>EPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessmei..
Washington DC 20460
600884020A4 .,,i
External Review Draft c,\
Research and Development
Air Quality Criteria Review
for Ozone and Other Draft
Photochemical
Oxidants
(Do Not
Cite or Quote)
Volume IV of V
NOTICE
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
-------
EPA-600/8-84-020A
Do Not June 1984
Cite or Quote External Review Draft
Air Quality Criteria
for Ozone and Other
Photochemical Oxidants
Volume IV
NOTICE
This document is a preliminary draft. It has not been formally released by EPA and should not at
this stage be construed to represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
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
U.S. Environmental Protection Agency
Region V, Library
230 South Dearborn Street
Chicago, Illinois 60604
-------
NOTICE
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Environmental Fraction Agen«
-------
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 1983 and early 1984.
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. Separate chapters are 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 proper-
ties, chemistry, 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 appreisal of effects of ozone and other oxidants; effects
observed in controlled human exposures; effects observed in field and epidemio-
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.
m
0190LG/B May 1984
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CONTENTS
Page
VOLUME I
Chapter 1. Summary and Conclusions 1-1
VOLUME II
Chapter 2. Introduction 2-1
Chapter 3. Precursors to Ozone and Other Photochemical
Oxidants 3-1
Chapter 4. Chemical and Physical Processes in the Formation
and Occurrence of Ozone and Other Photochemical
Oxidants 4-1
Chapter 5. Properties, Chemistry, and Measurement of Ozone
and Other Photochemical Oxidants 5-1
Chapter 6. Concentrations of Ozone and Other Photochemical
Oxidants in Ambient Air 6-2
VOLUME III
Chapter 7. Effects of Ozone and Other Photochemical Oxidants
on Vegetation 7-1
Chapter 8. Effects of Ozone and Other Photochemical Oxidants
on Natural and Agroecosystems 8-1
Chapter 9. Effects of Ozone and Other Photochemical Oxidants
on Nonbiological Materials 9-1
VOLUME IV
Chapter 10. Toxicological Effects of Ozone and Other
Photochemical Oxidants 10-1
VOLUME V
Chapter 11. Controlled Human Studies of the Effects of Ozone
and Other Photochemical Oxidants 11-1
Chapter 12. Field and Epidemiological Studies of the Effects
of Ozone and Other Photochemical Oxidants 12-1
Chapter 13. Evaluation of Integrated Health Effects Data for
Ozone and Other Photochemical Oxidants 13-1
iv
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Chapter 10: 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.
Northrop Services, Inc.
Environmental Sciences
P.O. Box 12313
Research Triangle Park, NC 27709
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
0190LG/B
May 1984
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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. Srnolko
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
VI
0190LG/B May 1984
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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
and 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
University of California
Santa Barbara, CA 93106
Stress
Dr. George J. Jakab
Department of Environmental
Health Sciences
Johns Hopkins School of Hygiene
and Public 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
0190LG/B
vn
May 1984
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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
Dr. William F. McDonnell
Health Effects Research Laboratory
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
Baltimore, ND 21205
Ms. Beverly E. Til ton
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
0190LG/B
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May 1984
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CHAPTER 10
TOXICOLOGICAL EFFECTS OF OZONE AND OTHER PHOTOCHEMICAL OXIDANTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
10.1 INTRODUCTION
10.2 REGIONAL DOSIMETRY IN THE RESPIRATORY TRACT
10.2.1 Absorption in Experimental Animals
10.2.1.1 Nasopharyngeal Absorption
10.2.1.2 Lower Respiratory Tract Absorption
10.2.2 Ozone Dosimetry Models
10.2.2.1 Modeling Nasal Uptake
10.2.2.2 Lower Respiratory Tract Dosimetry Models ...
10.2.3 Predictions of Lower Respiratory Tract Ozone
Dosimetry Modeling
10.2.3.1 Illustration of Dosimetry Simulations
10.2.3.2 Comparisons of Simulations to Experimental
Data
10.2.3.3 A Use of Predicted Doses
10.3 EFFECTS OF OZONE ON THE RESPIRATORY TRACT
10.3.1 Morphological Effects
10.3.1.1 Sites Affected
10.3.1.2 Sequence in which Sites are Affected
as a Function of Concentration and
Duration of Exposure
10.3.1.3 Structural Elements Affected
10.3.1.4 Considerations of Degree of Suscepti-
bility to Morphological Changes
10.3.2 Pulmonary Function Effects
10.3.2.1 Short-Term Exposure I
10.3.2.2 Long-Term Exposure
10.3.2.3 Airway Reactivity
10.3.3 Biochemically Detected Effects
10.3.3.1 Introduction
10.3.3.2 Antioxidant Metabolism
10.3.3.3 Oxidative and Energy Metabolism
10.3.3.4 Monooxygenases
10.3.3.5 Lactate Dehydrogenase and Lysosomal Enzymes
10.3.3.6 Protein Synthesis
10.3.3.7 Lipid Metabolism and Content of the Lung ...
10.3.3.8 Lung Permeability
10.3.3.9 Proposed Molecular Mechanisms of Effects ...
10.3.4 Effects on Host Defense Mechanisms
10.3.4.1 Mucociliary Clearance
10.3.4.2 Alveolar Macrophages
XT
xii
xiii
10-1
10-3
10-4
10-4
10-5
10-6
10-6
10-6
10-9
10-10
10-14
10-14
10-15
10-15
10-15
10-38
10-39
10-42
10-48
10-48
10-53
10-57
10-64
10-64
10-65
10-80
10-83
10-87
10-90
10-95
10-97
10-100
10-107
10-107
10-112
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TABLE OF CONTENTS (continued).
10.5
10.6
10.3.4.3 Interaction with Infectious Agents
10.3.4.4 Immunology
10.3.5 Tolerance
10.4 EXTRAPULMONARY EFFECTS OF OZONE
10.
10.
4.1
4.2
Effects
10.4.3
10.
10.
4.4
4.5
10.4.6
Central Nervous System and Behavioral
Cardiovasculcir Effects
Hematological and Serum Chemistry Effects ...
10.4.3.1 Animal Studies - In Vivo Exposures
10.4.3.2 In Vitro Studies
10.4.3.3 Changes in Serum
10.4.3.4 Interspecies Variations
Reproductive and Teratogenic Effects
Chromosomal and Mutational Effects
10.4.5.1 Chromosomal Effects of Ozone
10.4.5.2 Mutational Effects of Ozone
Other Extrapulmonary Effects
10.4.6.1 Liver
10.4.6.2 The Endocrine System
10.4.6.3 Other Effects
EFFECTS OF OTHER PHOTOCHEMICAL OXIDANTS
10.5.1 Peroxyacetyl Nitrate
10.5.2 Hydrogen Peroxide
10.5.3 Complex Pollutant Mixtures
SUMMARY
10.6.1
10.6.
10.6.
2
3
Introduction
Regional Dosimetry in the Respiratory Tract
Effects of Drone on the Respiratory Tract
10.6.3.1 Morphological Effects
Lung Function
Biochemically Detected Effects of Ozone
Host Defense Mechanisms
Tolerance
10
10
10
10
3.2
3.3
3.4
3.5
10.6.4
Extrapulmonary Effects
10.6.4.1 Central Nervous System and Behavioral
10.6.4.2 Cardiovascular Effects
Effects.
10.
10.
10.
10.
6.4.3
6.4.4
6.4.5
6.4.6
Hematological and Serum Chemistry Effects
Reproductive and Teratogenic Effects
Chromosomal and Mutational Effects
Other Extrapulmonary Effects
10.6.5 Effects of other Photochemical Oxidants
10.7 REFERENCES
APPENDIX A
10-119
10-127
10-130
10-138
10-138
10-143
10-144
10-145
10-151
10-155
10-156
10-157
10-160
10-160
10-172
10-174
10-174
10-181
10-187
10-188
10-188
10-189
10-192
10-199
10-199
10-200
10-201
10-201
10-203
10-205
10-210
10-212
10-213
10-213
10-214
10-214
10-216
10-217
10-218
10-219
10-221
A-l
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LIST OF TABLES
Table
10-1 Morphological effects of ozone
10-2 Effects of ozone on pulmonary function: short-term exposures ..
10-3 Effects of ozone on pulmonary function: long-term exposures ...
10-4 Effects of ozone on pulmonary function: airway reactivity
10-5 Changes in the lung antioxidant metabolism and oxygen
consumpti on by ozone
10-6 Monooxygenases
10-7 Lactate dehydrogenase and lysosomal enzymes
10-8 Effects of ozone on lung protein synthesis
10-9 Effects of ozone exposure on lipid metabolism and content of
the 1ung
10-10 Effects of ozone on lung permeability
10-11 Effects of ozone on host defense mechanisms: deposition and
clearance
10-12 Effects of ozone on host defense mechanisms: macrophage
alterati ons
10-13 Effects of ozone on host defense mechanisms: interactions with
infectious agents
10-14 Effects of ozone on host defense mechanisms: mixtures
10-15 Effects of ozone on host defense mechanisms: immunology
10-16 Tolerance to ozone
10-17 Central nervous system and behavioral effects of ozone
10-18 Hematology: animal-~iji vivo exposure
10-19 Hematology: animal--i_n vitro exposure
10-20 Hematology: humna—j_n vitro exposure
10-21 Reproductive and teratogenic effects of ozone
10-22 Chromosomal effects from iji vitro exposure to high ozone
concentrati ons
10-23 Chromosomal effects from ozone concentrations at or below
1960 ug/m3 (1 ppm)
10-24 Mutational effects o'* ozone
10-25 Effects of ozone on the 1 iver
10-26 Effects of ozone on xhe endocrine system, gastrointestinal
tract, and uri ne
10-27 Effects of complex pollutant mixtures
Page
10-16
10-49
10-54
10-58
10-66
10-84
10-88
10-91
10-96
10-98
10-109
10-113
10-122
10-125
10-128
10-132
10-139
10-146
10-152
10-153
10-158
10-161
10-163
10-167
10-175
10-182
10-193
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LIST OF FIGURES
Figure Page
10-1 Predicted tissue dose for several trachea! 03 concentrations
for rabbit and guinea pig 10-11
10-2 Tissue dose versus morphometric zone for rabbit and guinea
pig and tissue dose versus airway generation for human at a
trachea! 03 concentration of 510 pg/m3 (0.26 ppm) 10-12
10-3 Intracellular compounds active in antioxidant metabolism of
the 1 ung 10-65
xii
0190PT/A 5/1/84
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LIST OF ABBREVIATIONS
A-V
ACh
AChE
AM
AMP
ATPase
ATPS
BTPS
CC
Cdyn
CHEM
CL
C
CMP
CMS
CO
COHb
COLD
COMT
co2
CPK
CV
DL'
DLCO
DNA
E
ECG, EKG
EEC
ERV
FEF
max
FEF
Atrioventricular
AcetyIcholine
Acety1choli nesterase
Alveolar macrophage
Adenosine monophosphate
Adenosine triphosphatase
ATPS condition (ambient temperature and pressure, saturated
with water vapor)
BTPS conditions (body temperature, barometric pressure,
and saturated with water vapor)
Closing capacity
Dynamic lung compliance
Gas phase chemiluminescence
Lung compliance
Static lung compliance
Cytidine monophosphate
Central nervous system
Carbon monoxide
Carboxyhemoglobi n
Chronic obstructive lung disease
Catechol-o-methyl-transferase
Carbon dioxide
Creatine phosphokinase
Closing volume
Diffusing capacity of the lungs
Carbon monoxide diffusion capacity of the lungs
Deoxyribonucleic acid
Elastance
Electrocardiogram
Electroencephalogram
Expiratory reserve volume
The maximal forced expiratory flow achieved
during an FVC.
Forced expiratory flow
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May 9, 1983
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LIST OF ABBREVIATIONS (continued)
Mean forced expiratory flow between 200 ml and 1200 ml of
the FVC [formerly called the maximum expiratory flow rate
(MEFR)].
FEF25-75% Mean forced expiratory flow during the middle half of the
FVC [formerly called the maximum mid:expiratory flow rate
(MMFR)].
FEF75% Instantaneous forced expiratory flow after 75% of the FVC
has been exhaled.
FEV Forced expiratory volume
FIVC Forced inspiratory vital capacity
fR Respiratory frequency
FRC Functional residual capacity
FVC Forced vital capacity
G Conductance
G-6-PD Glucose-6-phosphate dehydrogenase
Gaw Airway conductance
GMP Guanosine monophosphate
GS-CHEM Gas-solid chemiluminescence
GSH Glutathione
GSSG Glutathione disulfide
Hb Hemoglobin
Hct Hemcitocrit
HO- Hydroxy radical
H20 Watfcr
1C Inspiratory capacity
IRV Inspiratory reserve volume
IVC Inspiratory vital capacity
K Average mucous production rate per unit area
LDH Lactate deyhydrogenase
LDrn Lethal dose (50 percent)
LM Light microscopy
LPS Lipopolysaccharide
MAO Monamine oxidase
xiv
0190PT/E May 9, 1983
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LIST OF ABBREVIATIONS (continued)
MAST
max Vr
max V02
MBC
MEFR
MEFV
MetHb
MMFR or MMEF
MNNG
MPO
MVV
NBKI
(NH4)2S04
N02
NPSH
AN2, dN2
°2
o2-
°3
P(A-a)02
PABA
PAco2
PaC02
PAN
PA°2
PaO.
