; Mustafa et al., 1973). Rats on a vitamin E-deficient diet experience an
increase in enzyme activities at 196 ug/m (0.1 ppm) ozone as compared to
Q
392 ug/m (0.2 ppm) in animals on normal diets (Chow et al., 1981; Mustafa and
Lee, 1976; Mustafa, 1975). Research on these enzymes has shown that there is
no significant difference in effects from continuous versus intermittent
exposure; this, along with concentration-response data, suggests that the con-
centration of ozone is more important than duration of exposure in causing
these effects (Chow et al., 1974; Schwartz et al., 1976; Mustafa and Lee,
1976).
/
Duration of exposure still plays a role, however. During exposures up to
1 or 4 weeks, antioxidant metabolism and 0^ consumption generally do not
change on the first day of exposure; by about day 2, increases are observed and
by about day 4 a plateau is reached (Mustafa and Lee, 1976; DeLucia et al.,
1975a). Recovery from these effects occurs by 6 days post-exposure (Chow
et al., 1976b). This plateauing of effects in the presence of exposure does
not result in long-term tolerance. If rats are re-exposed after recovery is
observed, the increase in enzyme activities is equivalent to that observed in
animals exposed for the first time (Chow et al., 1976b).
The influence of age on responsiveness is also similar for antioxidant
metabolism and oxygen consumption (Elsayed et al., 1982a; Tyson et al., 1982;
Lunan et al., 1977). Suckling neonates (5 to 20 days old) generally exhibited
a decrease in enzyme activities; as the animals grew older (up to about 180 days
old), enzyme activities generally increased with age. Species differences may
exist in this response (Mustafa and Lee, 1976; Mustafa et al. , 1982; Chow
et al. , 1975; DeLucia et al. , 1975a). Studies in which monkeys have been
compared to rats did not include a description of appropriate statistical
considerations applied (if any); thus, no definitive conclusions about respon-
siveness of monkeys versus rats can be made.
The mechanism responsible for the increase in antioxidant metabolism and
oxygen consumption is not known. The response is typically attributed, however,
to concurrent morphological changes, principally the loss of type 1 cells and
an increase in type 2 cells that are richer in the enzymes measured.
9-233
-------
Monooxygenases constitute another class of enzymes investigated after
ozone exposure. These enzymes function in the metabolism of both endogenous
(e.g., biogenic amines, hormones) and exogenous (xenobiotic) substances. The
substrates acted upon are either activated or detoxified, depending on the
3
substrate and the enzyme. Acute exposure to 1470 to 1960 (jg/m (0.75 to
1 ppm) ozone decreased cytochrome P-450 levels and enzyme activities related
to both cytochrome P-450 and P-448. The health impact of these changes is
uncertain since only a few elements of a complex metabolic system were measured.
The activity of lactate dehydrogenase is increased in lungs of vitamin E-
3
deficient rats receiving a short-term exposure to 196 ug/m (0.1 ppm) ozone
(Chow et al., 1981). Higher levels caused a similar response in rats, but not
in monkeys, on normal diets (Chow et a!,, 1974, 1977). This enzyme is frequent-
ly used as a marker of cellular damage because it is released upon cytotoxicity.
It is not known, however, whether the increase in this enzyme is a direct
reflection of cytotoxicity or whether it is an indicator of an increased
number of type 2 cells and macrophages in the lungs.
An increase in a few of the measured- activities of lysosomal enzymes has
3
been shown in the lungs of rats exposed to > 1372 ug/m (0.7 ppm) ozone (Dillard
et'al., 1972; Castleman et al.} 1973a; Chow et al,s 1974). This response is
most likely the result of an increase in inflammatory cells in the lungs
rather than an induction of enzymes, since lysosomal enzymes in alveolar
macrophages decrease after jr\ vivo or jji vitro exposure to ozone (Hurst et al.,
1970; Hurst and Coffin, 1971).
As discussed previously, long-term exposure to high 0, concentrations
causes mild lung fibrosis (i.e., local increase of collagen in centriacinar
interalveolar septa). This morphological change has been correlated with
biochemical changes in the activity of prolyl hydroxylase (an enzyme that
catalyzes the production of hydroxyproline) and in hydroxyproline content (a
component of collagen that is present in excess in fibrosis) (Last et al.,
1979; Bhatnagar et al., 1983). An increase in collagen synthesis has been
3
observed, with 980 |jg/m (0.5 ppm) 03 being the minimally effective concentra-
tion tested (Hussain et al., 1976a,b; Last et al., 1979), During a prolonged
exposure, prolyl hydroxylase activity increases by day 7 and returns to control
levels by 60 days of exposure. When a short-term exposure ceases, prolyl
hydroxylase activity returns to normal by about 10 days post-exposure, but
hydroxyproline levels remain elevated 28 days post-exposure. Thus, the product
9-234
-------
of the increased synthesis, collagen, remains relatively stable. One study
(Costa et al. , 1983) observed a small decrease in collagen levels of rats at
o
392 and 1568 (jg/m (0.2 and 0.8 ppm) 03 after an intermittent exposure for 62
days.
The effects of 0., on increasing collagen content may be progressive;
i.e., after a 6-week intermittent exposure of rats to 0.64 or 0.96 ppm 03
ceased, collagen levels 6 week post-exposure were elevated over the levels
immediately after exposure (Last et al., 1984b). Also, there appears to be
little difference between continuous and intermittent exposure in increasing
collagen levels in rat lungs (Last et al, 1984b). Thus, the intermittent clean
air periods were not sufficient to permit recovery.
Although the ability of 03 to initiate peroxidation of unsaturated fatty
acids in vitro is well established, few in vivo studies of lung lipids have
been conducted. Generally, ozone decreases unsaturated fatty acid content of
the lungs (Roehm et al., 1972) and decreases incorporation of fatty acids into
lecithin (a saturated fatty acid) (Kyei-Aboagye et al., 1973). These altera-
tions, however, apparently do not alter the surface-tension-lowering properties
of lung lipids that are important to breathing (Gardner et al., 1971; Huber
et al., 1971).
One of the earliest demonstrated effects of ozone was that very high
concentrations caused mortality as a result of pulmonary edema. As more
o
sensitive techniques were developed, lower levels (510 ug/m , 0.26 ppm) were
observed to increase the protein content of the lung (Hu et al., 1982). Since
some of the excess protein could be attributed to serum proteins, the interpre-
tation was that edema had occurred. This effect was more pronounced several
hours after exposure ceased. At higher concentrations, a loss of carrier-
mediated transport from the air side of the lung to the blood side was observed
(Williams et al., 1980). These changes imply an effect on the barrier function
of the lung, which regulates fluxes of various substances with potential
physiological activities across the alveolar walls.
The biochemical effects observed in experimental animals exposed to 03
are summarized in Figure 9-7 and Table 9-31 (see Section 9.6.1 for criteria
used in developing this summary).
9.6.3.4 Host Defense Mechanisms. Reports over the years have presented
substantial evidence that exposure to ozone impairs the antibacterial activity
of the lung, resulting in an impairment of the lung's ability to kill inhaled
9-235
-------
NJ
U)
CTl
a
a
«
o
0
U
o
U
i
O
XX*
0 0
0.1-
0,2-
0.3-
0.4-
0.5-
0.6-
0.7-
0.8-
0.9-
1.0
c
i
<
i
i
<
3
i C
i
i
i <
(
i 1
*
> C
1
(
5 <
1
i
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1
5 <
<
l
\ \
<
i
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1
C
3
i
3
1 1
) l
i
1
1
3 0
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1
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l t
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t
Figure 9-7. Summary of biochemical changes in experimental animals
exposed to ozone. See Table 9-31 for reference citations of studies
summarized here.