PEF
PEFV
PG
PHa
PHA
PL
PMN
Kl-coulometric (Mast meter)
Maximum ventilation
Maximal aerobic capacity
Maximum breathing capacity
Maximum expiratory flow rate
Maximum expiratory flow-volume curve
Methemoglobin
Maximum mid-expiratory flow rate
N-methy1-N'-ni trosoguani di ne
Myeloperoxidase
Maximum voluntary ventilation
Neutral buffered potassium iodide
Ammonium sulfate
Nitrogen dioxide
Non-protein sulfhydryls
Nitrogen washout
Oxygen
Oxygen radical
Ozone
Alveolar-arterial oxygen pressure difference
para-aminobenzoic acid
Alveolar partial pressure of carbon dioxide
Arterial partial pressure of carbon dioxide
Peroxyacetyl nitrate
Alveolar partial pressure of oxygen
Arterial partial pressure of oxygen
Peak expiratory flow
Partial expiratory flow-volume curve
Prostaglandin
Arterial pH
Phytohemaggluti ni n
Trarispulmonary pressure
Polymorphonuclear leukocyte
0190PT/E
xv
May 9, 1983
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LIST OF ABBREVIATIONS (continued)
PPD Purified protein derivative
Pgt Static transpulmonary pressure
PUFA Polyunsaturated fatty acid
R Resistance to flow
Raw Airway resistance
RBCs Red blood cells
Rcol-| Collateral resistance
R|_ Total pulmonary resistance
RQ, R Respiratory quotient
Rt- 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 Thyroid-stimulating hormone
TV Tidal volume
UFA Unsaturated fatty acid
UMP Uridine monophosphate
UV Ultraviolet photometry
xvi
0190PT/E May 9, 1983
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LIST OF ABBREVIATIONS (continued)
V. Alveolar ventilation
V./Q Ventilation/perfusion ratio
VC Vital capacity
VCOp Carbon dioxide production
Vr, Physiological dead space
Vrv Dead-space ventilation
Vr, . Anatomical dead space
Vr Minute ventilation
Vr Expired volume per minute
Vr Inspired volume per minute
L
V, Lung volume
Vm3V Maximum expiratory flow
lUaX
VCL Oxygen uptake
V00, Oo, Oxyqen consumption
12s
I Irradiated iodine
5-HT 5-hydroxytryptamine
6-P-GD 6-phosphogluconate dehydrogenase
xv ii
0190PT/E May 9, 1983
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MEASUREMENT ABBREVIATIONS
g gram
hr/day hours per day
kg ki1ogram
kgm/mi n ki1ogram-meter/mi n
L/min liters/min
pptn parts per million
mg/kg milligrams per kilogram
o
mg/m milligrams per cubic meter
min minute
ml milliliter
mm millimeter
3
pg/m micrograms per cubic meter
pm micrometers
uM micromole
sec second
xvi n
0190PT/E May 9, 1983
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10. TOXICOLOGICAL EFFECTS OF OZONE AND OTHER PHOTOCHEMICAL OXIDANTS
10.1 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 have been studied, and many of the effects
of ozone are reasonably well described. In general, emphasis has been placed
on recent studies at 1960 ug/m3 (1 ppm) of ozone or less. Higher concentra-
tions have been cited when the data add to an understanding of mechanisms.
Concentrations of 1 ppm or greater cannot be studied ethically in man because
of the toxicity of even short-term exposures.
Primary emphasis has been placed on 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 these 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.
Eirphasis 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. Technical considerations for inclusion of a specific
study included an evaluation of the exposure methods; the analytical method
used to determine the chamber ozone concentration; the calibration of the
0190PT/B 10-1 5/1/84
-------
ozone monitoring equipment and the analytical methods used (wherever possible);
the species, strain, age and physical characteristics of the the subject
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 judgments
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 DO
are next 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 protective role 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 beyond those now
well documented in the respiratory system.
A brief discussion of the available literature on the effects of other oxi-
dants 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 in this area,
but its brevity does not necessarily reflect a general lack of importance.
0190PT/B 10-2 5/1/84
-------
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.
10.2 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 the goal is dosimetry, which is the specification of the
quantity of inhaled material, in this instance ozone (00, 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 03 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 Oo ab-
sorption in the species and in different-sized, 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
research, and to test our understanding of 03 absorption in the respiratory
tract.
0190PT/B 10-3 5/1/84
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10.2.1 Absorption in Experimental Animals
There have been very few experiments in which measurements of the regional
uptake of (L or other reactive gases have been determined. Of the several
results published, only one is concerned with the uptake of 0, in the lower
respiratory tract; the others deal with nasopharyngeal uptake.
10.2.1.1 Nasopharyngeal Absorption. Nasopharyngeal removal of 03 lessens
the quantity of 0., delivered to the lung and must be accounted for when
estimating the 0, 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 0^ 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. Their data, however, indicate that the trachea! concentra-
tion increases with increased nose or mouth concentrations. They also demon-
strated that the concentration of 0, 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 Qg in the nasopharynx of acutely
and chronically exposed dogs. Beagles chronically exposed (18 months) to 1 to
3 ppm of 03 under various daily exposure regimes had significantly higher tra-
cheal concentrations of 0, than animals tested after 1 day of exposure to cor-
responding regimes. Moorman et al. (1973) suggested that the differences were
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,
0190PT/B 10-4 5/1/84
-------
they had significantly lower trachea! values than those continuously exposed.
The average tracheal 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 0^ 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 tracheal 0- concentrations significantly greater than
those observed with acute exposure.
Nasopharyngeal 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 tracheal
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 tracheal and chamber concentrations is in agreement with Yokoyama and
Frank (1972).
10.2.1.2 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 03 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 10.3).
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
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
03 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.
0190PT/B 10-5 5/1/84
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10-2.2 Ozone Dosimetry Models
10.2.2.1 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 (MAS, 1977) concluded that the model predicts 99 percent penetration
for 0~. This is much more than that measured by Yokoyama and Frank (1972) or
by Miller et al. (1979). NAS (1977) discusses several possible reasons for
the differences, but considers the major factor 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
assumption of quasi-steady-state flow, mass balance, and that the flux of a
trace gas at the air-mucus interface is proportional 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 0^
uptake data of Yokoyama and Frank (1972) and concluded that the uptake coef-
ficient (average mass transfer coefficient) for (k, as well as for the other
gases considered, increases with increasing air flow rate.
10.2.2.2 Lower Respiratory Tract Dosimetry Models. There are two models for
which published results are available. The model of McJilton et al. (1972)
has never been formally published; however, its features have been discussed
in detail (National Academy of Sciences, 1977; Morgan and Frank, 1977) and
simulation results for 0, absorption in each generation of the human lower
respiratory tract are available (National Academy of Sciences, 1977; Morgan
and Frank, 1977). A detailed description of the formulation of the mathemati-
cal model of Miller and co-workers is found in Miller (1977) and an overview
is given in Miller (1979). Results of simulations of the respiratory tract
absorption of 03 in humans, rabbits, and guinea pigs are in Miller (1977,
1979), and Miller et al. (1978).
0190PT/B 10-6 5/1/84
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Because both models were developed to simulate the local absorption of
03, they have much in common. 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-, and others
in the lower respiratory tract (see Scherer et al., 1972; Paiva, 1973; Chang
and Farhi, 1974; Yu, 1975; Pack et al., 1977; Bowes et al., 1982). The approx-
imation is appropriate for 0., as well.
McJilton et al. considered only molecular diffusion for CL transport in
the liquid lining the airway (National Academy of Sciences, 1977; Morgan and
Frank, 1977). In addition to this process, Miller et al. (1978) included the
effect of chemical reactions on CL transport in the liquid lining. By using
«J
•their respective assumptions, equations based on mass balance considerations
were developed for (L transport in the lining. The liquid-phase equations
were coupled to the gas-phase equations based on physical considerations:
continuity of 0, transfer across the gas-liquid interface was required and the
gas and liquid phase interfacial concentrations were related by Henry's law
constant.
Airway or morphometric zone models such as those of Weibel (1963) and
Kliment (1973) were used in both models to define the lengths, radii, surface
areas, cross-sectional areas, and volumes of the airways and air spaces of
each generation or zone. A sinusoidal breathing pattern was used; however,
dimensions were held constant throughout the breathing cycle. The physical
properties of the liquid lining were assumed to be those of water. The lining
thickness depended on generation or zone, being thicker in those with lower
numbers than in those with higher numbers.
The flux of 03 from the lumen to the liquid lining was defined in terms
of a mass-transfer coefficient which depended on gas-phase and liquid-phase
transfer coefficients. McJilton et al. (National Academy of Sciences, 1977)
made the assumption that the mass transfer was liquid-phase controlled and
estimated the transfer coefficient from empirical data on the physical proper-
ties (not chemical) of the liquid lining and of 03- In the Miller model
(Miller, 1977) 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. It was combined with the liquid-phase
0190PT/B 10-7 5/1/84
-------
mass transfer coefficient (which depended on the chemical and physical proper-
ties of the liquid and of 0~) to obtain the overall transfer coefficient. No
assumption was necessary or made about which phase, if either, controlled the
transfer.
Ozone concentration in tissue and at the liquid-tissue interface was
assumed to be zero (Miller, 1977; National Academy of Sciences, 1977). The
National Academy of Sciences (1977) and Miller (1977) believe this boundary
condition means that CU reacts (chemically) instantaneously when it reaches
the tissue. Miller et al. (1978) define the tissue dose as that quantity of
0, per unit area reacting with the tissue at the liquid-tissue interface.
There are major differences in the two models. Ozone is known to react
chemically with constituents of the liquid lining. This factor was not included
in the McJilton et al. model. Chemical reactions were taken into account in
the mucous-serous lining In the Miller model. The inclusion resulted in
significant differences between the tissue dose pattern curves in the tracheo-
bronchial region predicted by the two models. The McJilton et al. model
predicts a dose curve (equivalent to a tissue dose curve because of no mucous
reactions) in the conductive zone that has its maximum at the trachea and
decreases distally to the thirteenth or fourteenth generation (see Figure 7-5
in National Academy of Sciences, 1977). By contrast, the Miller model predicts
the tissue dose to be a minimum at the trachea and to increase distally to the
pulmonary region.
Miller and co-workers took into account the reaction of 0., with the
unsaturated fatty acids (UFA) and ami no acids in the mucous-serous lining.
Reactions of 0~ with other components (such as carbohydrates) were not included
in the model because of insufficient information (Miller, 1977; Miller et al.,
1978). The CL-UFA and CL-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 specification of the production
rate of the UFA and amino 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 03, and the assumption that the production rate decreased distally
(Miller, 1977).
Although the instantaneous reaction scheme is a good preliminary approach
to treating CL reactions in the mucous-serous lining, its use is not completely
0190PT/B 10-8 5/1/84
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justifiable. Second-order rate constants of 0- with some of the mucous UFA
indicate that although they are large (Razumovskii and Zaikov, 1972), they are
less than the diffusion-limited rates necessary for the instantaneous reactions
and scheme. Experimental evidence (Mudd et al., 1969) suggests that the
reactions of 0- with amino acids are very rapid. Rate constants for these
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 Miller model also 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 et al. did not take this factor into account
(Morgan and Frank, 1977). However, this may not be an important difference in
the two models. Pack et al. (1977) and Engle and Mack!em (1977) reported
results that indicate an insensitivity of airway concentrations to the effective
dispersion coefficient.
10.2.3 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 a possible use for dosimetry modeling.
The following discussion of modeling results of lower respiratory tract
absorption is based on simulations using the Miller model. This is because
the model includes the important effects of 0~ reactions in the mucous-serous
lining and because simulations of 0, absorption in laboratory animals are
available.
Simulations of CL absorption in different animals can be carried out by
modifying input parameters of the computer program that solves the mathema-
tical equations. These input parameters, which characterize an animal, include
the number and dimensions of the airways, tidal volume, and length of time of
one breath. The airway and alveolar dimensions of Weibel (1963) were used for
the simulation of 0_ 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). In general, more than one generation in man corresponds to one
zone in an animal. However, each zone is characterized by the number of
airways and their lengths and diameter. The zone models were used because
0190PT/B 10-9 5/1/84
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they were the only complete (tracheobronchial and pulmonary regions) "airway"
models available at the time. Also, similar zone models, such as that of
Landahl (1950), have been used successfully in human aerosol deposition studies.
To illustrate simulation results, three aspects of the simulations by
Miller and co-workers are considered: 1) the effect of various tracheal
concentrations on the tissue dose pattern (tissue dose as a function of zone
or generation) in guinea pigs and rabbits; 2) the similarity between the dose
patterns of guinea pigs, rabbits, and humans; and 3) the effect on simulations
due to the uncertainty of the knowledge of the production rates of the mucous-
serous components that react with (L and of the knowledge of the mucous-serous
thickness.
10.2.3.1 Illustration of Dosimetry Simulations. Figure 10-1 is a set of
plots of the tissue dose for one breath versus zone for various tracheal 0.,
concentrations for rabbit and guinea pig simulations. (Ambient concentrations
would be roughly twice the tracheal concentration according to Miller et al.,
1979.) 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 maximum dose of 03. Although the model predicts uptake of 03 by respiratory
tissue (zones 6, 7, and 8) for all tracheal concentrations studied (62.5 to
3
4000 ug/m ), the penetration of 0., to the tissue in the airways lined by mucus
depended on the tracheal concentration and the specific animal species. For
example, as illustrated in Figure 10-1 for the tracheal 0- concentration of
O -5
1000 ug/m , no 03 reaches the airway tissue of the rabbit until zone 3, whereas
00 is predicted to penetrate to the guinea pig airway tissue in all zones.
3
However, at the two lowest tracheal concentrations plotted, 250 and 62.5 jjg/m ,
no penetration occurs until zones 4 and 6, respectively, for both animals.
The similarity of the predicted dose patterns in rabbits and guinea pigs
extends to the simulation of 0, uptake in humans. Figure 10-2 compares the
3
tissue dose for the three species for a tracheal concentration of 500
(0.26 ppm). Because of the different airway models used, the plot of the data
for the laboratory animals differs from that of Figure 10-1. In Figure 10-2
the guinea pig and rabbit tissue dose are plotted in the form of a histogram.
This a"i lows a plot of the human data, even though there is more than one
generation corresponding to one zone.
0190PT/B 10-10 5/1/84
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(A.D. = alveolar duct; A.S. = alveolar sac).
Source: Adapted from Miller et al. (1978).
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Source: Adapted from Miller et al. (1978).
10-12
-------
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, 0., penetrates to the
tissue everywhere in the pulmonary region (zones > 5 and generations > 16);
however, 0, 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.
The characteristics of the ozone tissue dose pattern for the three species,
as discussed here, are general to the simulations presented by Miller and
co-workers (Miller, 1977; Miller et al., 1978; Miller, 1979): 1) the maximum
tissue dose occurs in the first surfactant-lined zone or generation; 2) 0-
penetrates to tissue everywhere in the pulmonary region (zones > 5), decreas-
ing distally from the maximum; and 3) the onset of 0- penetration to mucous-
lined tissue, as well as dose in general, depends on trachea! Q~ concentra-
tion, animal species, and the breathing pattern. These general characteristics
of tissue dose pattern are independent of the two airway models used. Whether
or not this would be so if more recent and detailed airway models (e.g.,
Phalen et al., 1978) were used or if (L uptake were simulated in other species
would be of interest in understanding CL absorption.
Sensitivity studies were carried out with two major parameters, the
average mucous production rate per unit area (K ) and the mucous-serous thick-
ness (Miller, 1977; Miller et al., 1978; Miller, 1979). The parameter KQ is
related to the chemical reaction scheme used by Miller and co-workers; as K
increases, so does the reactivity. The study showed that if K overestimated
the correct value, there would be no difference in the predicted respiratory
bronchiolar (zone 6 or generation 17) dose for any of the three species studied.
If KQ underestimated the correct value, the results were not as clear; they
depended on the tracheal concentration and species. The guinea pig and rabbit
were the least sensitive species; they required a twenty-five and a fifty-fold
3
underestimate of KQ for 325 and 750 ug/m tracheal 0, concentration, respec-
tively, before significant variations in respiratory bronchiolar tissue dose
occurred. Significant differences occurred in the human dose for a ten- and a
twenty-five-fold underestimate (corresponding to 325 and 750 ug/m , respective-
ly). Decreasing KQ also allowed more 03 to penetrate to the tissue in the
conducting airways, and increasing RQ resulted in less 03 penetrating to the
conducting airway tissue. In the other sensitivity study, a 25 percent in-
crease in the mucous lining thickness decreased the conducting airway dose but
had no effect on respiratory region tissue dose (see Figures 2 and 8 and
discussion in Miller, 1977).
0190PT/B 10-13 5/1/84
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10.2.3.2 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 0» in the lower respiratory tract. Yokoyama and Frank
(1972) observed 80 to 87 percent uptake of 0- by the lower respiratory tract
of dogs. Miller et al. (1978) predict a 60 percent uptake for humans; however,
because of differences between two species, comparing the two 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 10.3.1.1). Of interest
is that for the animals simulated by Miller and co-workers, the maximum tissue
dose of 0^ occurs in this junction and decreases distally. Thus, in the
pulmonary region, the model results are in qualitative agreement with experi-
mental observations.
Damage is also observed in the trachea and bronchi of animals (see Sections
10.3.1.1.1.2 and 10.3.1.1.1.3). In the animals modeled, the Miller model
either predicts significantly less tissue dose in these regions 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 conducting airways (see Figure 10-2).
A factor complicating an understanding of the above is the possibility of 03
reaction products being toxic. Other factors confounding interpretation are
cell sensitivity and animal differences. Also, much of the reported damage in
the trachea and bronchi is associated with the cilia of ciliated cells, which
in the Miller model are not part of the tissue. The cilia extend into the
hypophase (perciliary) portion of the mucous-serous layer, and the Miller
dosimetry model does not distinguish the cilia of the ciliated cells as a
separate component of this layer. Thus, zero or relatively low predicted
tissue dose should not be interpreted as predicting no cilia damage. Likewise,
the frequent reporting of cilia being damaged following 0_ exposure should not
be interpreted necessarily as an indication of 0., tissue dose since the defini-
tion of tissue applied does not include cilia.
10.2.3.3 A Use of Predicted Doses. Miller (1979) discusses how doses predicted
by the model 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. The procedure is to simulate tissue
0190PT/B 10-14 5/1/84
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dose for several species for the same time (Miller, 1979) for a range of
trachea! CL concentrations. The doses for a specific zone or generation, for
each species, are plotted versus tracheal or ambient 0- concentration. By
using such plots and information on nasopharyngeal removal (Miller et al.,
1979), the ambient concentration necessary to produce the same dose in differ-
ent species can be estimated (see Miller, 1979). Also, the relative quantity
of OT 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.
10.3 EFFECTS OF OZONE ON THE RESPIRATORY TRACT
10.3.1 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 10-1), it appears likely that many of the postexposure
biological processes of animals could also occur in man.
10.3.1.1 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 submucosa 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
0190PT/B 10-15 5/1/84
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TABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE
Exposure
Ozone . duration
concentration Measurement ' and
ug/m3 ppm method protocol Observed effect(s)
Species Reference
196
392
o
i
0.1
0.2
UV,
NBKI
7 days, Two of six fed with "basal" vitamin E diet had increased cen-
continuous 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.
Rat Plopper et al., 1979
196
392
392
686
980
1568
392
686
392
980
1568
0.1
0.2
0.2
0.35
0.5
0.8
0.2
0.35
0.2
0.5
0.8
UV,
NBKI
MAST,
NBKI
UV,
NBKI
UV,
NBKI
uv,
NBKI
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
Five of six fed E-deficient "basal" diet had centriacinar AMs
and bronchiclar 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
surface area. Lung weight and alveolar number did not
change. Decrease in lung tissue elasticity. Parenchyma
appeared "normal" by LM.
Respiratory bronchiolitis at all concentrations. Increased
AMs. Bronchi olar epithelium both hyperplastic and hypertrophic.
Increased alveolar type 2 cells. Random foci of short, blunt
cilia or absence of cilia (LM, SEM, TEM).
All exposed monkeys had LM & EM lesions. Trachea and bronchi
had areas of shortened or less dense cilia. RBs had AM
accumulation and cuboidal cell hyperplasia. Alveoli off RBs
had AM accumulations and increased type 2 cells. RB walls of
the 0.35-ppm group were often thickened due to mild edema and
cellular infiltration.
Exposed groups gained less weight. Focal areas of missing or
damaged cilia in trachea and bronchi. TB nonciliated (Clara)
cells were shorter and 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.
Rat
Rat
Monkey
(Rhesus
and
Bonnet)
Monkey
(Bonnet)
Rat
Chow et al . , 1981
Bartlett et al . , 1974
Dungworth et al., 1975b
Castleman et al. , 1977
Schwartz et al . , 1976
-------
TABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration Measurement '
ug/m3
392
980
1568
392
980
1960
ppm method
0.2 UV,
0.5 NBKI
0.8
1
i
0.2 NO,
0.5 NBKI
1.0
Exposure
., duration
b j
and
protocol
20, 50, or
90 days;
8 hr/day
4 days,
3 hr/day,
exercised in
a rotating
case alter-
nate 15 min
Observed effect(s)c
Epithelial changes and PAM accumulations at 90 days were
similar to 7-day exposures, but less severe. 0.5- and 0.8-ppm
groups had increased centriacinar PAMs at all times. 0.2 ppm
and controls could not be separated by "blind" LM examination,
nor were there distinguishing EM 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. Nonci Hated cells were flattened
lumenal surfaces that occasionally occurred in clusters. Proximal
alveoli of 20- and 90-day 0.8-ppm groups had thicker blood/air
barriers.
Exercised control mice have significantly smaller body weights.
Both unexercised and exercised mice exposed to 0.5 or 1.0 ppm
had smaller body weights and larger lung weight. Exercised
mice exposed to 0.2 ppm also had larger lung weights. Other
pathology not studied.
Species Reference
Rat Boorman et al., 1980
Mouse Fukase et al . , 1978 i
(male,
5 weeks
old, l
ICR-JCL)!
392 0.2 CHEM
1960 1.0
9800 5.0
7, 14, 30, 60, Short-term exposures produced a slight degree of tonsil epithelial
90 days; con- detachment. Cell infiltration below the epithelium was slight.
tinuous Long-term exposures caused slight edema of the lacunar epithelium
which was destroyed or detached in places. Lymphocyte infiltra-
tion also occurred.
10 days, con- Tonsil epithelium had a high degree of detachment. Cell satura-
tinuous tion occurred below the epithelium. Some protrusion of the tonsil
into the oral cavity.
3 hr 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.
Rabbit Ikematsu, 1978
-------
TABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Exposure
Ozone b duration
concentration Measurement ' and
ug/mj
490
510
980
1960
588
ppm method
0.25 NO
0.26 MAST,
0.50 NBKI
1.0
0.3 NBKI
protocol
6 weeks ,
12 hr/day
4.7-6.6 hr,
endotracheal
tube
16 days,
3 hr/day
Observed effect(s)c Species Reference
Centriacinar alveoli had more type 1 and 2 alveolar epithelial Rat
cells and macrophages. Type 1 cells were smaller in volume,
covered less surface, and thicker. Type 1 cell damage and
occasional sloughing seen by TEM.
Desquamation of ciliated epithelium. Focal swelling or Cat
sloughing of type 1 cells.
SEM, but not LM, showed swollen cilia with hemispheric Rat
extrusions and surface roughness. Some adhesion of
Barry et al. , 1983
j Boatman et al . , 1974.
i „ —
Sato et al. , 1976a
o
I
I—>
CO
severely injured cilia occurred. 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
and attachment of round bodies. All responses were pronounced
in vitamin E-deficient rats. Some rats had chronic respiratory
disease.
588
588
686
980
1372
1470
1960
686
980
0.3 NBKI
0.3 UV
0.35 ND
0.50
0.70
0.75
1.00
0.35
0.50
28 weeks,
5 days/week,
3 hr/day
6 weeks,
5 days/week,
7 hr/day
1, 2, 4, 5, 6
or 8 days, con-
tinuous
4 days, contin-
uous, followed
by 0.50, 0.70,
0.75 or 1.00
for 1-4 days
No morphological differences noted between vitamin E-defi- Rat ; Sato et al., 1980 '
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 Sherwin et al., 1983'
Dividing cells were labeled with tritiated thymidine and Rat Evans et al., 1976b
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 03 (0.35 ppm) did not maintain
tolerance. Groups exposed to higher initial concentration
(0.50 ppm) demonstrated tolerance.
-------
TABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued).
Ozone
concentration Measurement '
pg/nr1 ppm method
784 0.4 MAST
784 0.4 NBKI
980 0.5 NO *
D
J 1
980 0.5 UV,
or or NBKI
1568 0.8
980 0.5 UV,
NBKI
980 0.5 UV,
1568 0.8 NBKI
Exposure
, 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 24 hr/
day
7, 21, and
35 days,
continuous
7, 28, or
90 days,
continuous,
8 hr/day
Observed effect(s)c
All (exposed and control) lungs showed some degree of inflam-
matory infiltrate possibly due to intercurrent 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 framework of the alveoli was
difficult. Small pulmonary arteries had thickened tunica medias,
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.
Principal lesion was a "low-grade respiratory bronchiolitis"
characterized by "intraluminal accumulations of macrophages
and hypertrophy and hyperplasia of cuboidal bronchiolar
epithelial cells." Conducting airway lesions not apparent
by LM, but parallel linear arrays of uniform shortening and
reduction of density of cilia by SEM. Kulschitzky-type cells
appeared more numerous in exposed.
Species
Rabbit
(New
Zealand)
Rabbit
Rat
(young
males)
Rat
Mouse
(Swiss-
Webster;
60 days
old; 35-40 g)
Monkey
(Bonnet)
Reference
P'an et al. , 1972
Boatman et al . , 1983
Stephens et al. , 1974b
Brummer et al. , 1977
Zitnik et al., 1978
Eustis et al. , 1981
-------
ABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
ro
o
Ozone
concentration Measurement '
ug/fnj ppm method
980 0.5 UV,
1568 0.8 NBKI
980 0.5 UV,
to to NBKI
3920 2.0
>
Exposure
u duration
and
protocol Observed effect(s) Species Reference
7 days, All exposed monkeys had lesions. Lesions similar in 0.5- Monkey Mellick et al., 1975,
8 hr/day 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 eosinophilis and debris.
Nonciliated cuboidal bronchiolar cells were larger, more
numerous, and sometimes stratified. Proximal alveolar
epithelium thickened by increased numbers of type 2 cells.
Progressive decrease in intensity of lesions from proximal to
distal orders of RBs.
7 days, Elevated collagen synthesis rates and histologically Rat Last et al . , 1979
24 hr/day discernible fibrosis was present at all levels of 03.
0.5 ppm Minimal or no thickening of walls or evidence of
fibrosis. Increased number of cuboidal cells and
macrophages present.
0.5
to
1.5
14 days
and
21 days,
24 hr/day
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 0,
CHEM,
NBKI
6 months, Only 03 caused pulmonary lesions. Only LM histopathology,
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
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 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
ug/m3 ppm
Measurement
method
a.b
Exposure
duration
and
protocol
Observed effect(s)
Species
Reference
980
0.5
0.5 0,
uv,
NBKI
o
i
ro
3, 50, 90, H2S04 did not potentiate effects of 03 alone. Fixed lung
or 180 days; volumes were increased at 180 days, but decreased at 62 days
continuous, postexposure. After 50, 90, 94 180 days all 03 exposure
plus 62 days rats had "bronchiolization of alveoli" or formation of an RB
postexposure, between the TB and AOs. Centriacinar inflammatory cells were
24 hr/day 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 mild
interstitial thickening of alveolar septa in the centriacinar
region (LM, SEM, TEM).
Rat Moore and Schwartz, 1981
1058
1725
0.54
0.88
ND
1058 0.54
2, 4, 8, 12, Severe loss of cilia from TB after 2 hr. TB surface more
or 48 hr uniform in height than controls. Necrotic ciliate cells in
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
minimal 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. Macrophages
accumulated in proximal alveoli. Endothelium appeared
relatively normal.
Repair started at 20 hr. Type 2 cells divide, cuboidal
epithelium lines proximal alveoli whi :'e 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, SEM, TEM).
6 months, No mention in either the results or discussion of the 6 months
24 hr/day at 0.54-ppm group.
Rat ^tephens et al. , 1974a
-------
TABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
o
i
Ozone
concentration Measurement '
ug/m3
1058
1725
1764
490
1176
2548
1372
1568
1372
ppm method
0.54 ND
0.88
0.9 03
•«-
0.9 N02
0.25 03
+
2.5 N02
0.6 1 NO
1.3
0.7 UV,
to NBKI
0.8
0.7 ND
Exposure
. duration
b and
protocol
4 hr
to
3 weeks
60 days
6 months
1 or 2 days,
6 hr/day or
7 hr/day
7 days ,
continuous
24 hr,
continuous
r~
Observed effect(s)
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 N02 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.
In situ cytochemical studies of lungs from 03 exposed and
control rats. Ozone-exposed rats had increased acid phosphatase,
both in lysosomes and in the cytoplasm, in nonciliated bronchiolar
(Clara) cells, alveolar macrophages, type 1 and 2 cells, and
fibroblasts.
Exposure end: General depletion of cilia from TB surface.
Nonciliated cells were shorter and contained
Species
Rat
(month
old)
Rat
(month
old)
Rat
(month
old)
Mouse
(young)
Rat
Rat
Reference
Freeman et al. , 1974
!
Bils, 1970
Castleman et al. , 1973a
Evans et al . , 1976a
Post exposure:
(0-4 days)
fewer dense granules, less SER, and more free
ribosomes.
TB returned towards normal.
-------
TABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
(jg/ra3 ppm
1568 0.8
0
i
ro
CO
1568 0.8
1568 0.8
Exposure
. duration
Measurement ' and
method protocol
UV, 6, 10, 20
NBKI days
exposure,
24 hr/day
or
20 days
exposure +
10 days
postexposure
UV, 7 days,
NBKI continuous
with samples
at 6, 24,
72 and
168 hr.