-------
TABLE 9-31. SUMMARY TABLE: BIOCHEMICAL CHANGES
IN EXPERIMENTAL ANIMALS EXPOSED TO OZONE
Effect/response
concentration, ppm
References
Increased 02
consumption
Increased lysosomal
enzyme activities
Increased lung
hydroxyproline
and prolyl
hydroxylase
activity
Altered mucus
glycoprotein
secretions
Increased alveolar
protein and
permeability
changes
Increased LDH
activity
Increased NADPH
cytochrome c
reductase
activity
Increased GSH
metabolism
[0.1], 0.2
[0.1], 0.2, 0.35, 0.5, 0.8
0.2, 0.5, 0.8
0.2, 0.5, 0.8
0.45
0.8
0.8
[0.2], [0.5], 0.8
0.7, 0.8
0.7, 0.8
[0.2], 0.5, 0.8
0.2, 0.8
0.45, 0.8
0.5, 0.64, 0.96
0.5
0.8
[0.2], [0.4], 0.5, 0.6, 0.8
0.5, 0.6, 0.8
0.6, 0.8
[0.1], 0.26, 0.51, 1.0
[0.25], 0.5, 1.0
0.6, 1.0
1.0
[0.1]
[0.5], 0.8
0.8
0.2, 0.35, 0.8
0.2, 0.5, 0.8
0.2, 0.5, 0.8
[0.1]
0.1, 0.2
0.2, 0.35, 0.5,
0.2, 0.5, 0.8
0.2, 0.5, 0.8
0.2, 0.5, 0.8
0.2, 0.5, 1.0
0.32
0.45
0.5
0.8
Mustafa (1975)
Mustafa and Lee (1976)
Mustafa et al. (1973)
Schwartz et al. (1976)
Mustafa et al. (1982)
Chow et al. (1976b)
Elsayed et al. (1982a)
Chow et al. (1974)
Oil lard et al. (1972)
Castleman et al. (1973a,b)
Hussain et al. (1976a,b)
Costa et al. (1983)
Bhatnagar et al. (1983)
Last et al. (1979, 1984b)
Last and Greenberg (1980)
Hesterberg and Last (1981)
Last and Kaizu (1980)
Last and Cross (1978)
Last et al. (1977)
Hu et al. (1982)
Alpert et al. (1971a)
Williams et al. (1980)
Reasor et al. (1979)
Chow et al. (1981)
Chow et al. (1977)
Chow and Tappel (1973)
Mustafa and Lee (1976)
Schwartz et al. (1976)
DeLucia et al. (1972, 1975a,b)
Chow et al. (1981)
Plopper et al. (1979)
Mustafa and Lee (1976)
Chow et al. (1974)
DeLucia et al. (1972, 1975a,b)
Schwartz et al. (1976)
Fukase et al. (1975)
Moore et "al. (1980)
Mustafa et al. (1982)
Chow et al. (1975)
9-237
-------
TABLE 9-31. SUMMARY TABLE: BIOCHEMICAL CHANGES
IN EXPERIMENTAL ANIMALS EXPOSED TO OZONE (continued)
Effect/response
03 concentration, ppm
References
0.5, 1.0
0.7, 0.75, 0.8
Fukase et al. (1978)
Chow and Tappel (1972, 1973)
Increased NPSH
Decreased
unsaturated
fatty acids
0.8
0.8
0.9
0.9
0.1, 0.2
0.2, 0.5, 0.8
0.45
0.8
0.5-
Elsayed et al. (1982a,b;
1983)
Chow et al. (1976b)
Tyson et al. (1982)
Lunan et al. (1977)
Plopper et'al. (1979)
DeLucia et al . (1975b)
Mustafa et al. (1982)
Chow et al. (19765)
Roehm et al . , 1972
microorganisms. Suppression of this biocidal defense of the lung can lead to
microbial proliferation within the lung, resulting in mortality. The mortality
response is concentration-related and is significant at concentrations as low
as 157 to 196 |jg/m3 (0.08 to 0.1 ppm) (Coffin et al., 1967; Ehrlich et al.,
1977; Miller et al., 1978a; Aranyi et al., 1983). The biological basis for
this response appears to be that ozone or one of its reactive products can
impair or suppress the normal bactericidal functions of the pulmonary defenses,
which results in prolonging the life of the infectious agent, permitting its
multiplication and ultimately, in this animal model, resulting in death. Such
infections can occur because of 0, effects on a complex host defense .system
involving alveolar macrophage functioning, lung fluids, and other immune
factors.
The data obtained in various experimental animal studies indicate that
short-terra ozone exposure can reduce the effectiveness of several vital defense
systems including (1) the ability of the lung to inactivate bacteria and
viruses (Coffin et al., 1968; Coffin and Gardner, 1972b; Goldstein et al.,
1974a, 1977; Warshauer et §_!,._,. 1974j_Bergers et alj; 1983. Schwartz and Christman,
1979; Ehrlich et al., 1979); (2) the mucociliary transport system (Phalen
et al., 1980; Frager et al., 1979; Kenoyer et al., 1981; (3) the immunological
system (Campbell and Hilsenroth, 1976; Fujimaki et al., 1984; Thomas et al.,
9-238
-------
1981b; Aranyi et al. , 1983; and (4) the pulmonary macrophage (Dowel! et al. ,
1970; Goldstein et al., 1971a,b, and 1977; Hadley et al., 1977; McAllen et al.,
1981;-Witz-et al., 1983; Hurst et al,, 1570; Hurst and Coffin, 1971; Amoruso
et al., 1981). Studies have also indicated that the activity level of the
test subject and the presence of other airborne chemicals are important vari-
ables that can influence the determination of the lowest effective concen-
tration of the pollutant (Gardner et al., 1977; Aranyi et al., 1983; Ehrlich,
1980, 1983; Grose et al., 1980, 1982; Phalen et al.," 1980; Goldstein et al. ,
1974a; Illing et al. , 1980).
Ciliated cells are damaged by 0™ inhalation, as demonstrated by major
morphological changes in these cells, including necrosis and sloughing, or by
the shortening of the cilia in cells attached to the bronchi. Sufficient
ciliated cell damage should result in decreased transport of viable and non-
viable particles from the lung. Rats exposed to 784, 1568, 1960, or 2352
3
ug/m (0.4, 0.8, 1.0, or 1.2 ppm) for times as short as 4 hr have decreased
short-term clearance of particles from the lung (Phalen et al., 1980; Frager
et al. , 1979; Kenoyer et al., 1981). Short-term clearance is mostly due to
mucus transport of particles, and the decreased short-term clearance is an
anticipated functional result predicted from morphological observations. The
mucous glycoprotein production of the trachea is also altered by 0- exposure.
Mucous glycoprotein biosynthesis, as measured ex vivo in cultured trachea!
explants from exposed rats, was inhibited by short-term continuous exposure to
1568 ug/m3 (0.8 ppm) of 03 for 3 to 5 days (Last and Cross, 1978; Last and
Kaizu, 1980; Last et a!., 1977). Glycoprotein synthesis and secretion recovered
to control values after 5 to 10 days of exposure and increased to greater than
control values after 10 days of exposure. With this increase in production of
mucus, investigators have found that the velocity of the trachea! mucus was
3
significantly reduced following a 2 hr exposure to 1960 M9/m d-0 PPm) (Abraham
at al., 1980).
A problem remains in assessing the relevance of these animal data to
humans. Green (1984) reviewed the literature and compared the host antibacterial
defense systems of the rodent and man and found that these two species had
defenses that are very similar and thus provide a good basis for a qualitative
extrapolation. Both defenses consist of an aerodynamic filtration system, a
fluid layer lining the respiratory membranes, a transport mechanism for "removing
foreign particles, microorganisms, and pulmonary cells, and immune secretions
9-239
-------
of lymphocytes and plasma cells. In both rodents and humans, these components
act in concert to maintain the lung free of bacteria.
If the animal models are to be used to reflect the toxicological response
occurring in humans, then the endpoint for comparison of such studies should
be morbidity rather than mortality. A better index of 0- effect in humans
might be the increased prevalence of infectious respiratory illness in the
community. Such a comparison may be proper since both mortality from respira-
tory infections (animals) and morbidity from respiratory infections (humans)
can result from a loss in pulmonary defenses (Gardner, 1984). Whether the
microorganisms used in the various animal studies are comparable to the organ-
isms responsible for the respiratory infections in a community still requires
further investigation.
Ideally, studies of pulmonary host defenses should be performed in man,
using epidemiological or volunteer methods of study. Unfortunately, such
studies have not been reported yet. Attention must therefore be paid to the
results of host-defense experiments conducted with animals.
In the area of host defense of the lung against infection, present know-
ledge of the physiology, metabolism, and function have come primarily from the
study of various animal systems, but it is generally accepted that the basic
mechanisms of action of these defense cells and systems function similarly in
both animals and man. There are also human data to support this statement,
especially in such areas as immunosuppression, ciliostasis, and alveolar
macrophages. The effects seen in animals represent alterations in basic
biological systems. One can assume that similar alterations in basic defense
mechanisms could occur in humans since they possess equivalent pulmonary
defense systems. It is understood, however, that different exposure levels
may be required to produce similar responses in humans. The concentration of
0, at which effects become evident can be influenced by a number of factors,
such as preexisting disease, virulence of the infectious agent, dietary factors,
concurrent exposure to other pollutants, exercise, or the presence of other
environmental stresses, or a combination of these. Thus, one could hypothesize
that humans exposed to CU could experience effects on host defense mechanisms.
At the present time, however, one cannot predict the exact concentration at
which effects may occur in man nor the severity of the effects.
The effects of 0- on host defense mechanisms in experimental animals are
summarized in Figure 9-8 and Table 9-32 (see Section 9.6.1 for criteria used
in developing this summary).
9-240
-------
*'
ID
a
a
o
o
i
01
o
N
O
/v
<&?
fP ^ (
<
(
i
i i
** »*>'
X
I
1
1 j
I
1
1 ,
AW
s
\
1
1 1
<
1
t 1 1
>'
•
(
1 1
<
1 <
i
\
1 <
!
IIT nl ii 1 t
X''
(
(
1 (
)
<
1
(
1
\
(
<
1
$
(
1 ., 1
^5^
•
•
i <
>
•
i
<
*
*
i
4
1
»xs
1
1
1
t
J?
Figure 9-8. Summary of effects of oione on host defense mechanisms
in experimental animals. See Table 9-32 for reference citations of
studies summarized here.
-------
TABLE 9-32. SUMMARY TABLE: EFFECTS OF OZONE ON HOST DEFENSE
MECHANISMS IN EXPERIMENTAL ANIMALS
Effect/response
03 concentration, ppm
References
Delayed mucociliary
clearance; accelerated
alveolar clearance,
ciliary beating
frequency
Inhibited bactericidal
activity
Altered macrophage
membrane
Decreased macrophage
function
Altered no. of defense
cells
Increased suscepti-
bility to infection
[0.1]
0.4, 0.8, 1.0
[0.5]
[0.5], 1.0
0.8
1.2
0.4
0.4
0.5
0.62
0.