UV, 4, 8, 12,
NBKI 18, 26, 36,
50, and post
48 and
168 hr,
continuous
Observed effect(s)c Species Reference
SEM 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 Lum et al., 1978
changed after 03 exposure; fewer ciliated and more non-
ciliated secretory cells.
Degeneration and necrosis of RB type 1 cells predominates Monkey Castleman 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
macrophages increased during exposure, but marked clusters
of K cells at 26-36 hr.
-------
'ABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
pg/m3 ppm
1568 0.8
Exposure
. duration
Measurement ' and
method protocol
UV, 3 days, 1st Exposure:
NBKI continuous
0, 2, 6, 9,
16, and 30
days post-
exposure;
2nd 3 days
continuous
after 6, 13,
and 27 days Postexposure:
postexposure 6 days:
Observed effect(s)c
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.
Species Reference
Rat Plopper et al., 1978
(Sprague-
Dawley;
70 days
old)
Most obvious lesions were not present. TB epithelium
o
i
ro
had usual pattern. Clumps of macrophages had cleared
from the lumen. Most proximal alveoli lined by normal
type 1 and 2 cells.
30 days: Lungs indistinguishable from controls.
2nd 3-Oay 6 or 27 days after the end of the 1st. Lesions
Exposure: same as 1st exposure.
1666 ,0.85 NO
Similar
exposure
regimen
for 14 ppm
N02, but not
mixtures.
1960 1.0 NBKI
1, 2, 3
days,
continuous
~60 weeks,
~5 days/week,
6 hr/day
(268 expo-
posures)
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- j
Dawley; ',
1, 5, 10,
15, 20, 25,
30, 35, and
40 days old)
Mouse, Stokinger et al., 1957
Hamster,
Rat
-------
TABLE 10-1. MORPHOLOGICAL EFFECTS OF OZONE (continued)
Ozone
concentration
(jg/m3
1960
to
1960
1960
1960
5880
ppm
1.0
to
3. 0
1 0
1.0
1.0
2.0
3.0
Measurement3'
method
NO
A
B
C
D
E
Expasure
duration
and
protocol
18 months,
8-24 hr/
day
= 8 hr/day
= 16 hr/day
= 24 hr/day
= 8 hr/day
= 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.
C 1 ppm, 24 hr/day: More extensive fibrosis of
centriacinus. Thickened AO walls. More "extra"
o
1
ro
en
RB and
AO.
D & More fibrosis. Epithelial hyperplasia and squamous
E metaplasia.
1960
3920
1-0 CHEM,
and NBKI
o n
mixtures
with
H2S04
2 or 7 days, Results:
6 hr/day 03 alone:
1 ppm:
2 ppm:
03 plus
H2S04
Rat Cavender et al. , 1977
Lesions limited to centriacinus. and
Hypertrophy and hyperplasia of TB epithelium. Guinea
Centriacinar alveoli had increased type 2 pig
cells, increased macrophages, and thickened
walls. Some edema in all animals.
Lesions less severe at 7 days than at ? days.
This adaptation was more rapid in rats than
guinea pigs.
Same plus loss of cilia in bronchi.
No additive or synergistic morphological
changes.
Measurement method MAST - Kl-coulometric (Mast meter), CHEM = gas phase chemiluminescence), NBKI = neutral buffered potassium iodide,
UV = UV photometry; ND = not described.
Calibration method NBKI = neutral buffered potassium iodide.
Abbreviations used: LM = light microscope, EM = electron microscope; SEM = 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 endoplasmic reticulum, RER = rough endoplasmic reticulum.
-------
and glands associated with the sense of smell. Significant morphological
differences exist among the various animal species used for 0- exposures as
»J
well as between most of them and man (Schrieder 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 "scrubbing" has been studied (Yokoyama and Frank, 1972; and Miller et
al., 1979).
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:
Morphology (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 bron-
chiolar epithelial cells, which in some species can appropriately be called
"Clara" cells. Subepithelial tissues are sparse and do not contain glands.
The sensitive cell of the upper and lower conducting airways is the cili-
ated cell, which is primarily responsible for physical clearance or removal of
inhaled foreign material from conducting airways of the respiratory system
(see Section 10.3.4). The effects of (L on this sensitive 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 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).
0190PT/B 10-26 5/1/84
-------
10.3.1.1.1 Ai rways
10.3.1.1.1.1 Upper airways (nasal cavity, pharynx, and larynx). The
effects of 0- on the upper extrathoracic airways have received little attention.
The effect of upper airway scrubbing on the level of 0- 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
ambient concentrations between 196 and 3920 pg/m (0.1 and 2.0 ppm). Earlier,
Yokoyama and Frank (1972) studied nasal uptake in dogs. They found uptake to
3
vary with flow rate as well as with 0^ concentration. At 510 to 666 (jg/m
(0.26 to 0.34 ppm) of 0^, 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 ±
2.7 percent. At 1529 to 1568 ug/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.
The effects of 392, 1960, and 9800 pg/m3 (0.2, 1.0, and 5.0 ppm) of 03 on
the tonsils, the primary lymphoreticular structures of the upper airways, were
studied. In palatine tonsils from rabbits exposed to 392 |jg/m (0.2 ppm) of
0- continuously for 1 and 2 weeks, Ikematsu (1978) reported epithelial detach-
ment and disarrangement and a slight cellular ^filtration. The significance
of these observations to the function of immune mechanisms in host defense is
unknown.
10.3.1.1.1.2 Trachea. Tracheal epithelial lesions have been described
3
in several species following exposure to less than 1960 (jg/m (1 ppm) of 0,..
Boatman et al. (1974) exposed anesthetized, paralyzed cats to 510, 980, or
3
1960 pg/m (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 tracheal concentrations than in usual exposures. They reported desquama-
3
tion of ciliated epithelium at 1960 pg/m (1 ppm) of 0~, but none at 510 or
980 jjg/'m3 (0.26 or 0.5 ppm).
0190PT/B 10-27 5/1/84
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In rats exposed to 960 or 1568 ug/m (0.5 or 0.8 ppm) of 0-, 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
'3
electron microscope (SEM) and were not obvious in rats exposed to 392 ug/m
(0.2 ppm) of GO for the same times. Cavender et al. (1977), when using only
light microscopy (LM), studied tracheas from rats and guinea pigs exposed for
7 days to 1960 or 3920 ug/m (1 or 2 ppm) of 03 or sulfuric acid (H2$04) or
both. The article does not state the hours of exposure per day. Tracheal
lesions, which consisted of reduced numbers of cilia and goblet cells, were
reported only for guinea pigs exposed to 0.,. Animals exposed to both pollutants
had lesions similar to those exposed to 03 alone.
By using SEM and transmission electron microscopy (TEM), Castleman et al.
(1977) described shortened and less dense cilia in tracheas from bonnet mon-
3
keys exposed to 392 or 686 ug/m (0.2 or 0.35 ppm), 8 hr/day for 7 days.
Lesions occurred as randon 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.
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 156o ug/m (0.5 or 0.8 ppm) of
03 in the same exposure regimen (Dungworth et al., 1975b; Mellick et al.,
1977).
3
After mice were exposed to 1568 ug/m (0.8 ppm) of 03 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 surface of the tracheas was
examined by SEM by Ibrahim et al. (1980). Short and normal-length cilia were
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
0190PT/B 10-28 5/1/84
-------
clusters of nonciliated cells were still present. Earlier, Penha and Wer-
thamer (1974) observed metaplasia of the tracheal epithelium from mice exposed
3
to high concentrations of 03 (4900 ug/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.
10.3.1.1.1.3 Bronchi. Bronchial lesions were studied in many of the
same animals as those whose tracheal lesions are described above and were
reported as generally similar to the tracheal lesions. At low concentrations
(Castleman et al., 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 pg/m (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 Mel lick
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. Sato et al. (1976a,b)
studied bronchi from vitamin E-deficient and control rats exposed to 588 pg/m
(0.3 ppm) of 03 3 hr/day for up to 16 days. Using LM, they did not see bron-
chial 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) exposed vitamin E-deficient
3
and supplemented rats to 588 ng/m (0-3 ppm) of 0- 3 hr/day, 5 days/week for 7
months following which they did not see clear differences with SEM or TEM.
10.3.1.1.1.4 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
bronchroles). 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
0190PT/B 10-29 5/1/84
-------
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 (Castleman 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 03 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.
Terminal bronchiolar lesions in rats due to inhalation of < 1960 pg/m (<
1.0 ppm) of DO for 2 hr to 1 week have been des ribed by Stephens et al.
•j
(1974a), Evans et al. (1976a,c), Schwartz et al. (1976), and others
(Table 10-1). Ciliated cells are the most sensitive 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., 19766.; 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 granularity. 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
0190PT/B 10-30 5/1/84
-------
and precursors of basal bodies indicative of ciliogenesis (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 genera-
tion, 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/m (0.54 or 0.88 ppm) of 03 for 4 hr to 3 weeks.
In addition to the centriacinar accumulations of macrophages and the hyperplasia
of the distal airway epithelium, they reported an increase in connective
tissue elements and collagen-like strands which formed bridges across alveolar
3
openings. In the 1725-ug/m (0.88-ppm) 03 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 i
and one of 1690 |jg/m (0.9 ppm) nitrogen dioxide, or to a mixture of 490 ug/m
(0.25 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 expo-
sures (Freeman et al., 1972). Freeman et al. (1974) did not report emphysema
in rats exposed to 03 alone, only in those rats exposed to the 0.9/0.9 mixture.
The topic of emphysema is discussed later (Section 10.3.1.4.2).
Results differ in three studies of long-term (3- to 6-month) exposures of
3
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
using only LM to study effects in rats exposed for 6 months to 980 (jg/m3 (0.5
ppm) for 6 hr/day, Cavender et al. (1978) did r t find exposure-related lesions.
Using SEM and TEM to supplement the LM technique, Boorman et al. (1980) and
Moore and Schwartz (1981) reported significant bronchiolar lesions following
exposure to 980 or 1568 ug/m3 (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
luminai 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 bronchiolar 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.
0190PT/B 10-31 5/1/84
-------
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. 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 morphology persisted, the changed
segment reduced in length after the 180-day-exposed rats had breathed filtered
air for 62 postexposure days. The addition of HpSO. to these concentrations
of 0., for the same exposure times did not potentiate the lesions seen in the
O
03-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
3
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 03 levels (980
or 1568 pg/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.
Guinea pigs were exposed by Cavender et al. (1977, 1978) continuously to
1 or 2 ppm of 0,. for 2 or 7 days in acute studies and to 980 ng/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-c, .rway 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
HUSO, aerosol and 03-
10.3.1.1.2 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.
0190PT/B 10-32 5/1/84
-------
10.3.1.1.2.1 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 0, 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).
Short-term exposures of monkeys to 392, 686, 980, or 1568 ug/m (0.2, 0.35,
0.5, or 0.8 ppm) of 03 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 07 concentration.
o
The pathogenesis of the lesions due to 1568 ug/m (0.8 ppm) of 03 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, 8, and 12 hr of
exposure. Type 1 cell necrosis, which resulted in bare basal lamina, 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
stem cells in renewal epithelium, and both contributed to the hyperplasia seen
at the latter exposure times. The inflammator^ exudate included both fibrin
and a variety of leukocytes in the early phases. In the latter phases, the
inflamnatory 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.
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 al. , 1981). After the 90-day exposure, the percent of
cuboidal respiratory bronchiolar epithelial cells was 90 percent rather than
0190PT/B 10-33 5/1/84
-------
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 seven-fold those of controls. This study did not include a
postexposure period.
In an earlier study, Freeman et al. (1973) exposed female beagle dogs to
2
1960 ug/m (1 ppm) of 0~ 8, 16, or 24 hr/day for 18 months. Dogs exposed to
3
1960 ug/m (1 ppm) of 03 for 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
and proximal alveoli. Epithelial hyperplasia was seen sporadically in the
bronchiolar-ductal zone. Other dogs in that study exposed to 3920 or 5880
ug/m (2 or 3 ppm) of 0- 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.
10.3.1.1.2.2 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 al., 1974b; Schwartz et al., 1976; Mellick et al.,
1977). 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 th.ckening of the interalveolar
septa. In animals with respiratory bronchioles (e.g., dog, monkey) the alve-
oli involved at low concentrations are those opening directly into the respira-
tory 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
3
al., 1977). In monkeys exposed tp 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.
0190PT/B 10-34 5/1/84
-------
Type 1 cell damage and loss have been reported in rats after a 2-hr ex-
3
posure to 392 (jg/m (0.2 ppm) of 0~ (Stephens et al., 1974a). 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
3
980 ug/m (0.35 or 0.5 ppm) of 0, (Evans et al., 1976b). Although the labeling
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 follow-
3
ing 90 days of exposure to 1568 ug/m (0.8 ppm) by Boorman et al. (1980).
They interpreted these intermediate cells as due to delay or arrest of the
transformation 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
3
exposed to 490 ug/m (0.25 ppm) of 03 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. Sherwin
et al. (1983) found increased numbers of lactate dehydrogenase (LDH)-positive
cells, presumed to be type 2 alveolar epithelial cells, by automated LM morph-
3
ometry of lungs from mice exposed to 588 ug/m (0.3 ppm) of 03 for 6 weeks.
Moore and Schwartz (1981) reported nonciliated bronchiolar cells lined some
alveoli opening into the transformed airways located between terminal bronchi-
oles and alveolar ducts of rats exposed to 1568 ug/m (0.8 ppm) of 03 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
3
early as after 4 hr of 1568 ug/m (0.8 ppm) of J, exposure (Castleman et al.,
1980). In rats, the numbers of inflammatory cells per centriacinar alveolus
q
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 \ig/m (0.5 ppm) of 03 and after 62 days of filtered
air following 180 days of exposure. The addition of H?S04 aerosol did not
result in larger increases. In monkeys, the intensity of the response was
greater than in rats exposed to the same 03 concentration (1568 ug/m3, 0.8
ppm) in the same regimen (8 hr/day) for 7, 28, or 90 days (Eustis et al.,
0190PT/B 10-35 5/1/84
-------
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).
The interalveolar septa of centriacinar alveoli are thickened following
exposure to ambient concentrations of 0~. 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
in rats exposed to 1568 pg/m (0.8 ppm) of 0- 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. This study is especially
important, because it is quantitative and concerns rats exposed only 8 hr/day
3
to 1568 ug/m (0.8 ppm) of 0,.
3
Moore and Schwartz (1981), after exposing rats to 980 ug/m (0.5 ppm) of
03 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
dogs exposed to 1960 to 5880 ug/m3 (1 to 3 ppm) of 03 8 to 24 hr/day for 18
months.