0.
0.99
0.1, 1.0
0.5
0.5
0.5, 1.0
0.25, 0.5
0.5
0.5, 0.67
0.5, 0.67
0.8
1.0
1.0
0.35,
0.35
0.2
0.2,
0.2,
0.2, 0.5,
0.25
0.5
0.5, 0.88
0.5
0.5, 0.88
0.5, 0.8
0.54, 0.88
0.8
1.0
1.0
0.08
0.08, 0.1
0.1
0.5, 0.8
0.8
Grose et al. (1980)
Kenoyer et al. (1981)
Friberg et al. (1972)
Abraham et al. (1980)
Phalen et al. (1980)
Frager et al. (1979)
Coffin and Gardner (1972b)
Goldstein et al. (1972b)
Friberg et al. (1972)
Goldstein et al. (1971b)
Bergers et al. (1983)
Warshauer et al. (1974)
Goldstein et al. (1971a)
Gardner et al. (1971)
Dowel! et al. (1970)
Hadley et al. (1977)
Goldstein et al. (1977)
Hurst et al. (1970)
Hurst and Coffin (1971)
Alpert et al. (197Jb)
Coffin et al. (1968)
Coffin and Gardner (1972b)
Schwartz and Christman (1979)
Shingu et al. (1980)
McAllen et al. (1981)
Plopper et al. (1979)
Dungworth et al. (1975b)
Castleman et al. (1977)
Boorman et al. (1977, 1980)
Barry et al. (1983)
Zltnik et al. (1978)
Stephens et al. (1974a)
Last et al. (1979)
Brummer et al. (1977)
Eustis et al. (1981)
Freeman et al. (1974)
Castleman et al. (1980)
Freeman et al. (1973)
Cavender et al. (1977)
Coffin et al. (1967)
Miller et al. (1978a)
Ehrlich et al. (1977)
9-242
-------
TABLE 9-32. SUMMARY TABLE: EFFECTS OF OZONE ON HOST DEFENSE
MECHANISMS IN EXPERIMENTAL ANIMALS (continued)
Effect/response
Increased suscepti-
bility (cont'd)
Altered immune
activity
03 concentration, ppm
0.1
0.1, 0.3
[0.2], 0.4, 0.7
0.3
0.5
[0.64]
0.7, 0.9
1.0
0.1
0.5, 0.8
0.5, 0.8
0.59
0.8
References
Aranyi et al . (1983)
111 ing et al. (1980)
Bergers et al. (1983)
Abraham et al . (1982)
Wolcott et al. (1982)
[Sherwood et al. (1984)]
Coffin and Blommer (1970)
Thomas et al. (1981b)
Aranyi et al . (1983)
Osebold et al. (1979, 1980)
Gershwin et al. (1981)
Campbell and Hilsenroth
(1976)
Fujimaki et al. (1984)
9.6.3.5 Tolerance. Examination of responses to short-term, repeated exposures
to 0, clearly indicates that with some of the parameters measured, animals
have an increased capacity to resist the effects of subsequent exposure. This
tolerance persists for varying times, depending on the degree of development
of the tolerance. Previous exposure to low concentrations of 0, will protect
against the effects of subsequent exposure to lethal doses and the development
of lung edema (Stokinger et al., 1956; Fairchild, 1967; Coffin and Gardner-,
1972a; Chow, 1984). The prolongation of mucociliary clearance reported for 03
can also be eliminated by pre-exposure to a lower concentration (Frager et
al., 1979). This effect is demonstrated for a short period of time and is
lost as soon as the mucus secretion rate returns to normal. However, not all
of the toxic effects of 0~, such as reduced functioning activity of the pulmonary
defense system (Gardner et al., 1972); hyperplasia of the type 2 cells (Evans
et al., 1971, 1976a,b); increased susceptibility to respiratory disease (Gardner
and Graham, 1977); loss of pulmonary enzymatic activity (Chow, 1976, Chow
et al., 1976b); and inflammatory response (Gardner et al., 1972) can be totally
prevented by prior treatment with low levels of 0,,. Dungworth et al. (1975b)
and Castleman et al. (1980) have attempted to explain tolerance by careful
examination of the morphological changes that occur with repeated 03 exposures.
These investigators suggest that during continuous exposure to 0., the injured
9-243
-------
cells attempt to initiate early repair of the specific lesion. The repair
phase results in a reduction of the effect first observed but lasts only for a
short time since,the recovered cells are as sensitive to re-exposure to 0- as
the pre-exposed counterpart (Plopper et a!., 1978). This information is an
important observation because it implies that the decrease in susceptibility
to 0, persists only as long as the exposure to 0, continues. The biochemical
studies of Chow et al. (1976b) support this conclusion.
At this time, there are a number of hypotheses proposed to explain the
mechanism of this phenomenon (Mustafa and Tierney, 1978; Schwartz et al.,
1976; Mustafa et al., 1977; Berliner et al., 1978; Gertner et al., 1983b;
Bhatnagar et al., 1983). Evidence by Nambu and Yokoyama (1983) indicates that
although the pulmonary antioxidant system (glutathione peroxidase, glutathione
reductase, and glucose-6-phosphate dehydrogenase) may play an active role in
defending the lung against ozone, it does not explain the mechanism of toler-
ance in that the development of tolerance does not coincide with the described
biochemical enhancement of the antioxidant system in the lungs of rats.
From this literature, it would appear that tolerance, as seen in animals,
may not be the result of any one single biological process, but instead may
result from a number of different events, depending on the specific response
measured. Tolerance does not imply complete or absolute protection, because
continuing injury does still occur, which could potentially lead to nonrever-
sible pulmonary changes.
Tolerance may not be long-lasting. During 0- exposure, the increase in
antioxidant metabolism reaches a plateau and recovery occurs a few days after
exposure ceases. Upon re-exposure, effects observed are similar to those that
occurred during the primary exposure (Chow et al., 1976b).
9.6.4 Extrapulmonary Effects of Ozone
It is still believed that CL, on contact with respiratory system tissue,
immediately reacts and thus is not absorbed or transported to extrapulmonary
sites to any significant degree. However, several studies suggest that possibly
products formed by the interaction of 0- and respiratory system fluids or
tissue can produce effects in lymphocytes, erythrocytes, and serum, as well as
in the parathyroid gland, the heart, the liver, and the CNS. Ozone exposure
also produces effects on animal behavior that may be caused by pulmonary
consequences of Q~, or by nonpulmonary (CNS) mechanisms. The mechanism by
9-244
-------
which 0, causes extrapulmonary changes is unknown. Mathematical models of 0,
dosimetry predict that very little 03 penetrates to the blood of the alveolar
capillaries. Whether these effects result from 0- or a reaction product of 0-,
which penetrates to the blood and is transported is the subject of speculation.
9.6.4.1 Central Nervous System and Behavioral Effects. Ozone significantly
affects the behavior of rats during exposure to concentrations as low as
3
235 |jg/m (0.12 ppm) for 6 hr. With increasing concentrations of 0,, further
decreases in unspecified motor activity and in operant learned behaviors have
been observed (Konigsberg and Bachman, 1970; Tepper et a!., 1982; Murphy
et al . , 1964; and Weiss et a!., 1981). Tolerance to the observed decrease in
motor activity may occur on repeated exposure. At low 0, exposure concentra-
3
tions (490 pg/m , 0.25 ppm), an increase in activity is observed after exposure
q
ends. Higher 0_ concentrations (980 ug/m , 0.5 ppm) produce a decrease in
rodent activity that persists for several hours after the end of exposure
(Tepper et al. , 1982, 1983).
The mechanism by which behavioral performance is reduced is unknown.
Physically active responses appear to- enhance the effects of Q3, although this
may be the result of an enhanced minute volume that increases the effective
concentration delivered to the lung. Several reports indicate that it is
unlikely that, animals have reduced physiological capacity to respond, prompt-
ing Weiss et al. (1981) to suggest that 03 impairs the inclination to respond.
Two studies indicate that mice will respond to remove themselves from an
atmosphere containing greater than 980 |jg/m (0.5 ppm) (Peterson and Andrews,
1963, Tepper et al., 1983). These studies suggest that the aversive effects
of 03 may be due to lung irritation. It is unknown whether lung .irritation,
odor, or a direct effect on the CNS causes change in rodent behavior at lower
03 concentrations.
9.6.4.2 Cardiovascular Effects. Studies on the effects of 03 on the cardio-
vascular system are few, and to date there are no reports of attempts to con-
firm these studies. The exposure of rats to 0, alone or in combination with ,
3
cadmium (1176 |jg/m , 0.6 ppm 03) resulted in measurable increases in systolic
pressure and heart rate (Revis et al., 1981). No additive or antagonistic
response was observed with the combined exposure. Pulmonary capillary blood
3
flow and PaO decreased 30 rnin following exposure of dogs to 588 MS/I" (°-3
ppm) of 0-j (Friedman et al., 1983). The decrease in pulmonary capillary blood
flow persisted for as long as 24 hr following exposure.
9-245
-------
9.6.4.3 Hematological and Serum Chemistry Effects. The data base for the
effects of 0^ on the hematological system Is extensive and indicates that 0.,
or one of its reactive products can cross the blood-gas barrier, causing
changes in the circulating erythrocytes (RBC) as well as significant differ-
ences in various components of the serum.
Effects of On on the circulating RBCs can be readily identified by exa-
mining either morphological and/or biochemical endpoints. These cells are
structually and metabolically well understood and are available through rela-
tively non-invasive methods, which makes them ideal candidates for both human
and animal studies. A wide range of structural effects have been reported in
a variety of species of animals, including an increase in the fragility of
3
RBCs isolated from monkeys exposed to 1470 ug/m (0.75 ppm) of 03 4 hr/day for
4 days (Clark et a!., 1978). A single 4-hr exposure to 392 M9/m3 (0.2 ppm)
also caused increased fragility as well as sphering of RBCs of rabbits (Brinkman
et a!., 1964). An increase in the number of RBCs with Heinz bodies was detected
3
following a 4-hr exposure to 1666 pg/m (0.85 ppm). The presence of such
inclusion bodies in RBCs is an indication of oxidant stress (Menzel et a!.,
1975a).
These morphological changes are frequently accompanied by a wide range of
2
biochemical effects. RBCs of monkeys exposed to 1470 |jg/m (0.75 ppm) of 0,
for 4 days also had a decreased level of glutathione (GSH) and decreased
acetylcholinesterase (AChE) activity, an enzyme bound to the RBC membranes.
The RBC GSH activity remained significantly lower 4 days postexposure (Clark
et al,, 1978).
Animals deficient in vitamin E are more sensitive to 0-. The RBCs from
these animals, after being exposed to 0,, had a significant increase in the
activity of GSH peroxidase, pyruvate kinase, and lactic dehydrogenase, but had
3
a decrease in RBC GSH after exposure to 1568 pg/m (0.8 ppm) for 7 days (Chow
and Kaneko, 1979). Animals with a vitamin E-supplemented diet did not have
any changes in glucose-6-phosphate dehydrogenase (G-6-PD), superoxide dismutase,
3
or catalase activities. At a lower level (980 |jg/m , 0.5 ppm), there were no
changes in GSH level or in the activities of GSH peroxidase or GSH reductase
(Chow et al., 1975). Menzel et al. (1972) also reported a significant increase
in lysis of RBCs from vitamin E-deficient animals after 23 days of exposure to
3
980 ug/m (0.5 ppm). These effects were not observed in vitamin E-supplemented
rats. Mice on a vitamin E-supplemented diet and those on a deficient diet
9-246
-------
3
showed an increase in G-6-PD activity after an exposure of 627 ijg/m (0.32 ppm)
of Q~ for 6 hr. Decreases observed in ACHE activity occurred in both groups
(Moore et al., 1980).
Other blood changes are attributed to 0-. Rabbits exposed for 1 hr to
3
392 ug/m (0.2 ppm) of 0« showed a significant drop in total blood serotonin
3
(Veninga, 1967). Six- and 10-month exposures of rabbits to 784 pg/m (0.4 ppm)
of 0~ produced an increase in serum protein esterase and in serum trypsin
inhibitor. This latter effect may be a result of thickening of the small
pulmonary arteries. The same exposure caused a significant decrease in albumin
levels and an increase in alpha and gamma globulins (P'an and Jegier, 1971,
1976; P'an et al., 1972; Jegier, 197-3). Chow et al. (1974) reported that the
serum lysozyme level of rats increased significantly after 3 days of continuous
exposure to 0., but was not affected when the exposure was intermittent (8 hr/day,
3
7 days). The 03 concentration in both studies was 1568 yg/m (0.8 ppm) of Q-.
Short-term exposure to low concentrations of 03 induced an immediate
change in the serum creatine phosphokinase level in mice. In this study, the
DO doses were expressed as the product of concentration and time. The C x T
value for this effect ranged from 0.4 to 4.0 (Veninga et al., 1981).
A few of the hematological effects observed in animals (i.e., decrease in
GSH and AChE activity and the formation of Heinz bodies) following exposure to
03 have also been seen following j_n vitro exposure of RBCs from humans (Freeman
and Mudd, 1981; Menzel et al., 1975b; Verweij and Van Steveninck, 1981). A
common effect observed by a number of investigators is that 03 inhibits the
membrane ATPase activity of RBCs (Koontz and Heath, 1979; Kesner et al., 1979;
Kindya and Chan, 1976; Freeman et al., 1979; Verweij and Van Steveninck,
1980). It has been postulated that this inhibition of ATPase could be related
to the spherocytosis :and increased fragility of RBCs seen in animal and human
cells.
Although these i_n vitro data are useful in studying mechanisms of action,
it is difficult to extrapolate these data to any effects observed in man. Not
only is the method of exposure not physiological, but the actual concentration
of 0, reaching the RBC cannot be determined with any accuracy.
9.6.4.4 Cytogenetic and Teratogenic Effects. Uncertainty still exists regard-
ing possible reproductive, teratogenic, and mutational effects of exposure to
ozone. Based on various rn vitro data, a number of chromosomal effects of
ozone have been described for isolated cultured cell lines, human lymphocytes,
9-247
-------
and microorganisms (Fetner, 1962; Hamelin et al., 1977a,b, Hamelin and Chung,
1975a»b, 1978; Scott and Lesher, 1963; Erdman and Hernandez, 1982; Guerrero
et a!., 1979; Dubeau and Chung, 1979, 1982), The interpretation, relevance,
and predictive values of such studies to human health are questionable since
(1) the concentrations used were many-fold greater than what is found in the
ambient air (see Chapter 10); (2) extrapolation of i_n vitro exposure concen-
trations to human exposure dose is not yet possible; and (3) direct exposure
of isolated cells to ozone is highly artifactual since it bypasses all the
defenses of the host that would normally be functioning in protecting the
individual from the inhaled gas. Furthermore, the direct exposure of isolated
cells jm vitro to ozone may result in chemical reactions between ozone and
culture media that might not occur jjn vivo.
Important questions still exist regarding ijn vjvo cytogenetic effects of
ozone in rodents and humans. Zelac et al. (1971a,b) reported chromosomal
abnormalities in peripheral leukocytes of hamsters exposed to 0, (0.2 ppm).
Combined exposures to ozone and radiation (227-233 rads) produced an additive
effect on the number of chromosome breaks in peripheral leukocytes. These
specific findings were not confirmed by Gooch et al. (1976) or by Tice et al.
(1978), but sufficient differences in the various experimental protocols make
a direct comparison difficult. The latter group did report significant increases
in the number of chromatid deletions and achromatic lesions resulting from
exposure to 0.43 ppm ozone.
Because the volume of air inspired during pregnancy is significantly
enhanced, the pregnant animal may be at greater risk to low levels of ozone
exposure. Early studies on the possible teratogenic effects of ozone have
suggested that exposures as low as 0.2 ppm can reduce infant survival rate and
cause unlimited incisor growth (Brinkman et al,, 1964; Veninga, 1967). Kavlock
et al. (1979, 1980) found that pregnant rats exposed to 1.0 and 1.49 ppm ozone
showed a significant increase in embryo resorption rate, slower growth, slower
development of righting reflexes, and delayed grooming and rearing behavior,
but no increase in neonatal mortality was observed.
9.6.4.5 Other Extrapulmonary Effects. A series of studies was conducted to
show that 03 increases drug-induced sleeping time in a number of species of
animals (Gardner et al., 1974; Graham, 1979; Graham et al., 1981, 1982a,b,
3
1983, 1985). At 1960 pg/m (1.0 ppm), effects were observed after 1, 2, and 3
days of exposure. As the concentration of Q., was reduced, increasing numbers
of daily 3-hr exposures were required to produce a significant effect. At the
9-248
-------
3
lowest concentration studied (196 (jg/m , 0.1 ppm), the increase was observed
at days 15 and 16 of exposure. It appears that this effect is not specific to
the strain of mouse or to the three species of animals tested, but it is
sex-specific, with females being more susceptible. Recovery was complete
within 24 hr after exposure. Although a number of mechanistic studies have
been conducted, the reason for this effect on pentobarbital-induced sleeping
time is not known. It has been hypothesized that some common aspect related
to liver drug metabolism is quantitatively reduced (Graham et al., 1983).
Several investigators have attempted to elucidate the involvement of the
endocrine system in Og toxicity. Most of these studies were designed to
investigate the hypothesis that the -survival rate of mice and rats exposed to
lethal concentrations of 0™ could be increased by use of various thyroid
blocking agents or by thyroidectomy. To follow up these findings, demons and
Garcia (1980a,b) and demons and Wei (1984) investigated the effects of a
3
24-hr exposure to 1960 \ig/m (1.0 ppm) of 0, on the hypothalamo-pituitary-thyroid
system of rats. These three organs regulate the function of each other through
various hormonal feedback mechanisms. Ozone caused decreases in serum concen-
tration of thyroid stimulating hormone (TSH), in circulating thyroid hormones
(T- and T*) and in protein-bound iodine. No alterations were observed in many
other hormone levels measured. - Thyroidectomy prevented the effect of 0., on
TSH and T. and hypophysectomy prevented effects on T-, unless the animals were
supplemented with T. in their drinking water. The thyroid gland itself was
altered (e.g., edema)- by 0-. The authors hypothesyzed that 03 alters serum
binding of these hormones.