In several biochemical studies, Last and colleagues have shown that 0~ is
collagenic. In one of these (Last et al., 1979), Me biochemical observations
were correlated with histological observations of slides stained for collagen
and reticulin. Elevated collagen synthesis rates were found at all concentra-
tions and times studied. Mildly increased amounts of collagen were seen
morphologically in centriacinar alveolar duct septa from most rats exposed to
3
980 ug/m (0.5 ppm) of 0, 24 hr/day for 14 or 21 days. More severe lesions
3
were seen in rats exposed to 1568 to 3920 ug/m (0.8 to 2.0 ppm) of 0, 24 hr/day
for 7, 14, or 21 days.
Only one published report addressed the biologically important question
of the morphological effects that follow multiple sequential exposures to 03
with several days of clean air interspersed between 0- exposures (i.e., a
0190PT/B 10-36 5/1/84
-------
multiple episode exposure regime). Plopper et al. (1978) exposed rats to 1568
3
ug/m (0.8 ppm) 0- continuously for 3 days, held them in the chambers breathing
filtered air until postexposure day 6, 13, or 27, when they were again exposed
3
to 1568 pg/m (0.8 ppm) of 0, 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 0_ 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 the first exposure.
10.3.1.1.3 Vasculature, blood, and lymphatics. Although edema is the apparent
cause of death due to inhalation of high concentrations of 0-, there is very
3
little morphological evidence of pulmonary vascular damage due to < 1960 ug/m
U 1-0 ppm) of Oo exposure. Bils (1970) reported capillary endothelial damage
3
in mice less than 1 month of age exposed for 7 hr to 1960 ug/m (1 ppm) of 03>
but this experiment has not been confirmed by others. Boatman et al. (1974)
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 03 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 endothelium in both groups
exposed to 784 ug/m (0.4 ppm) of 0, 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 ^30 or 1764 ug/m (0.5 or 0.9
3
ppm) of 0, 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 0_ 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.
0190PT/B 10-37 5/1/84
-------
3
The earlier report of arterial lesions in rabbits exposed to 784 ug/m
(0.4 ppm) of 03 6 hr/day, 5 days/week for 10 months by P'an et al. (1972) has
not been confirmed by other investigators, because no other long-term exposure
of rabbits has been reported. Possibly, susceptibility to arterial lesions
was specific to this animal species, or these rabbits may have had intercurrent
infectious 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."
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 0-, blood capillary endothelial damage has not been reported, and
<3
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.
10.3.1.2 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) found minimal type 1 alveolar epithelial cell damage
3
after a 2-hr exposure to 392 ug/m (0.2 ppm) of 0- and more significant damage
3
2 hr after exposure to 980 ug/m (0.5 ppm) of 03- Boatman et al. (1974) found
lesions in both the conducting airways and paren iyma of cats exposed to 510,
3
980, or 1960 ug/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).
Severa' 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, Mel lick
0190PT/B 10-38 5/1/84
-------
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 |jg/m (0.5 or 0.8 ppm) of 0- 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 pg/m (0.2
and 0.35 ppm) of 03 8 hr/day for 7 days. In the centriacinar region, this
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 0~.
•3
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 al., 1974a,b; Castleman et al., 1980). Repair of the damage,
as indicated by DMA 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 dayi 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).
10.3.1.3. Structural Elements Affected
10.3.1.3.1 Relative sensitivity of cell types. The ciliated cell of airways
and the type 1 (squamous) alveolar epithelial cell are the cells most sensitive
to the effect of inhaled C<3 (Stephens et al., 1974a,b; Schwartz et al. , 1976;
Mel lick et al., 1977; Castleman et al., 1980; Eustis et al. , 1981). The
amount of damage to these sensitive cells varies with their position in the
0190PT/B 10-39 5/1/84
-------
respiratory system. As mentioned earlier, 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 cells in terminal bronchioles of animals without respiratory
bronchioles (i.e., rats) are severely damaged by even low concentrations of 03
(Stephens et al., 1974a; Schwartz et al., 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 al., 1977).
In a similar manner, type 1 alveolar epithelial cells located in the
centriacinar region are subject to damage by low concentrations of 03, 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; Castle-
man et al., 1980).
Although type 2 alveolar epithelial cells are relatively resistant to 03,
some in centriacinar locations develop mild lesions detectable with the TEM
(Castleman et al., 1980). 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 03 lesion
(Evans et al., 1976b).
Although nonciliated cuboidal bronchiolar cells are more resistant to 03
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 03 damage (Schwartz et al., 1976). Following exposure to 1960
ug/m3 (< 1 ppm) of 03, their height is reduced and their luminal surface is
more granular (Schwartz et al., 1976). The red ction in height appears to be
due to a loss of smooth endoplasmic reticulum (Schwartz et al., 1976).
Several investigators report that type 3 alveolar epithelial cells, the
brush cells, are not damaged by less than 1 ppm of 03 (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 much less sensitive 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). In the earlier reports, damaged endothelial
cells were those located immediately deep to denuded basal lamina and resulted
0190PT/B 10-40 5/1/84
-------
from sloughing of type 1 epithelial cells in the centriacinar 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 CL 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). Only one morphometric
evaluation of the centriacinar interalveolar septa is in the open literature
(Boorman et al., 1980), and it does not provide detail concerning specific
cell types and extracellular materials.
Mucous-secreting cells in conducting airways appear relatively resistant
to 0.,. Boatman et al. (1974), in studies of cats exposed to < 1960 ug/m (g
1.0 ppm) of 0,, 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
mucous cells from monkeys exposed to 392 or 686 ug/m (0.2 or 0.35 ppm) of 03
8 hr/day for 7 days. Mellick et al. (1977) mention that mitochondria! 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 ug/m (0.5 or 0.8 ppm)
of 0- 8 hr/day for 7 days. Schwartz et al. (1976), who reported mucigen
droplets being released from the apical surfaces of mucous cells and mucous
droplets trapped among cilia, did not find charjes suggesting damage to organ-
el les 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. No reports of damage to conducting airways other
than to ciliated and mucous cells were found.
10.3.1.3.2 Extracellular elements (structural proteins). Although physiologic
and biochemical changes following 0- exposure suggest changes in the extracell-
ular 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. Three studies
provide morphological evidence of mild fibrosis (i.e., local increase of
0190PT/B 10-41 5/1/84
-------
collagen) in centriacinar interalveolar septa following prolonged exposure to
3
< 1960 ug/m (< 1 ppm) of 03 (Last et a!., 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). Additional evidence
of more collagen or changes in collagen location is in the report of dogs
exposed to 1960 or 5880 ug/m (1 or 3 ppm) of 03 for 18 months (Freeman et
al., 1973).
10.3.1.3.3 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
(^ 1.0 ppm) of 0, for short or long exposure periods (Schwartz et al., 1976;
Cavender et al. , 1978; Mel lick 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 0- for several hours
to 1 week. Interstitial edema is not reported following longer-term (i.e.,
3
weeks to months) exposure to < 1960 ug/m (< 1 ppm) or less (Cavender et al.,
1977; Eustis et al., 1981; Boorman et al., 1980; Moore and Schwartz, 1981;
Zitnik et al., 1978). Biochemical indicators of edema are described in
Section 10.3.3.
10.3.1.4 Considerations of Degree of Susceptibility to Morphological Changes
10.3.1.4.1 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 respr .d 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.
10.3.1.4.1.1 Vitamin E deficiency. Rats maintained on vitamin E-deficient
diets tended to develop more morphological lesions following 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
3
average U. S. human adult intake were exposed to 196 or 392 |jg/m (0.1 or
0.2 ppm) of OT 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
0190PT/B 10-42 5/1/84
-------
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 pg/m (0.1
ppm) Oo for the same period, LM revealed no increased centriacinar macrophages.
O
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
o
exposed to 196 pg/m (0.1 ppm) of 0-j 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
they were exposed either to filtered air or to 196 yg/m (0.1 ppm) of 03
continuously for 7 days. The morphology of six rats from each diet and exposure
group was studied using SEH, None of the filtered-air control animals had
lesions. Of the rats exposed to 0^, five of the six on the vitamin E-deficient
O
diet, four of six on the ceficient 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 0., 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 pg/m (1.3 ppm) of 0, 3 hr daily for 16 consecutive days or
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 03 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, Og-exposed rats. They did find mild centriacinar
0- 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 M9/m (0-9 ppm) of 03 for 1, 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
0190PT/B 10-43 5/1/84
-------
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 10.3.3.
10.3.1.4.1.2 Acje___at sta_rt__of__gxp_os.ure. Although most exposures use
young adult experimenter animals, there are a few reports of exposures of very
young animals (i ». , either before weartinc or very soon thereafter).
Bartlett ^t al. (197'--) exposG-5 3- to 4-week-old rats to 392 pg/m (0.2
ppm) of Q, for 30 days. Lung volumes, • ut not body weights, were significantly
greater in the exposed rats. Light w>;:"o$>cepy of paraffin sections of conven-
tionally fixed lungs did net rovudl drr';:eri:ices between exposed and control
rats in the lung parenchyma cr '.en; i •>;• bro.,-:,n"o seas with the exception of two
control animals which had 'lesi>.'.v-. -j'= "•*,yp1r&i in wine pneumonia." Morphometry
was done on thick sections art by nar.d with a razor blade from the dorsal and
lateral surfaces of aJr-d'^'Osj h.-j.^b >. a'.".",*-jr ;han c;; the paraffin sections of
conventionally fix-^d 'uncjs. Hor,.i'.o,T,et.ry tiaincj these nonrandom samples revealed
significantly increased mean ah/ec'-a" chord "engths and alveolar surface area,
but no difference in alveolar nu^bess.
Freeman et al. (1974: 9*pr,s«.G irontrrolri rats to ]058 or 1725 |jg/m' (0.54
or 0.88 ppm) of 0^ for period-. ;f iVorr. 4 irr to 3 weeks. In addition to the
centriacinar accumulation? of iTi<:.C'Ophoget> and hyperp'lasia of distal airway
epithelium seen by ethers foT'.ovM.c exposures of young adult animals, they
reported an increase in connect-t c!. H974) studied month-old rats exposed
3 3
to 1764 jjg/m (0.3 ppiiij of 0- *••• t:}u ir^/in' (0.5 ppm) nitrogen dioxide combined.
,';
After (50 days of expos-jr?, ^r,fJ -^'-..>. vvi5 the yoss and microscopic appearance
of advanced experimental e,nphy-:.tyi of \,he type xbey earlier described following
nitrogen dioxide exposure (frecmsr; ei. al. , 1372). Although others have reported
larger fixed lung volumes in .:?xpo~,ee; young adult rats (Moore and Schwartz
1981), reports of emphysema !V;'uvr.nc; 0,, exposures are uncommon and are discus-
sed in the next subsection of chir. document.
0190PT/B 10-44 5/1/84
-------
Stephens et al. (1978) exposed rats at 1, 5, 10, 20, 25, 30, 35, and
3
40 days of age to 1666 ug/m (0.85 ppm) of 0, continuously for 24, 48, or
72 hr. Rats exposed to 0~ before weaning at 20 days of age developed little
or no evidence of injury, as evaluated by light and electron microscopy.
Following weaning at 20 days of age, centriacinar lesions increased progressive-
ly, 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
ug/m (0.25 ppm) of 0, 12 hr/day for 6 weeks. They observed persistence of
the centriacinar damage to type 1 epithelial cells and increased centriacinar
macrophages. 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 discus-
sed 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. (1983) are discussed in Section 10.3.2.
Bils (1970) studied the effects of 1176 to 2548 [jg/m3 (0.6 to 1.3 ppm) of
03 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 0^, a conclusion
not supported by more recent studies, which deal mostly with other species.
Bils did note the lesions were more severe in the 4~day-old mice than in the
1- or 2-month-old mice.
10.3.1.4.1.3 Effect of pneumonectomy. Two to four weeks following
pneumonectomy of rabbits, the centralateral 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 pneuinonectomized and control rabbits to 784 (jg/m (0.4 ppm)
of 0-, 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 0~ exposure and that no difference existed between males and
females in this response.
10.3.1.4.2 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
0190PT/B 10-45 5/1/84
-------
3
following exposure to < 1960 |jg/m (< 1 ppm) of 0- 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.s 1980; Wright et al., 1983) 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). Thus, a restudy of these three reports in the 1978
document appears appropriate.
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/
week for 14.5 months to a mean concentration of slightly more than 1960 pg/m
(1 ppm) of 0-. 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 (CIBA, 1959) or enlarged air spaces accompanied by
destructive changes in alveolar walls (World Health Organization, 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). 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 the rats, the mild degree of emphysema in those
exposed "did not exceed the emphysema found in the unexposed control rats."
In the hamsters, "mild to moderate" emphysema was present in exposed animals,
but not 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 presentation at the seventh Aspen Conference on
Research in Emphysema, reviewed the lesions in the hamsters from this exposure
and described a "destructive process" that resulted in contraction of inter-
alveolar septa not associated with enlargement of air spaces.
0190PT/B 10-46 5/1/84
-------
The earlier 0., criteria document (U.S. Environmental Protection Agency,
«j
1978) cites Stephens et al. (1976), a "long abstract" that appears not to be
refereed. This brief article states "rats exposed continuously for long
3 3
periods (3-5 months) to 28,200 ug/m (15.0 ppm) N02 or 1568 ug/m (0.8 ppm)
0- develop a disease that closely resembles emphysema" but does not provide
O
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 NO^-exposed animals.
The fourth reference (Freeman et al., 1973) is to an exposure of dogs to 1960
to 5880 |jg/m (1 to 3 ppm) of 0., 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 group of month-old rats exposed continuously for
3
3 weeks to 1725 ug/m (0.88 ppm) of 03, half of which died and had "grossly
inflated, dry lungs." In this same study, they also exposed month-old rats to
3
a mixture of 1690 yg/m (0.9 ppm) of N02 and 03 continuously for 60 days, at
which time the lungs were grossly enlarged, and "both grossly and microscopical-
ly, the appearance of the lungs was characteristic of advanced experimental
emphysema" of the type they earlier reported followed N02 alone at much higher
concentrations (Freeman et al., 1972).
The third citation in the 0- criteria document (U.S. Environmental Protec-
tion Agency, 1978) is to P'an et al. (1972). These investigators exposed
rabbits to 784 ug/m (0.4 ppm) of 03 6 hr/day, 5 days/week for 10 months.
Tissues were apparently fixed by immersion rather than infusion via the trachea,
which Is not in accord with the American Thoracic Society's diagnostic standard
for emphysema and makes emphysema lesions much more difficult to accurately
evaluate. 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
congested that visualization of the mural framework of the alveoli was difficult."