The extrapulmonary effects of ozone in experimental animals are summarized
in Figure 9-9 and Table 9-33. Criteria used in developing the summary were
presented in Section 9.6.1.
9.6.5 Interaction of Ozone With Other Pollutants.
Combined exposure studies in laboratory animals have produced varied
results, depending upon the pollutant combination evaluated and the measured
variables. Additive and/or possibly synergistic effects of 03 exposure in
combination with N0? have been described for increased susceptibility to
bacterial infection (Ehrlich et al., 1977, 1979; Ehrlich, 1980, 1983), morpho-
logical lesions (Freeman et al., 1974), and increased antioxidant metabolism
(Mustafa et al., 1984). Additive or possibly synergistic effects from exposure
9-249
-------
f
M
U1
P
,^e
•&* V
0.1 _
0.2-
E 0.3-
o.
a
c 0.4-
,2
**>
S
c O.S-
u
c
Q
8 0.6-
0
c
o
a 0-7-
0.8-
0.9_
1 n
I \ i
I
t
(
i
i
<
; :
• i
<
i
^
(
<
i «
i
<
i
<
<
i
i
i
i
• <
i <
<
i
i
, •
i
t
i
i
> 1
» *
•
» i
•
• ,
i
I
i
i
»
•
i
•> i «« — ~
Figure 9-9. Summary of extrapulmonary effects of ozone in
experimental animals. See Table 9-33 for reference citations of studies
summarized here.
-------
TABLE 9-33. SUMMARY TABLE: EXTRAPULMONARY EFFECTS OF OZONE
IN EXPERIMENTAL ANIMALS
Effect/response
03 concentration, ppm
References
CNS effects
Hematological effects
Chromosomal, reproduc-
tive, terato "logical
effects
Liver effects
Endocrine system
effects
0
0.5, 0.7
0.05, 0.5
0.1 - 1.0
0.12 -
0.2, 0.
0.5
0.5
0.5
0.6
1.0
1.0
0.06, 0.12, 0.48
0.2
0.2, 1.0
0.25, 0.32, 0.5
0.4
0.4
0.5
0.64
0.75
0.8
0.8
0.85
0.86
1.0
1.0
1.0
0.1
0.2
0.24, 0.3
0.43
0.44
1.0
0.1, 0.25, 0.5, 1.0
0.82
1.0
0.75
0.75
0.75
0.75
1.0
1.0
Tepper et al
Murphy et al
Tepper et al
Konigsberg and Bachman (1970)
Weiss et al. (1981)
(1982)
(1964)
(1983)
Reynolds and Chaffee (1970)
Xintaras et al. (1966)
Peterson and Andrews (1963)
Fletcher and Tappel (1973)
Trams et al. (1972)
Calabrese et al. (1983a)
Brinkman et al. (1964)
Veninga (1967, 1970)
Veninga et al. (1981)
Moore et al. (1980; 1981a,b)
Jegier (1973)
P'an and Jegier (1972, 1976)
Menzel et al. (1972)
Larkin et al. (1983)
Clark et al. (1978)
Chow and Kaneko (1979)
Chow et al. (1974)
Menzel et al. (1975a)
Schlipkoter and Bruch (1973)
Dorsey et al. (1983)
Mizoguchi et al. (1973)
Christiansen and Giese (1954)
Brinkman et al. (1964)
Veninga (1967)
Zelac et al. (1971a)
Tice et al. (1978)
Kavlock et al. (1979)
Kavlock et al. (1980)
Graham (1979)
Graham et al. (1981, 1982a,b)
Veninga et al. (1981)
Gardner et al. (1974)
Atwal and Wilson (1974)
Atwal et al. (1975)
Atwal and Pemsingh (1981, 1984)
Pemsingh and Atwal (1983)
demons and Garcia (1980a,b)
demons and Wei (1984)
9-251
-------
to DO and HpSO- have also been reported for host defense mechanisms (Gardner
et a!., 1977; Last and Cross, 1978; Grose et al., 1982), pulmonary sensitivity
(Osebold et al. 1980), and collagen synthesis (Last et al., 1983), but not
for morphology (Cavender et al., 1977; Moore and Schwartz, 1981). Mixtures of
03 and (NH-)2 SO- had synergistic effects on collagen synthesis and morphometry,
including percentage of fibroblasts (Last et al., 1983, 1984a).
Combining 03 with other particulate pollutants produces a variety of
responses, depending on the endpoint measured. Mixtures of 03, Fe2(SO,)3,
HpSCL, and (NH-^SO. produced the same-effect on clearance rate as exposure to
0~ alone. However, when measuring changes in host defenses, the combination
of GO with NOp and ZnSO- or 03 with S02 and (NH-^SO, produced enhanced effects
that can not be attributed to 03 only.
However, since these issues are complex, they must be addressed experi-
mentally using exposure regimens for combined pollutants that are more represen-
tative of ambient ratios of peak concentrations, frequency, duration, and time
intervals between events.
The interactive effects of 03 with other pollutants are summarized in
Figure 9-10 and Table 9-34.
9.6.6 Effects of Other Photochemical Oxidants
There have been far too few controlled toxicological studies with the
other oxidants to permit any sound scientific evaluation of their contribution
to the toxic action of photochemical oxidant mixtures. When the effects seen
after exposure to 03 and PAN are examined and compared, it is obvious that the
test animals must be exposed to concentrations of PAN much greater than those
needed with 03 to produce a similar effect on lethality, behavior modification,
morphology, or significant alterations in host pulmonary defense system (Campbell
et al., 1967; Dungworth et al., 1969; Thomas et al., 1979, 1981a). The concen-
trations of PAN required to produce these effects are many times greater than
what has been measured in the atmosphere (0.047 ppm).
Similarly, most of the investigations reporting H202 toxicity have involved
concentrations much higher than those found in the ambient air, or the investi-
gations were conducted by using various i_n vitro techniques for exposure. Very
limited information is available on the health significance of inhalation expo-
sure to gaseous Hp02. Because H202 is highly soluble, it is generally assumed
that it does not penetrate into the alveolar regions of the lung but is instead
9-252
-------
NJ
UI
U)
o
«
s
o
\pp
V.Vf —
0.1-
0.2-
0.3-
0.4-
0.5-
0.6-
0.7-
0.8-
O.S-
1.0,
c
c
(
(
(
1 1
)
) • • <
•
•
1 (
<
I
1 (
1
I 0
o
1 0
1
) O
1
Figure 9-10. Summary of effects in experimental animals exposed to
ozone combined with other pollutants. See Table 9-34 for reference
citations of studies summarized here.
-------
TABLE 9-34. SUMMARY TABLE: INTERACTION OF OZONE
WITH OTHER POLLUTANTS IN EXPERIMENTAL ANIMALS
Effect/response
Pollutant concentrations
References
Increased
pulmonary
lesions
Increased
pulmonary
sensitivity
Increased anti-
oxidant metabolism
and QZ consumption
Altered mucus
secretion
Increased collagen
synthesis
Increased
susceptibility to
respiratory
infections
[0.25 ppm 03
+2.5 ppm N02]
[0.5 ppm Q3
+ 1 mg/m3 H2S04]
[0.5 ppm 03
+ 10 mg/m3 H2S04
0.64, 0.96 ppm 03
+ 5 mg/m3 (NH4)2 S04
0.9 ppm 03
+0.9 ppm N02
1.2 ppm 03
+ 5 mg/m3 (NH4)2S04
0.5 ppm 03
+ 1 mg/m3 H2S04
0.45 ppm 03
+4.8 ppm N02
0.5 ppm 03
+1.1 mg/m3 H2S04
[0.5], [0.8], 1.5 ppm 03
+ 5 mg/m3 (NH4)2S04
0.5 ppm 03
+ 1 mg/m3 H2S04
0.64, 0.96 ppm 03
+ 5 mg/m3 (NH4)2S04
0.05 ppm 03
+ 3760 pg/m3 (NH4)2S04
0.05 ppm 03
+ 100-400 Mg/m3 N02
+ 1.5 mg/m3 ZnS04
0.1 ppm 03
+0.9 mg/m3 H2S04
(sequential exposure)
0.1 ppm 03
+4.8 mg/m3 H2S04
0.1 ppm 03
+ 940 M9/m3 N02
0.1 ppm 03
+13.2 mg/m3 S02
+1.0 mg/m3 (NH4)2S04
Freeman et al. (1974)
Moore and Schwartz (1981)
Cavender et al. (1978)
Last et al. (1984a)
Freeman et al. (1974)
Last et al. (1983)
Osebold et al. (1980)
Mustafa et al. (1984)
Last and Cross (1978);
Last and Kaizu (1980)
Last et al. (1983)
Last et al. (1983)
Last et al. (1984a)
Ehrlich et al. (1977, 1979);
Ehrlich (1980)
Ehrlich et al. (1983)
Gardner et al. (1977)
Grose et al. (1982)
Ehrlich (1980)
Aranyi et al. (1983)
9-254
-------
TABLE 9-34. SUMMARY TABLE: INTERACTION OF OZONE
WITH OTHER POLLUTANTS (continued)
Effect/response Pollutant concentrations References
Altered upper [0.1 ppm 03 Grose et al. (1980)
respiratory +1.1 mg/m3 H2S04]
clearance (sequential exposure)
mechanisms 0.4 ppm 03 Goldstein et al. (1974b)
+7.0 ppm N02
0.5 ppm 03 Last and Cross (1978)
+ 3 mg/m3 H2S04
[0.8 ppm 03 Phalen et al. (1980)
+3.5 mg/m3
(Fe2(S04)3
+ H2S04
+ (NH4)2S04}]
deposited on the surface of the upper airways (Last et al., 1982). Unfortu-
nately, there have not been studies designed to look for possible effects in
this region of the respiratory tract.
A few i_n vitro studies have reported cytotoxic, genotoxic, and biochemical
effects of H202 when using isolated cells or organs (Stewart et al., 1981;
Bradley et al., 1979; Bradley and Erickson, 1981; Speit et al. , 1982; MacRae
and Stich, 1979). Although these studies can provide useful data for studying
possible mechanisms of action, it is not yet possible to extrapolate these
responses to those that might occur in the mammalian system.
Field and epidemiological studies have shown that human health effects
from exposure to ambient mixtures of oxidants and other airborne pollutants
can produce human health effects (Chapter 11). Few such studies have been
conducted with laboratory animals, because testing and measuring of such
mixtures is not only complicated, but extremely costly. In these studies, the
investigators attempted to simulate the photochemical reaction products pro-
duced under natural conditions and to define the cause-effect relationship.
Exposure to complex mixtures of oxidants plus the various components
found in UV-irradiated auto exhaust indicates that certain effects, such as
histopathological changes, increase in susceptibility to infection, a variety
of altered pulmonary functional activities were observed in this oxidant
atmosphere which was not reported in the nonirradiated exhaust (Murphy et al.,
9-255
-------
1963; Murphy, 1964; Nakajima et a"L, 1972; Hueter et al., 1966). Certain
other biological responses were observed in both treatment groups, including a
decrease in spontaneous activity, a decrease in infant survival rate, fertil-
ity, and certain pulmonary functional abnormalities (Hueter et al., 1966;
Boche and Quilligan, 1960; Lewis et al., 1967).
Dogs exposed to UV-irradiated auto exhaust containing oxidants either
with or without SO showed significant pulmonary functional abnormalities that
f{
had relatively good correlation with structural changes (Hyde et al., 1978;
Gillespie, 1980; Lewis et al., 1974). There were no significant differences
in the magnitude of the response in these two treatment groups, indicating
that oxidant gases and SO did not interact in any synergistic or additive
A
manner.
9-256
-------
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APPENDIX A: GLOSSARY OF PULMONARY TERMS AND SYMBOLS*
Acetylcholine (ACh): A naturally occurring substance in the body having
important parasympathetic effects; often used as a bronchoconstrictor.
Aerosol: Solid particles or liquid droplets that are dispersed or suspended
in a gas, ranging in size from 10 to 10 micrometers (urn).
Air spaces: All alveolar ducts, alveolar sacs, and alveoli. To be contrasted
with AIRWAYS.
Airway conductance (Gaw): Reciprocal of airway resistance. Gaw = (I/Raw).
Airway resistance (Raw): The (frictional) 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.
Allergen: A material that, as a result of coming into contact with appropriate
tissues of an animal body, induces a state of allergy or hypersensitivity;
generally associated with idiosyncratic hypersensitivities.
Alveolar-arterial oxygen pressure difference [P(A-a)02]: The difference in
partial pressure of 02 in the alveolar gas spaces and that in the systemic
arterial blood, measured in torr.
Alveolar-capillary membrane: A fine membrane (0.2 to 0.4 pm) separating
alveolus from capillary; composed of epithelial cells lining the alveolus,
a thin layer of connective tissue, and a layer of capillary endothelial
cells.
Alveolar carbon dioxide pressure (P/fiQ?)' 'Partial. pressure of carbon dioxide
in the air contained in the lung alveoli.
Alveolar oxygen partial pressure (P/vOo^ Partial pressure of oxygen in the
air contained in the alveoli or tne lungs.
Alveolar septum (pi. septa): A thin tissue partition between two adjacent
pulmonary alveoli, consisting of a close-meshed capillary network and
interstitium covered on both surfaces by alveolar epithelial cells.
^References: Bartels, H.; Dejours, P.; Kellogg, R. H.; Mead, J. (1973) Glossary
on respiration and gas exchange. J. Appl. Physio!. 34: 549-558.
American College of Chest Physicians - American Thoracic Society
(1975) Pulmonary terms and symbols: a report of the ACCP-ATS
Joint Committee on pulmonary nomenclature. Chest 67: 583-593.
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Alveolitis: (interstitial pneumonia): Inflammation of the lung distal to the
terminal non-respiratory bronchiole. Unless otherwise indicated, it is
assumed that the condition is diffuse. Arbitrarily, the term is not used
to refer to exudate in air spaces resulting from bacterial infection of
the lung.
Alveolus: Hexagonal or spherical air cells of the lungs. The majority of
alveoli arise from the alveolar ducts which are lined with the alveoli.
An alveolus is an ultimate respiratory unit where the gas exchange takes
place.
Anatomical dead space (V~ .): Volume of the conducting airways down to the
level where, during^ airbreathing, gas exchange with blood can occur, a
region probably situated at the entrance of the alveolar ducts.
Arterial oxygen saturation (Sa02): Percent saturation of dissolved oxygen in
arterial blood.
Arterial partial pressure of carbon dioxide (PaCOp): Partial pressure of
dissolved carbon dioxide in arterial blood.
Arterial partial pressure of oxygen (PaOp): Partial pressure of dissolved
oxygen in arterial blood.
Asthma: A disease characterized by an increased responsiveness of the airways
to various stimuli and manifested by slowing of forced expiration which
changes in severity 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 its duration.
Atelectasis: State of collapse of air spaces with elimination of the gas
phase.
ATPS condition (ATPS): Ambient temperature and pressure, saturated with water
vapor. These are the conditions existing in a water spirometer.
Atropine: A poisonous white crystalline alkaloid, Cy/H^NO-, from belladonna ,
and related plants, used to relieve spasms of smodtn muscles. It is an
anticholinergic agent.
Breathing pattern: A general term designating the characteristics of the
ventilatory activity, e.g., tidal volume, frequency of breathing, and
shape of the volume time curve.
Breuer-Hering reflexes (Hering-Breuer reflexes): Ventilatory reflexes originat-
ing in the lungs. The reflex arcs are formed by the pulmonary mechanore-
ceptors, the vagal afferent fibers, the respiratory centers, the medullo-
spinal pathway, the motor neurons, and the respiratory muscles. The af-
ferent link informs the respiratory centers of the volume state or of the
rate of change of volume of the lungs. Three types of Breuer-Hering re-
flexes have been described: 1) an inflation reflex in which lung inflation
tends to inhibit inspiration and stimulate expiration; 2) a deflation
reflex in which lung deflation tends to inhibit expiration and stimulate
inspiration; and 3) a "paradoxical reflex," described but largely disre-
garded by Breuer and Hering, in which sudden inflation may stimulate
inspiratory muscles.
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Bronchiole: One of the finer subdivisions of the airways, less than 1 mm 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 bronchiolitis obliterans may be used.
Bronchitis: A non-neoplastic 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 etiol-
°fly> its chronicity, the presence of associated airways dysfunction, or
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 mucous 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 has sometimes been accepted as a criterion
for the diagnosis.
Bronchoconstrictor: An agent that causes a reduction in the caliber (diame-
ter) of airways.
Bronchodilator: An agent that causes an increase in the caliber (diameter) of
airways.
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.
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 man the normal temperature is taken as 37°C, the pressure
as the barometric pressure, and the partial pressure of water vapor as 47
torr.
Carbachol: A parasympathetic stimulant (carbamoyjcholine chloride, CgH-jj-ClNpOp)
that produces constriction of the bronchial smooth muscles.
Carbon dioxide production (VC02): Rate of carbon dioxide production by organ-
isms, tissues, or cells. Common units: ml C02 (STPD)/kg*min.
Carbon monoxide (CO): An odorless, colorless, toxic gas formed by incomplete
combustion, with, a strong affinity for hemoglobin and cytochrome; it
reduces oxygen absorption capacity, transport, and utilization.
Carboxyhemoglobin (COHb): Hemoglobin in which the-iron is associated with
carbon monoxide. The affinity of hemoglobin for CO is about 300 times
greater than for 0^-
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Chronic obstructive lung disease (COLD): 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 pulmonary disease
(COPD).
Closing capacity (CC): Closing volume plus residual volume, often expressed
as a ratio of TLC, i.e. (CC/TLCX).
Closing volume (CV): The volume exhaled after the expired gas concentration
is inflected from an alveolar plateau during a controlled breathing
maneuver. Since 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. Closing volume is often
expressed as a ratio of the VC, i.e. (CV/VC%).
Collateral resistance (R ,-,): Resistance to flow through indirect pathways.
See COLLATERAL VENTItWlON and RESISTANCE.
Collateral ventilation: Ventilation of air spaces via indirect pathways,
e.g., through pores in alveolar septa, or anastomosing respiratory bron-
chioles.