This is more reaction than reported in other species exposed to this compara-
tively low 0, concentration. The rabbits were not specified pathogen free nor
O
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.
0190PT/B 10-47 5/1/84
-------
10.3.2 Pulmonary Function Effects
10.3.2.1 Short-Term Exposure. Results of short-term 0~ exposures of experi-
mental animals are shown in Table 10-2. These studies were designed to evalu-
ate the acute changes in lung function associated with 0., 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 0- exposure of the lung periphery have
been examined in dogs by Gartner et al. (1983a,b). A fiber-optic bronchoscope
with an outside diameter of 5.5 mm was wedged into a segmental airway and a
continuous flow of 196 or 1960 ug/m (0.1 or 1.0 ppm) of 03 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
3
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
(1.0 ppm) of 0,, but decreased again to control levels during continued expo-
3
sure to 196 ug/m (0-1 PPm) °f 63- Based on these observations, the authors
reported that tolerance appears to develop in the collateral airways to locally
3
delivered 03 at concentrations of 196 but not 1960 (jg/m (0.1 but not 1.0 ppm)
of 03.
Amdur et al. (1978) measured breathing 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 Q-. Accelerated respiration rates with no significant
o
changes in tidal volume were measured during exposures to all 0^ 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
to 804 or 1568 ug/m (0.41 or 0.8 ppm) of 03> 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 0.,. 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.
0190Z4/A 10-48 5/1/84
-------
TABLE 10-2. EFFECTS OF OZONE ON PULMONARY FUNCTION: SHORT-TERM EXPOSURES
O
i
Ozone
concentration Measurement :
ug/mj
196
1960
431
804
1568
470 to
2156
510
980
1960
666
1333
2117
2646
980
ppm method
0.1 MAST
1.0
0.22 CHEM,
0.41 NBKI
0.80
0.24 to NBKI
1.1
0.26 MAST
0.5
1.0
0.34 NBKI
0.68
1.08
1.35
0.5 NBKI
. Exposure
' duration
& protocol Observed effects0 Species
30 min Collateral system resistance increased Dog
rapidly during exposure, falling to
control levels at 0.1 ppm tout con-
tinuing to increase at 1.0 ppm of 03.
2 hr Concentrati on- dependent increase in fR Guinea pig
for all exposure levels. No change in (200-300 g)
R. , TV, or MV. Decreased C.dyn during
exposure to 0.4 and 0.8 ppm of 03.
12 hr Premature airway closure at 6 hr, and Rabbit
1 and 3 days following exposure, reflec-
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.
2.0 to Concentration-dependent increase in R. Cat
6.5 hr 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.
2 hr Increased fg and decreased TV during Guinea pig
exposure to all 0, concentrations. (300-400 g)
Increased R during exposure to 1.08
and 1.35 ppfo of 03.
2 hr Slight increase in fg and R (to 113% Guinea pig
of control values) during exposure. (280-540 g)
Reference
Gertner et al. ,
1983a,b
Amdur et al . , 1978
Inoue et al. , 1979
Watanabe et al . ,
1973
Murphy et al. , 1964
Yokoyama, 1969
-------
TABLE 10-2. EFFECTS OF OZONE ON PULMONARY FUNCTION: SHORT-TERM EXPOSURES (continued)
o
i
en
o
Ozone
concentration Measurement
(jg/m3 ppm method
1470 0.75 CHEM
1960 1.0
3920 2.0
1960 1 NBKI
1960 1 NBKI
. Exposure
' duration
& protocol
Continuous
1, 3, 7 or
14 days
3 hr
6 hr/day,
7 to 8 days
Observed effects Speoes 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 ppm of 03).
Reduced TLC at air inflation pressure Rabbit Yokoyama, 1972, 1973
of 30 cm 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 MEFV curves.
Measurement method: MAST = Kl-coulometric (Mast meter); CHEM - gas phase chemiluminescence; NBKI = neutral buffered potassium iodide.
Calibration method: NBKI = neutral buffered potassium iodide.
cSee Glossary for the definition of pulmonary symbols.
-------
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 (jg/m (0.26, 0.50, or
1.00 ppm) of 0., 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
3 3
to 510 |jg/m (0.26 ppm) of 0-, after 63 min of exposure to 980 (jg/m (0.50 ppm)
3
of 0~, and after 49 min of exposure to 1960 ug/m (1 ppm) of 03- Dynamic lung
compliance was decreased during 0~ exposure in the Watanabe et al. (1973)
study, as it was in the A«ndur 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
during 2-hr 0, exposures. These investigators found concentration-related
3
increases in respiration rate during exposure to 666, 1333, 2117 or 2646
(0.34, 0.68, 1.08, or 1.35 ppm) of O,. Respiratory flow resistance was increased
(to 148 and 170 percent of pre-exposure values) in guinea pigs exposed to 2117
3
and 2646 ug/m (1.08 and 1.35 ppm) of 03 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 03 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
0190Z4/A 10-51 5/1/84
-------
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™.
Inoue et al. (1979) exposed rabbits to 470 to 2156 ug/m3 (0.24 to 1.1 ppm)
of 0- 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, 1 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
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 0~-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 O^-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 HLO, 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
0190Z4/A 10-52 5/1/84
-------
(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
3
to 1960 |jg/m (1 ppm) of 0~, 6 hr/day, for 7 to 8 days. He found increased
pulmonary resistance and decreased dynamic compliance in CL-exposed animals
compared to air-exposed control animals. Static pressure-volume curves and
maximum expiratory flow-volume curves were not significantly different between
the two groups.
10.3.2.2 Long-Term Exposure. Table 10-3 summarizes results of long-term 0~
exposures. Raub et al. (1983a) exposed neonatal and 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 0^ exposure. Peak inspiratory flow measured in these
animals during spontaneous respiration was significantly lower following
3
exposure to 235 or 490 ug/m (0.12 or 0.25 ppm) of 0~. Lung volumes measured
at high distending pressures were significantly higher in neonatal animals
3
exposed to 490 ug/m (0.25 ppm) of 0- 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) following exposure
3
of young rats (3- to 4-week-old) to 392 ug/m (0.2 ppm) of 0, 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
3
fixative) after 180 days cf continuous exposure to 980 ug/m (0.5 ppm) of 0^.
Yokoyama and Ichikawa (1974) found no change in lung static pressure-volume
curves in mature rats exposed to 882 |jg/m (0.45 ppm) of 0., 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 HpO was also reported following 0- exposure, which is
similar to the fixed lung volume changes reported by Moore and Schwartz (1981).
Morphology and morphometry of paired lungs or lungs from animals similarly
exposed to 03 is reported in Section 10.3.1.4.
0190Z4/A 10-53 5/1/84
-------
TABLE 10-3. EFFECTS OF OZONE ON PULMONARY FUNCTION: LONG-TERM EXPOSURES
o
en
Ozone
concentration
ug/mj
157
235
490
392
392
1568
3920
784
882
980
1568
ppm
0.08
0.12
0.25
0.2
0.2
0.8
2.0
0.4
0.45
0.5
0.8
Exposure
Measurement ' duration
method & protocol
CHEM 6 weeks,
12 hr/day,
7 days/week
MAST, 28 to 32 days,
N6KI continuous
UV or CHEM 62 exposures,
NBKI 6 hr/day,
5 days/week
NBKI 6 weeks,
7 hr/day,
5 days/week
MAST 6 to 7 weeks,
6 hr/day,
6 days/week
UV, 7, 28, or
NBKI 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
0_-exposed rats at high lung
vBlumes (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 and FEF25 and FEFto
were decreased at 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 quasi static compliance (not
related to concentration).
Species Reference
Rat Raub et al. , 1983
(neonate or
6-week-old
young adult)
Rat Bartlett et al. ,
(3 to 4 weeks) 1974
Rat Costa et al. , 1983
(10 weeks)
Rabbit Martin et al. ,
1983
Rat Yokoyama and
Ichikawa, 1974
Monkey Eustis et al . , 1981
(Bonnet)
-------
TABLE 10-3. EFFECTS OF OZONE ON PULMONARY FUNCTION: LONG-TERM EXPOSURES (continued)
o
i
01
Ozone
concentration
ug/nr1 ppm
1254 0.64
Exposure
Measurement ' duration
method & protocol
UV, 1 year,
NBKI 8 hr/day,
7 days/week
Observed effects Species
Following 6 months of exposure, venti- Monkey
lation was less homogeneous and R, (Bonnet)
was increased. Following 12 months
of exposure, R. remained elevated
and forced expiratory maneuvers showed
small airway disfunction (decreased
FEVj and FEFl2.s). During the 3-month
recovery period following exposure,
C.st decreased.
Reference
Wegner, 1982
Measurement method: MAST = Kl-coulometric (Mast meter); CHtM = gas phase chemiluminescence; UV - UV photometry.
Calibration method: NBKI = neutral buffered potassium iodide.
See Glossary for the definition of pulmonary symbols.
-------
PRELIMINARY DRAFT
Costa et al. (1983) evaluated lung function changes in rats exposed to
3
392, 1568, or 3920 ug/m (0.2, 0.8, or 2 ppm) of 0.,, 6 hr/day, 5 days/week for
62 exposure days. (This report will not discuss effects in animals exposed to
2 ppm of 0~). These investigators found increased pulmonary resistance (not
3
concentration related) in rats exposed to 392 or 1568 ug/m (0.2 or 0.8 ppm)
of 0-j. Lung volumes measured at high distending pressure (VC and TLC) were
3
increased following exposure to 1568 ng/m (0.8 ppm) of 0,. Similar changes
in lung distensibility were observed by Raub et al. (1983a), Bartlett et al.
(1974), Moore and Schwartz (1981), and Martin et al. (1983). Costa et al.
(1983) also observed decreased (not concentration-related) maximum expiratory
flows at low lung volumes (25 and 10 percent of VC) in rats exposed to 392 or
1568 |jg/m (0.2 or 0.8 ppm) of 0,. Changes in maximum flow at low lung volumes
O
indicate peripheral airway dysfunction and may be related to decreased airway
stiffness or narrowing of the airway lumen.
Eustis et al. (1981) evaluated lung function in bonnet monkeys (Macaca
radiata) exposed to 980 or 1568 ug/m (0.5 or 0.8 ppm) of 03 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, quasistatic lung compliance was significantly different in
Oo-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
were exposed to 1254 ug/m0 (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 (DITQ)>
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.
0190Z4/A 10-56 5/1/84
-------
PRELIMINARY DRAFT
Following six months of 0~ exposure, pulmonary resistance and frequency
dependence of pulmonary compliance were significantly increased. After 12
months5 the CL exposure had significantly increased pulmonary resistance and
O
inertance (related to the pressure required to accelerate air and lung tissue),
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 CL-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 0.,-exposed and control animals. However, the
decrease in compliance was significantly greater in CL-exposed animals than in
control animals. No other significant differences were measured following the
3-month recovery period, although values for 0,-exposed animals remained
substantially different from those for control animals, suggesting that full
recovery was not complete.
10.3.2.3 Airway Reactivity. Ozone potentiates the effects of drugs that con-
strict airway smooth muscle in mice, guinea pigs, dogs, sheep, and humans
(Table 10-4). Early experimental evidence for hyperreactivity to broncho-
constrictive drugs following 00 exposure was provided by Easton and Murphy
(1967). Although much of their work was done with very high Oq concentrations
3
(9800 to 11760 ng/m , 5 tc 6 ppm), they did show that mortality from a single
subcutaneous injection of histamine was higher in guinea pigs exposed to 980
or 1960 (jg/m (0.5 or 1 ppm) of 0~ 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.
Abraham et al. (1980) evaluated airway reactivity in sheep from measure-
ments of pulmonary resistance following inhaled carbachol aerosols. Carbachol
causes bronchoconstrictior 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
0190Z4/A 10-57 5/1/84
-------
TABLE 10-4. EFFECTS OF OZONE ON PULMONARY FUNCTION: AIRWAY REACTIVITY
o
-I
CDl
Ozone
concentration
ug/m3 ppm
196 to 0.1 to
1568 0.8
196 0.1
1568 0.8
196 0. 1
1960 1.0
980 0.5
980 0. 5
1568 0.8
980 0.5
2156 1.1
980 0.5
1960 1-
3920 2
Exposure
Measurement3 duration
method & protocol
CHEM 1 hr
CHEM 1 hr
MAST 10-30 min
CHEM 2 hr
MAST Continuous,
13 to 16 days
of 0- 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
Subcutaneous injection of histamine 2 hr following 0,
exposure caused a greater increase in R, following expo-
sure to 0.8 ppm of 03 and a greater decrease in C.dyn
following exposure to all 0., concentrations (magni-
tude of C. changes not related to 0., concentration).
Decreased diaphragm and lung cholinesterase activity;
parathi on-treated animals had increased peak airway
resistance compared to controls, but the difference was
not statistically significant.
Bilateral vagotomy: completely Dlocked increased peri-
pheral lung resistance from 0.1 ppm of 03 but not histamine;
only partially blocked response from 1.0 ppm of 03.
Hi stamine- 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
lavage 24 hr after exposure.
Repeated exposures to 0.5 or 0.8 ppm of 03 plus aerosolized
ovalbumin resulted in greater mortality from anaphy lactic
shock produced by intravenojection of ovalbumin compared
with effects of ovalbumin injection in mice repeatedly
exposed to ovalbumin aerosols but no 0,.
«J
Increased histamine- induced mortality immediately
following exposure to 0.5 or 1.1 ppm of 03.
Increased airway reactivity to aerosolized carbachol
24 hr but not immediately following exposure to
980 ug/m3 (0.5 ppm) of 0., with no change in R, ,
FRC, C.st, or tracheal mucous velocity. Increased
R, 24 nr following exposure and airway reactivity
immediately and 24 hr following exposure (1 ppm).
Species Reference
Guinea pig Gordon and Amdur,
(200-300 g) 1980
Guinea pig Gordon et a!., 1981
Bog Gertner et al. ,
1983a,b,c
Kaplan et al . ,
1981
Sheep Sielczak et al., 1983
Mouse Qsebold et al.,
1980
Guinea pig Easton and Murphy,
1967
Sheep Abraham et al., 1980
-------
TABLE 10-4. EFFECTS OF OZONE ON PULMONARY FUNCTION: AIRWAY REACTIVITY (continued)
Ozone
concentration Measurement
ug/m3 ppm method
1100 to 0.56 to CHEM
1666 0.85
Exposure
duration
& protocol Observed effects Species
2 hr Abnormal, rapid, shallow breathing in conscious dogs Dog
while walking on a treadmill following 0, exposures.