Compliance (C,,C .): 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, ex-
pressed in liters/cm HpO or ml/cm HpO. Since 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 full
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.
Conductance (G): The reciprocal of RESISTANCE. See AIRWAY CONDUCTANCE.
Diffusing capacity of the lung (D. , D.Op, DiCO?* D.CO): Amount of gas (0^,
CO, COg) commonly expressed as mr gas cSTTO) diffusing between alveorar
gas ana pulmonary capillary blood per torr mean gas pressure difference
per min, i.e., ml QgAmin-torr). Synonymous with transfer factor and
diffusion factor.
Dynamic compliance (Cjyn): The ratio of the tidal volume to the change in
intrapleural pressure between the points of zero flow at the extremes of
tidal volume in liters/cm H,0 or ml/cm H^O. Since at the points of zero
airflow at the extremes of "tidal volume, volume acceleration is usually
other than zero, and since, particularly in abnormal states, flow may
still be taking place within lungs between regions which 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.
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Elastance (E): The reciprocal of COMPLIANCE; expressed in cm H90/liter or cm
H20/ml. *
Electrocardiogram (ECG, EKG): The graphic record of the electrical currents
that are associated with the heart's contraction and relaxation.
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.
Expiratory reserve volume (ERV): The maximal volume of air exhaled from the
end-expiratory level.
FEV./FVC: A ratio of timed (t = 0.5, 1, 2, 3 s) forced expiratory volume
(FEV.) to forced vital capacity (FVC). The ratio is often expressed in
percent 100 x FEVt/FVC. It is an index of airway obstruction.
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 VC 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.
Forced expiratory flow (FEFx): Related to some portion of the FVC curve.
Modifiers refer to the amount of the FVC already exhaled when the measure-
ment is made. For example:
= instantaneous forced expiratory flow after 75%
of the FVC has been exhaled.
FEF9nn 19nn = mean forced expiratory flow between 200 ml
^uu-i^uu gnd 1200 ml Qf the pvc (formerly called the
maximum expiratory flow rate (MEFR).
FEF?r -jf-o/ = mean forced expiratory flow during the middle
"~/3A half of the FVC [formerly called the maximum
mid-expiratory flow rate (MMFR)].
FEF = the maximal forced expiratory flow achieved during
max an FVC.
Forced expiratory volume (FEV): Denotes the volume of gas which is exhaled in
a given time interval during the execution of a forced vital capacity.
Conventionally, the times used are 0.5, 0.75, or 1 sec, symbolized FEVg ,-,
FEVn 7r, FEV-, n. These values are often expressed as a percent of the'
forCed5vitaTLcaJpacity, e.g. (FEV-j^ Q/VC) X 100.
Forced inspiratory vital capacity (FIVC): The maximal volume of air inspired
with a maximally forced effort from a position of maximal expiration.
Forced vital capacity (FVC): Vital capacity performed with a maximally forced
expiratory effort.
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Functional residual capacity (FRC): The sum of RV and ERV (the volume of air
remaining in the lungs at the end-expiratory position). The method of
measurement should be indicated as with RV,
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 exchange ratio (R): See RESPIRATORY QUOTIENT.
Gas trapping: Trapping of gas behind small airways that were opened during
inspiration but closed during forceful expiration. It is a volume differ-
ence between FVC and VC.
Hematoerit (Hct): The percentage of the volume of red blood cells in whole
blood.
Hemoglobin (Hb): A hemoprotein naturally occurring in most vertebrate blood,
consisting of four polypeptide chains (the globulin) to each of which
there is attached a heroe group. The heme is made of four pyrrole rings
and a divalent iron (Fe -protoporphyrin) which combines reversibly with
molecular oxygen.
Histamine: A depressor amine derived from the amino 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.
Hypoxemia: A state in which the oxygen pressure and/or concentration in
arterial and/or venous blood is lower than its normal value at sea level.
Normal oxygen pressures at sea level are 85-100 torr in arterial blood
and 37-44 torr in mixed venous blood. In adult humans the normal oxygen
concentration is 17-23 ml 02/100 ml arterial blood; in mixed venous blood
at rest it is 13-18 ml 02/100 ml blood.
Hypoxia: Any state in which the oxygen in the lung, blood, and/or tissues is
abnormally low compared with that of normal resting man breathing air at
sea level. If the PQ2 is low in the environment, whether because of
decreased barometric pressure or decreased fractional concentration of
Og, the condition is termed environmental hypoxia. Hypoxia when referring
to the blood is termed hypoxemia. Tissues are said to be hypoxic when
their P02 is low, even if there is no arterial hypoxemia, as in "stagnant
hypoxia" which occurs when the local circulation is low compared to the
local metabolism.
Inspiratory capacity (1C): The sum of IRV and TV.
Inspiratory reserve volume (IRV): The maximal volume of air inhaled from the
end-inspiratory level.
Inspiratory vital capacity (IVC): The maximum volume of air inhaled from the
point of maximum expiration.
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Kilogram-meter/min (kg-m/min): The work performed each min to move a mass of 1
kg through a vertical distance of 1 m against the force of gravity.
Synonymous with kilopond-meter/min.
Lung volume (V,): Actual volume of the lung, including the volume of the
conducting airways.
Maximal aerobic capacity (max VO^): The rate of oxygen uptake by the body
during repetitive maximal respiratory effort. Synonymous with maximal
oxygen consumption.
Maximum breathing capacity (MBC): Maximal volume of air which can be breathed
per minute by a subject breathing as quickly and as deeply as possible.
This tiring lung function test is usually limited to 12-20 sec, but given
in liters (BTPS)/min. Synonymous with maximum voluntary ventilation (MVV).
Maximum expiratory flow (V ): Forced expiratory flow, related to the
total lung capacity or tne actual volume of the lung at which the measure-
ment is made. Modifiers refer to the amount of lung volume remaining
when the measurement is made. For example:
V -.cw - instantaneous forced expiratory flow when the
max /a* lung 1g flt ?5% Qf Us TLC>
V ~ n = instantaneous forced expiratory flow when the
max j.u volume is 3-0 -|-jters
Maximum expiratory flow rate (MEFR): Synonymous with ^^ 200-1200"
Maximum mid-expiratory flow rate (MMFR or MMEF): Synonymous with ^05-75%-
Maximum ventilation (max V>): The volume of air breathed in one minute during
repetitive maximal respiratory effort. Synonymous with maximum ventilatory
minute volume.
Maximum voluntary ventilation (MVV): The volume of air breathed by a subject
during voluntary maximum hyperventilation lasting a specific period of
time. Synonymous with maximum breathing capacity (MBC).
Methemoglobin (MetHb): Hemoglobin in which iron is in the ferric state.
Because the iron, is oxidized, methemoglobin is incapable of oxygen trans-
port. Methemoglobins are formed by various drugs and occur under pathol-
ogical conditions. Many methods for hemoglobin measurements utilize
methemoglobin (chlorhemiglobin, cyanhemiglobin).
Minute ventilation (Vp): Volume of air breathed in one minute. It is a
product of tidal volume (VT) and breathing frequency (fR). See VENTILA-
TION. ' . °
Minute volume: Synonymous with minute ventilation.
Mucociliary transport: The process by which mucus is transported, by ciliary
action, from the lungs.
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Mucus: The clear, viscid secretion of mucous membranes, consisting of mucin,
epithelial cells, leukocytes, and various inorganic salts suspended in
water.
Nasopharyngeal: Relating to the nose or the nasal cavity and the pharynx
(throat).
Nitrogen oxides: Compounds of N and 0 in ambient air; i.e., nitric oxide (NO)
and others with a higher oxidation state of N, of which N02 is the most
important toxicologically.
Nitrogen washout (AN-, dN«): The curve obtained by plotting the fractional
concentration of N? "in expired alveolar gas vs. time, for a subject
switched from breattiing ambient air to an inspired mixture of pure Op. A
progressive decrease of N« concentration ensues which may be analyzed
into two or more exponential components. Normally, after 4 min of pure
0? breathing the fractional N7 concentration in expired alveolar gas is
dSwn to less than 2%.
Normoxia: A state in which the ambient oxygen pressure is approximately 150 ±
10 torr (i.e., the partial pressure of oxygen in air at sea level).
Oxidant: A chemical compound that has the ability to remove, accept, or share
electrons from another chemical species, thereby oxidizing it.
Oxygen consumption (VO?J Q0?): Rate of oxygen uptake of organisms, tissues,
or cells. Common unitf: ml 02 (STPD)/(kg-min) or ml 02 (STPD)/(kg-hr).
For whole organisms the oxygen Consumption is commonly expressed per unit
surface area or, some power of the body weight. For tissue samples or
isolated cells QQ2 = pi Op/hr per mg dry weight.
Oxygen saturation (S0?): The amount of oxygen combined with hemoglobin,
expressed as a percentage of the oxygen capacity of that hemoglobin. In
arterial blood, SaO~.
Oxygen uptake ($02): Amount of oxygen taken up by the body from the environ-
ment, by the blood from the alveolar gas, or by an organ or tissue from
the blood. When this amount of oxygen is expressed per unit of time one
deals with an "oxygen uptake rate." "Oxygen consumption" refers more
specifically to the oxygen uptake rate by all tissues of the body and is
equal to the oxygen uptake rate of the organism only when the $„ stores
are constant.