Maximal 1~ to 3-hr postexposure, normal z4-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 .
Reference
Lee et al. , 1979
1313
O
i
0.67
CHEM
2 hr
Abnormal, rapid, shallow breathing during exposure to
air containing low 02 or high CQz immediately following
O- exposure. Abnormal breathing not affected by
inhaled atropine aerosols or inhaled isoproteronol
Dog Lee et al., 1980
aerosols but abolished by vagal cooling.
1372 to 0.7 to CHEM 2 hr
2352 1.2 j I
1960 1.0 ! UV 2 hr
4312 2.2
5880 3.0
Greater increase in R, caused by histamine aerosol Dog
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
bronchoconstrictlon) prevented 0.,-induced reactivity
to histamine.
Marked increase in airway responsiveness to ACh and Dog
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.
Lee et al . , 1977
Holtzman et al. ,
1983a,b
Fabbri et al . , 1984
Measurement method: MAST = Kl-coulometric (Mast meter); CHEM = gas phase chemiluminescence; NBKI = neutral buffered potassium iodide.
See Glossary for the definition of pulmonary symbols.
-------
but not significantly higher than pre-exposure values at 24 hr postexposure
3
but not immediately following a 2-hr exposure to 980 |jg/m (0.5 ppm) of Ov
O
This 0-, exposure did not affect resting end-expiratory lung volume (functional
0 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.
Gordon and Amdur (1980) evaluated airway reactivity to subcutaneously
3
injected histamine in guinea pigs following a 1-hr exposure to 196 to 1568 ug/m
(0.1 to 0.8 ppm) of 0-. Airway reactivity to histamine was maximal 2 to 6 hr
following 0^ exposure and returned to control levels by 24 hr following exposure.
The histamine-induced increase in pulmonary resistance was greater in guinea
3
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
0.,-exposed groups than in air-exposed controls, but there were no differences
in the the histamine-induced decreases in pulmonary compliance between any of
3
the 0» concentrations (from 196 to 1568 ug/m ; 0.1 to 0.8 ppm).
Gordon et al. (1981) studied the effect of 0^ 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 1,568 ug/m (0.1 or 0.8 ppm) of 03 for I hr.
After 2 hr, brain, lung, and diaphragm samples were analyzed for cholines-
terase activity. Brain cholinesterase activity was not affected, but lung
cholinesterase underwent a 17 percent decrease in activity at 196 pg/m (0.1 ppm)
3 3
and a 16 percent decrease at 1,568 fjg/m (0.8 ppm). Ozone at 1,568 ug/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 resistance
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 cholinesterase
inhibition by 0~ may contribute to the 0.,-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 Oo exposure, should then
increase the contraction of the bronchus. The persistence of this activity is
not known.
0190Z4/A 10-60 5/1/84
-------
Lee et al. (1977) evaluated airway reactivity in 0.,-exposed dogs from
changes in pulmonary resistance induced by histamine aerosol inhalation. Dogs
were exposed to 1372 to 2352 ug/m (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-
O
teronol aerosols prevented any change in resistance following histamine exposure.
This experiment showed that the increased resistance normally observed follow-
ing 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) de-
creased the response to histamine both before and following 0- exposure and
abolished the hyperreactive airway response. These experiments showed that
the increased sensitivity to histamine following 0, exposure was caused by
heightened activity of vagal bronchoconstrictor reflexes.
Although the work of Lee et al. (1977) suggests that stimulation of vagal
reflexes by histamine is responsible for the increased airway reactivity found
in dogs following ozone exposure, Kaplan et al. (1981) found that local respon-
ses 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 bron-
choscope, collateral airflow resistance increased both before and after bila-
teral cervical vagotomy. In follow-up studies that used similar techniques,
Gertner et al. (1983a,b,c) described the role of vagal reflexes in the response
of the lung periphery to locally administered histamine and Ov Collateral
3
resistance increased during separate 30-min exposures to either 196 |jg/m
-fi 3
(0.1 ppm) of 0., or 1.5 xlO mg/m of histamine. However, although parasym-
pathetic blockade (atropine or bilateral cervical vagotomy) prevented the re-
sponses to 0~, it did not prevent the responses to histamine (Gertner et al.,
3
1983b). In addition, a 30-min exposure to 196 ug/m (0.1 ppm) of 0- did not
affect the airway responsiveness to histamine, but when the 0, exposure was
3
increased to 1960 ug/m (1.0 ppm) for 10 min, histamine produced greater in-
creases in collateral resistance that were not abolished by parasympathetic
3
blockade (Gertner et al., 1983c). Exposure to 1960 |jg/m (1.0 ppm) of Q~ for
0190Z4/A 10-61 5/1/84
-------
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) 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 resis-
tance, 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 the lung may respond differently from the larger conducting
airways during exposure to G~ and that factors in addition to vagal broncho-
constrictor reflexes can produce an increased airway reactivity to histamine.
Holtzman et al. (1983a) reported the time course of 0,-induced airway
3
hyperreactivity in dogs exoosed to 1960 and 4312 (jg/m (1.0 and 2.2 ppm) of 0^
for 2 hrs. Airway responsiveness to acetylcholine in 7 dogs increased markedly
3
1 hr, to a lesser extent, 24 hr after exposure to 4312 ug/m (2.2 ppm) of 0-,
returning to control levels by 1 week after exposure. Ozone-induced increases
in airway responsiveness to histamine were similar following exposure to
3
1960 ug/m (1.0 ppm) of 03, but data were reported for only 2 dogs. The
authors suggested that the time course of the 0- 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-
3
mation in dogs 1 hr following a 2-hr exposure to 4116 ug/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 a!., 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
3
acetylcholine following a 2-hr exposure to 5880 ug/m (3.0 ppm) of 0,. The
3
results of trachea! lavage in sheep exposed to 980 jjg/m (0.5 ppm) of 03 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
0190Z4/A 10-62 5/1/84
-------
(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,
suggesting an association between an enhanced inflammatory response and 0.,-
induced bronchial reactivity reported previously in sheep (Abraham et al. ,
1980). The relationship between airway inflammation and airway reactivity in
other studies at lower concentrations of 0~ is not well understood.
Increased drug-induced bronchoconstriction is not the only indicator of
airway hyperreactivity following 0- 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 11). Lee et al.
(1979, 1980) showed that abnormal, rapid, shallow breathing in conscious dogs
3
immediately following 2-hr exposures to 1100 to 1666 (jg/m (0.56 to 0.85 ppm)
of Oo was a hyperreactive airway response. This abnormal breathing pattern
was elicited by mild exercise, histamine aerosol inhalation, or breathing air
with reduced oxygen (0^) or elevated carbon dioxide (C0?) concentrations. The
rapid, shallow breathing observed in dogs following 0., 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
following 03 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 0, exposure showed damage to the
respiratory epithelium (Section 10.3.1). Damage to the epithelium overlying
sensory receptors may be responsible for the increased receptor reactivity to
mechanical stimulation (increased ventilation with exercise, low 0?, or high
C02) or chemical (histamine) stimulation (Nadel, 1977; Boushey et al., 1980).
The rapid, shallow breathing observed in experimental animals during 0., expo-
sures (Amdur et al. 1978; Yokoyama, 1969; Murphy et al., 1964) may also be
related to increased sensory neural activity coming from the lungs, not to an
indirect effect of changes in airway diameter or lung distensibility as pre-
viously speculated.
0190Z4/A 10-63 5/1/84
-------
In their study of allergic lung sensitization, Osebold et al. (1980)
showed additional functional evidence for epithelial disruption caused by 0,
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 ug/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 0~ 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
aerosolized ovalbumin, 34 percent of the 0--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 ug/m (0.5 ppm) of 0~ 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 03 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
(1970) observed a similar increase in the allergic response of sensitized
guinea pigs to inhaled antigen following 30 min of exposure to 2 ppm 0.,.
10.3.3 Biochemically Detected Effects
10.3.3.1 Introduction. This section will include some studies involving con-
centrations above 1960 ug/m (1 ppm) of 03, 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- are similar across the classes of metabolism.
Lung permeability is discussed in this section, because it is typically detected
0190Z4/A 10-64 5/1/84
-------
biochemically. The final subsection presents hypotheses about the molecular
mechanism(s) of action of 0_, relying on the data presented earlier by metabolic
class.
10.3.3.2 Antioxidant Metabolism. Antioxidant metabolism of the lungs is
influenced by 0_ exposure. As shown in the schematic diagram below (Figure
O
10-3), this system consists of a number of enzymes. As shall be discussed, 03
has been shown to produce several reactive oxidant species iji vitro from com-
pounds found in the lung, as well as in other organs. It is reasonably certain
that 0.( can produce such reactive species in the lung after jj\ 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 0_.
-G.6.p v—NADP + -*-v^ GSH
G-6PD GSH GSH
HMP shunt reductase peroxidase
-A—GSSG**-A*-»» I
6_PG .*_/^-»*NADPH-/V— GSSG *•*-**-»» ROH
Figure 10-3. 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 = glutathione; GSSG -
glutathione disulfide; [O] = oxidizing moiety [i.e., hydrogen
peroxide, free radical, lipid peroxide]; GSH peroxidase =
glutathione peroxidase; GSH reductase = glutathione reductase;
and ROH = reduced form of [O]).
Source: U.S. Environmental Protection Agency (1978).
3
Typically, following exposures to levels of 0_ below 1960 pg/m (1 ppm),
«3
the activities of most enzymes in this system are increased (Table 10-5).
Whether this increase is due to direct mechanisms (e.g., de novo 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
0190Z4/A 10-65 5/1/84
-------
TABLE 10-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE
o
i
en
cr>
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
. 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
1 8 hr/day for
7 days
Observed effect(s)c Species Reference
With vit E-deficient diet, increased levels of GSH and Rat Chow et al., 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-PO 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 succinate-cytochrome C
reductase activity fairly proportional to 03 level.
Similar results for intermittent exposure groups.
1568 0.8
Continuous Increased rates of 02 consumption, reaching a peak
for 1 to 30 at day 4 and remaining at a plateau for the re-
days 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 10-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
o
i
Ozone
concentration Measurement3'
ug/m3
392
686
980
1568
392
980
1568
392
OOA
980
1568
392
980
-| f\ff\
1960
392
OOA
3OU
1960
2352-
1 c f\T)
-LO , U / L.
ppm method
0
0
0
0
0.
0.
0.
0.
0.
0.
0.
0.
1.
0.
.
1.
1.
.
2 I
35
5
8
i
i
2 NBKI
5
8
2 ! MAST,
5 NBKI
8
2 NBKI
5
0
2 NO
0
2-
k Exposure
duration and
protocol
8 hr/day for
7 days
Continuous
for 8 days or
8 hr/day for
7 days
8 or 24 hr/day
for 7 consecu-
tive days
3 hr/day for
4 days
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 ppm.
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 Mouse
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 in 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 10-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
O
I
Co
Ozone
concentration
ug/m3 ppm
392 0.2
980 0.5
1568 0.8
1568 0.8
3920 2
7840 4
392 0.2
980 0.5
1568 0.8
627 0.32
882 0.45
. 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) Species
All 03 levels: increase in NPSH levels; increased Rat
activities of G-6-PO, 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 Monkey
enzymes .
Loss of GSH; loss of SH from lung Rat
raitochrondrial and nricrosomal frac-
tions and inhibition of marker
enzyme activities from these
fractions.
Concentrati on- related increase in 02 consumption. . Rat
In both vit E-supplemented and nonsupplemented Mouse
groups: increased G-6-PO activities and GSH
levels; decreased ACHase activities.
Mice: increased levels/activities of TSH, NPSH, Mouse,
GSH peroxidase, GSH reductase, G-6-PD, 6-P-GD, 3 strains
Reference
DeLucia et al . ,
1975a
j
j
Mustafa et al . , i
1973
Moore et al. , 1980
Mustafa et al . ,
1982
882
0.45
UV
8 hr/day
for 7 days
isocitrate dehydrogenase, cytochrome c oxidase,
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.
03 and 4.8 ppm of N02 alone produced no significant
effects but 03 + N02 produced synergistic effects:
increased total and nonprotein sulfhydryls; increased
activities of succinate oxidase and cytochrome c
oxidase.
of rats
Mouse Mustafa et al. ,
1984
-------
TABLE 10-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
en
10
Ozone
concentration
ug/m3 ppm
882 0.45
980 0.5
1372 0.7
1568 0.8
1470 0.75
1568 0.8
1568 0.8
1568 0.8
3920 2
. Exposure
Measurement ' duration and
method protocol
NO 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
Continuous
for 7 days
NBKI Continuous for
3 days
I Continuous for
10 to 20 days
8 hr
Observed effect(s)c Species; Reference
Increased SOD activity at days 3 and 5, but not Rat
days 2 and 7 of exposure.
Bhatnagar et al. ,
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 monkeys.
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 vitamin E
levels in diet.
Increase in activities of GSH peroxidase, GSH Rat
reductase, G-6-PD, 6-P-GD, and pyruvate kinase
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
and G-6-PD; levels of NPSH; general protein synthesis;
and rate of mitochondrial succinate oxidation.
Decrease to control values 6 to 9 days postexposure.
Re-exposure using same regimen (6, 13, or 27 days
postexposure) resulted in similar elevations.
At the higher concentration: increase in the lung Rat
mitochondrial 03 consumption in oxidation of 2-
oxyglutarate and glycerol-1-phosphate and the number
of type 2 alveolar cells which are rich in mito-
chondria. No change in malonaldehyde. At the
lower concentration: increase in 02 consumption.
Chow and Tappel ,
1973
i
Chow et al., 1976b;
Mustafa et al . ,
1973
-------
TABLE 10-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
Ozone
concentration
ug/m3 ppm
1568 0.8
2940- 1.5-
7840 4
1568 0.8
. Exposure
Measurement ' duration and
method protocol
I < 24 hr
< 8 hr
I 10 days
Observed effect(s)c
NPSH level unaffected at 0.8 ppm of 0
or 1.5 ppm for < 8 hr; decreased at 2
8 hr or 4 ppm for 6 hr. At 4 ppm of
decreased level of GSH; no change in
Species
3 for < 24 hr or Rat
ppm of 03 for
03 for 6 hr,
GSSG level.
Lung SH levels unchanged. Increase in G-6-PO and Rat
and cytochrome c reductase activities. No change
in malonaldehyde levels.
Reference
DeLucia et al . ,
1975b
OeLucia et al. ,
1972
3920
o
I
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.
3920- 2- 30 min In vitro: decrease in SH levels; increase in
5880 3 malonaldehyde levels.
1568 0.8 NO Continuous Increased activity of superoxide dismutase.
for 7 days
1568 0.8 UV 92 hr 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
High mortality in 7- and 12-day-old rats.