Particulates: Fine solid particles such as dust, smoke, fumes, or smog, found
in the air or in emissions.
Pathogen: Any virus, microorganism, or etiologic agent causing disease.
Peak expiratory flow (PEF): The highest forced expiratory flow measured with
a peak flow meter.
Peroxyacetyl nitrate (PAN): Pollutant created by action of UV component of
sunlight on hydrocarbons and NO in the air; an ingredient of photochem-
ical smog.
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Physiological dead space (Vn): Calculated volume which accounts for the
difference between the pressures of C02 in expired and alveolar gas (or
arterial blood). Physiological dead space reflects the combination of
anatomical dead space and alveolar dead space, the volume of the latter
increasing with the importance of the nonuniformity of the
ventilation/perfusion ratio in the lung.
Plethysmograph: A rigid chamber placed around a living structure for the
purpose of measuring changes in the volume of the structure. In respira-
tory measurements, the entire body is ordinarily enclosed ("body plethys-
mograph") and the plethysmograph is used to measure changes in volume of
gas in the system produced 1) by solution and volatilization (e.g.,
uptake of foreign gases into the blood), 2) by changes in pressure or
temperature (e.g., gas compression in the lungs, expansion of gas upon
passing into the warm, moist lungs), or 3) by breathing through a tube to
the outside. Three types of plethysmograph are used: a) pressure, b)
volume, and c) pressure-volume. In type a, the body chambers have fixed
volumes and volume changes are measured in terms of pressure change
secondary to gas compression (inside the chamber, outside the body). In
type b, the body chambers serve essentially as conduits between the body
surface and devices (spirometers or integrating flowmeters) which measure
gas displacements. Type c combines a and b by appropriate summing of
chamber pressure and volume displacements.
Pneumotachograph: A device for measuring instantaneous gas flow rates in
breathing by recording the pressure drop across a fixed flow resistance
of known pressure-flow characteristics, commonly connected to the airway
by means of a mouthpiece, face mask, or cannula. The flow resistance
usually consists either of parallel capillary tubes (Fleisch type) or of
fine-meshed screen (Silverman-Lilly type).
Pulmonary alveolar proteinosis: A chronic or recurrent disease characterized
by the filling of alveoli with an insoluble exudate, usually poor in
cells, rich in lipids and proteins, and accompanied by minimal histologic
alteration of the alveolar walls.
Pulmonary edema: An accumulation of excessive amounts of fluid in the lung
extravascular tissue and air spaces.
Pulmonary emphysema: An abnormal, permanent enlargement of the air spaces
distal to the terminal nonrespiratory bronchiole, accompanied by destructive
changes of the alveolar walls and without obvious fibrosis. The term
emphysema may be modified by words or phrases to indicate its etiology,
its anatomic subtype, or any associated airways dysfunction.
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.
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Resistance flow (R): The ratio of the flow-resistive components of pressure
to simultaneous flow, in cm hLO/liter per sec. Flow-resistive components
of pressure are obtained by subtracting any elastic or inertia! 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,
TISSUE RESISTANCE, TOTAL PULMONARY RESISTANCE, COLLATERAL RESISTANCE.
Respiratory cycle: A respiratory cycle is constituted by the inspiration
followed by the expiration of a given volume of gas, called tidal volume.
The duration of the respiratory cycle is the respiratory or ventilatory
period, whose reciprocal is the ventilatory frequency.
Respiratory exchange ratio: See RESPIRATORY QUOTIENT.
Respiratory frequency (fp): The number of breathing cycles per unit of time.
Synonymous with breathing frequency (fg).
Respiratory quotient (RQ, R): Quotient of the volume of C0? produced divided
by the volume of 0,, consumed by an organism, an, organ, or a tissue during
a given period of Time. Respiratory quotients are measured by comparing
the composition of an incoming and an outgoing medium, e.g., inspired and
expired gas, inspired gas and alveolar gas, or arterial and venous blood.
Sometimes the phrase "respiratory exchange ratio" is used to designate
the ratio of C02 output to the Op uptake by the lungs, "respiratory
quotient" beirtg restricted to the actual metabolic CO,, output and 02
uptake by the tissues. With this definition, respiratory quotient and
respiratory exchange ratio are identical in the steady state, a condition
which implies constancy of the 02 and COp stores.
Shunt; Vascular connection between circulatory pathways so that venous blood
is diverted into vessels containing arterialized Wood (right-to-left
shunt, venous admixture) or vice versa (left-to-right shunt). Right-to-
left shunt within the lung, heart, or large vessels due to malformations
are more important in respiratory physiology. Flow from left to right
through a shunt should be marked with a negative sign.
Specific airway conductance (SGaw): Airway conductance divided by the lung
volume at which it was measured, i.e., normalized airway conductance.
SGaw = Gaw/TGV.
Specific airway resistance (SRaw): Airway resistance multiplied by the volume at
which it was measured. SRaw = Raw x TGV.
Spirograph: Mechanical device, including bellows or other scaled, moving
part, which collects and stores gases and provides a graphical record of
volume changes. See BREATHING PATTERN, RESPIRATORY CYCLE.
Spirometer: An apparatus similar to a spirograph but without recording facil-
ity.
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Static lung compliance (C, t): Lung compliance measured at zero flow (breath-
holding) over linear portion of the volume-pressure curve above FRC. See
COMPLIANCE.
Static transpulmonary pressure (P .): Transpulmonary pressure measured at a
specified lung volume; e.g., TS+JLC is static recoil pressure measured at
TLC (maximum recoil pressure).
Sulfur dioxide (SCL): Colorless gas with pungent odor, released primarily from
burning of fossil fuels, such as coal, containing sulfur.
STPD conditions (STPD): Standard temperature and pressure, dry. These are
the conditions of a volume of gas at 0°C, at 760 torr, without water
vapor. A STPD volume of a given gas contains a known number of moles of
that gas.
Surfactant, pulmonary: Protein-phospholipid (mainly dipalmitoyl lecithin)
complex which lines alveoli (and possibly small airways) and accounts for
the low surface tension which makes air space (and airway) patency possible
at low transpulmonary pressures.
Synergism: A relationship in which the combined action or effect of two or
more components is greater than the sum of effects when the components
act separately.
Thoracic gas volume (TGV): Volume of communicating and trapped gas in the
lungs measured by body plethysmography at specific lung volumes. In
normal subjects, TGV determined at end expiratory level corresponds to
FRC.
Tidal volume (TV): 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.
Tissue resistance (R+4): Frictional resistance of the pulmonary and thoracic
tissues.
2
Torr: A unit of pressure equal to 1,333.22 dynes/cm or 1.33322 millibars.
The torr is equal to the pressure required to support a column of mercury
1 mm high when the mercury' is of standard density and subjected to standard
acceleration. These standard conditions are met at 0°C and 45° latitude,
where the acceleration of gravity is 980.6 cm/sec . In reading a mercury
oarometer at other temperatures and latitudes, corrections, which commonly
exceed 2 torr, must be introduced for these terms and for the thermal
expansion of the measuring scale used. The torr is synonymous with
pressure unit mm Hg.
Total lung capacity (TLC): The sum of all volume compartments or the volume
of air in the lungs after maximal inspiration. The method of measurement
should be indicated, as with RV.
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Total pulmonary resistance (R,): Resistance measured by relating flow-dependent
transpulmonary pressure xo airflow at the mouth. Represents the total
(frictional) resistance of the lung tissue (R,.) and the airways (Raw).
RL = Raw+Rtr
Trachea: Commonly known as the windpipe; a cartilaginous air tube extending
from the larynx (voice box) into the thorax (chest) where it divides into
left and right branches.
Transpulmonary pressure (P,): Pressure difference between airway opening
(mouth, nares, or cannula opening) and the visceral pleura! surface, in
cm HpO. Transpulmonary in the sense used includes extrapulmonary struc-
tures, e.g., trachea and extrathoracic airways. This usage has come
about for want of an anatomic term which includes all of the airways and
the lungs together.
Ventilation: Physiological process by which gas is renewed in the lungs. The
word ventilation sometimes designates ventilatory flow rate (or ventila-
tory minute volume) which is the product of the tidal volume by the
ventilatory frequency. Conditions are usually indicated as modifiers;
i.e.,
VV = Expired volume per minute (BTPS),
. and
V,- =, Inspired volume per minute (BTPS).
Ventilation is often referred to as "total ventilation" to distinguish it
from "alveolar ventilation" (see VENTILATION, ALVEOLAR).
Ventilation, alveolar (V«): Physiological process by which alveolar gas is
completely removed and replaced with fresh gas. 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 times the ventilatory frequency constitutes the alveolar
ventilation.
Ventilation, dead-space (Vr,): Ventilation per minute of the physiologic dead
space (wasted ventilation), BTPS, defined by the following equation:
VD = VE(PaCD2 - PEC02)/(PaC02 - PjC02)
Ventilation/perfusion ratio (V./Q): Ratio of the alveolar ventilation to the
blood perfusion volume flow through the pulmonary parenchyma. This ratio
is a fundamental determinant of the 0~ and C0? 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.
Vital capacity (VC): The maximum volume of air exhaled from the point of
maximum inspiration.
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