1568
0.8
UV
Continuous Diet was constant vitamin E and deficient or sup-
for 5 days plemented (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 10-5. CHANGES IN THE LUNG ANTIOXIDANT METABOLISM AND OXYGEN CONSUMPTION BY OZONE (continued)
Ozone
concentration
ug/m3 ppm
1568 0.8
. Exposure
Measurement ' duration and
method protocol
Observed effect(s)
UV Continuous Diet was constant vitamin E and 0 ppm of Se or 1 ppro
for 5 days in
in
GSH
Se. +Se: increased G-6-PD, 6-P-GD; no change
GSH reductase or GSH peroxidase. -Se: decreased
reductase.
Species
Mouse
Reference
Elsayed et al . ,
1983
Both diet groups had increase in TSH and NPSH, and
lung Se levels after 03.
1568 0.8
1764 0.9
1764 0.9
NBKI continuous Vitamin E partially prevented increased activities
for 7 days of
of
Ho
G-6-PD, 6-P-GD, and malic enzyme. Activities
phosphofructokinase and pyruvate kinase increased.
effect on aldolase and malate dehydrogenase.
CHEM 96 hr Trend towards decreased activities of GSH reductase
GSH
by
peroxidase, G-6-PO before 18 days of age, followed
increases thereafter. For G-6-PD: no change at 5
Rat
Rat
(5-180
days old)
Chow and Tappel ,
1973
Tyson et al . ,
1982
and 10 days of age; decrease at 15 days, and Increase
at
MAST > 96 hr 96
25 and 35 days.
hr: No effect below 20 days of age; G-6-PD in-
creases thereafter up to 35 days, after which (40
anc
at
mal
age
50 days old) it decreases. When exposure started
25 or 32 (but not 10 to 15) days of age, the maxi-
increase in G-6-PD occurred at about 32 days of
under continuous exposure conditions.
Rat
(10-50
days old)
Lunan et al . ,
1977
Measurement method: MAST = Kl-coulometric (Mast meter); NBKI = neutral buffered potassium iodide; CHEM = gas solid chemiluminescence;
UV = UV photometry;, I = iodometric; ND = 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.
-------
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 10-5). 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 CL 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 0, levels (3920 and 7840 ug/m , 2 and 4 ppm) and
short-term (8 or 24 hr/day, 7 days) exposures to lower 0~ levels (392, 980,
3
1568 ug/m , 0.2, 0.5, 0.8 ppm) on rats. For nonprotein sulfhydryl levels
(principally glutathione, GSH), decreases in the level of GSH were progressive
3
with time of exposure (2 to 6 hr) to 7840 ug/m (4 ppm). For glutathione
disulfide (GSSG), 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
3
7-day continuous exposure to 1568 ug/m (0.8 ppm) of 0_, 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
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 10-5) 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 ug/m (0.2, 0.5, or 0.8 ppm) of 03 and found a
0190Z4/A 10-72 5/1/84
-------
concentrated-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 ug/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 10.3.1). 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
3
392, 980, or 1960 ug/m (0.2, 0.5, or 1.0 ppm) of 0-. Exercise enhanced the
effect. At the lower 03 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 ug/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 had increased by day 3 and reached a peak at 10 days and
then began to return toward control values. GSH peroxidase activity continued
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
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
metabolism. Rats were exposed continuously for 3 days to 1568 ug/m (0.8 ppm)
of 0~, allowed to remain unexposed for 6, 13, or 27 days, and then re-exposed
for 3 days to the same 0- level (Chow et al., 1976b). Immediately after
0190Z4/A 10-73 5/1/84
-------
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 0, toxicity has been
extensively studied, because it typically reduces the toxicity of 0~ 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 ug/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
3
(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 0., 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
ug/m (0.8 ppm) of 07 continuously for 7 days also had changes in 6-phospho-
3
gluconate dehydrogenase activity. Rats were exposed to 1372 to 31,360 ug/m
(0.7 to 16 ppm) of Qo 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
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
ug/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
0190Z4/A 10-74 5/1/84
-------
fed high- or low-PUFA diets. High supplemental levels (105 mg/kg) of vitamin E
acetate were protective and delayed the LT50 to 0- by an average of 15 days.
Although these experiments demonstrate clearly the protective effect of vitamin
E against (L toxicity, they do not support the hypothesis that changes in
fatty acid composition of the lung will increase (L 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
10.3.1) 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
days to 196 or 392 |jg/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
of the responses in the higher vitamin E group at 392 pg/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 ug/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:
0, 11, or 110 ppm (Chow et al., 1981). Rats were exposed to 196 M9/m3 (0.1
ppm) of 0~ continuously for 7 days. In the 0-ppm vitamin E group, 0~ 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.
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 10.3.1), 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.
0190Z4/A 10-75 5/1/84
-------
3
Chow and Tappel (1972) exposed rats continuously to 1372 ng/m (0.7 ppm)
of 0- for 5 days. The animals had been maintained on diets with different
levels of dl-a-tocopherol 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. , 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 a-tocopherol) from the effect of 0~ (1568 ug/m , 0.8 ppm; 7
O
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
ug/m (0.8 ppm) of 0,. continuously for 5 days. In these mice, Se deficiency
•3
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 P3 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
0190Z4/A 10-76 5/1/84
-------
influence the effects of 0~ 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 sparring action
between Se (i.e., GSH peroxidase activity) and vitamin E might explain similar
effects of 0- 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 (jg/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 ug/m (0.5 ppm) of 0~ 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
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
3
after exposures for 8 hr/day for 7 days to levels as low as 392 ug/m (0.2
0190Z4/A 10-77 5/1/84
-------
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
Long Evans) were compared after a 5-day continuous exposure to 882 ug/m (0.45
ppm) of 0 (Mustafa et al., 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 the
increased activities 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 0_, an innate difference in species sensitivity, or differences in
experimental design (e.g., small sample sizes, insufficient concentration-
response studies).
Age-dependent responsiveness to 0--induced changes in GSH systems has
been observed. Tyson et al. (1982) exposed rats (5 to 180 days old) to 1764
(jg/m (0.9 ppm) of 0_ continuously for 96 hr, except for suckling neonates (5
O
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
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
•3
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-
0190Z4/A 10-78 5/1/84
-------
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.
Ten- to 50-day-old rats were exposed continuously to 1764 ug/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.
3
Elsayed et al. (1982a) exposed rats of various ages to 1568 ug/m (0.8
ppm) of 0~ continuously for 92 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-5-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
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 0^. The
decreased activities observed in the neonates are reminiscent of the decreased
0190Z4/A 10-79 5/1/84
-------
activities that occur at higher 0~ 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 10-5).
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.
3
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 Q* 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 (0~-), a toxic oxidant species thought to be formed from
07 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 mitochondrial fractions of the lungs (Mustafa
et al. , 1977). In a more complex exposure regimen in which rats were exposed
3
for 3 days to 1568 ug/m (0.8 ppm) and then various days of combinations of
2940 ug/m (1.5 ppm) and 5880 ug/m (3 ppm) of 0~, SOD activity also increased.
Bhatnager et al. (1983) studied the time course of the increase in SOD activity
3
after continuous exposure of rats to 882 ug/m (0.45 ppm) of 0.,. 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.
10.3.3.3. Oxidative and Energy Metabolism. Mitochondrial enzyme activities
are typically studied to evaluate effects on Op consumption, which is a funda-
mental parameter of cellular metabolism. Mitochondria are cellular organelles
that are the major sites of 0? utilization and energy production. Many of the
enzymes in mitochondria have functional sulfhydryl groups, which are known to
be affected by 0~, and mitochondrial membranes have unsaturated fatty acids
that are also susceptible to 0-. The patterns of 0, effects on 0? consumption,
as will be discussed, are quite similar to effects on antioxidant metabolism
(Section 10.3.3.2).
0190Z4/A 10-80 5/1/84
-------
Mustafa et al. (1973) showed in rats that acute (8-hr) exposure to a high
3
concentration of 0- (3920 pg/rn , 2 ppm) decreases (L consumption using the
substrates succinate, croxoglutarate, 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 mitochondrial fraction. No
change in malonaldehyde levels was found. When rats were exposed to high CL
3
levels (5880 ug/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 ug/m , 0.8 ppm)
caused an increase in 0^ 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 activity 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 0? 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 ug/m3 (0.2, 0.5, or 0.8 ppm) of 03.
Succinate oxidase activity increased linearly with 0., concentration. No major
differences were apparent between continuous and intermittent exposures. No
statistical analyses were reported. Concentration-dependent morphological
effects were also observed (Section 10.3.1). 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 0? consumption of rats increased with in-
creasing 03 level (392, 980, 1568 |jg/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
3
to 1568 ug/m (0.8 ppm) of 03 continuously for 30 days, and 02 consumption was
measured as the activities of succinate oxidase, 2-oxoglutarate oxidase, and
glycerol-1-phosphate oxidase. On day 1, 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
0190Z4/A 10-81 5/1/84
-------
30 days of exposure. Mitochondria! succi nate-cytochrome c reductase exhibited
a similar pattern under similar CL levels for 7 days of exposure. Equivalent
3
results occurred in rats during a 7-day continuous exposure to 1568
(0.8 ppm) of 0, (DeLucia et al . , 1975a).
3
In rats, recovery from an ozone-induced (1568 yg/m , 0.8 ppm; 3 days,
continuous) increase in succi nate oxidase activity occurred by 6 days post-
exposure (Chow et al . , 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 09 consumption.
3
After 7 days of continuous exposure to 196 or 392 pg/rn (0.1 or 0.2 ppm) of
0~, the lung homogenates of rats maintained on diets with either 11 or 66 ppm
O
of vitamin E were examined for changes in 0^ consumption (succinate oxidase
activity) (Mustafa, 1975; Mustafa and Lee, 1976). In the 11-ppm vitamin E
group, increases in 09 consumption occurred at both 0-. levels. In the 66-ppm
3
vitamin E group, only 392 [jg/m (0.2 ppm) of 0, caused an increase. Mitochon-
dria were isolated from the lungs and studied. Neither dietary group had
03-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 mitochondrial 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 10.3.3.2),
responsiveness to effects of 0Q on 09 consumption is age-related (Table 10-5).
3
Elsayed et al . (1982a) exposed rats of various ages to 1568 pg/m (0.8 ppm) of
0- continuously for 92 hr. The 0,- induced increase in the activities of
O O
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.
Species and strain differences were observed after a 5-day continuous
exposure to 882 |jg/m3 (0.45 ppm) of 03 (Mustafa et al . , 1982). Mice and
Sprague Dawley rats (but net 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.
0190Z4/A 10-82 5/1/84
-------
Rats have also been compared to two strains of monkeys after a 7-day (8
hr/day) exposure to various concentrations of 03 (Mustafa and Lee, 1976).
Rats were exposed to 392, 980, or 1568 ug/m3 (0.2, 0.5, or 0.8 ppm) of DS and
exhibited increases in succinate oxidase activity. Rhesus monkeys exposed to
3
either 980 or 1568 pg/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 0.,, succinate
oxidase activity increased at the two higher 0- levels. The number of animals
o
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).
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 0^ consumption is typically attributed to concurrent
morphological changes (Section 10.3.1) 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 al., 1981; Schwartz et al., 1976; DeLucia
et al. , 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 pg/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
the number of mitochondria, rather than an increased activity within a given
mitochondrion, in exposed lungs is the probable dominant cause.
10.3.3.4 Monooxygenases. Multiple microsomal enzymes function in the metab-
olism of both endogenous (e.g., biogenic amines, hormones) and exogenous
(xenobiotic) 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 0- exposure (Table 10-6).
0190Z4/A 10-83 5/1/84
-------
TABLE 10-6. MONOOXYGENASES
Ozone
concentration Measurement '
o
i
oo
-pa
MQ/rn3
392
980
1568
1470
5880
19,600
1470
5880
19,600
1568
3920
1568
3920
392
1568
1568
1568
1568
ppm method
0.2 MAST
0.5 NBKI
0.8
0.75 I
3.0
10.0
0.75 I
3.0
10.0
0.8 NO
2.0
0.8 I
2.0
0.2 I
0.5
0.8
0.8
0.8 I
. Exposure
duration and
protocol
Continuous or
8 hr/day for 7
days
3 hr
3 hr
Continuous for
7 days
8 hr
Continuous
for 7 days
8 hr
Continuous or
8 hr/day
8 hr/day
Continuous for
7 days
Observed effect(s) Species
Concentration-related linear Rat
increase in NAOPH cytochrome c
reductase activity. No
difference between continuous
and intermittent.
Decreased activity of benzpyrene Hamster
hydroxylase in lung parenchyma.
Decreased activity of banzpyrene Rabbit
hydroxylase in tracheobronchial
mucosae.
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 10-6. MONOOXYGENASES (continued)
Ozone
concentration
i — *
0
OD
ug/m3
1960
1960
ppm
1.0
1.0
b Exposure
Measurement ' duration and
method protocol Observed effect(s)
ND 90 min Decreased levels of lung cyto-
chrome P-450. Maximal decrease
at 3.6 days postexposure.
MAST 24 hr 50% decrease in benzphetamine
N-demethylase activity 1 day
Species
Rabbit
Rat
Reference
Goldstein et al . ,
1975
Montgomery and
Niewoehner, 1979
5880 3
NBKI
10 min before
lung perfusion
and continuous
throughout
experiment.
postexposure; return to control
levels by 1 wk postexposure.
Stimulation of cytochrome
bs-mediated lipid desaturation.
Decreased enzymatic conversion of
arachidonic acid to prostaglandins
when using isolated ventilated per-
fused lung.
Rat
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.
-------
Monoamine oxidase (MAO) activity has been investigated (Mustafa et al.,
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 |jg/m (0.8 ppm) continuously for 7 days.
Substrates used included rramylamine, benzyl amine, 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
3
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 mitochondrial enzymes and antioxidant metabolism (Sections 10.3.3.2
and 10.3.3.3).
The cytochrome P-450-dependent enzymes have been studied because of their
function in drug and carcinogen metabolism. Palmer et al. (1971, 1972) found
that hamsters exposed to 1,470 ug/m (0.75 ppm) of 0., for 3 hr had lower
benzo(a)pyrene hydroxylase activity. Goldstein et al. (1975) showed that
3
rabbits exposed to 1,960 |jg/m (1 ppm) of 0- 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-demethylase in
3
the lung was lowered by a 24-hr exposure to 1,960 ug/m (1 ppm) of 03 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 1 week. Cytochrome bg-mediated lipid desaturation was
stimulated by 03 4, 7, and 14 days postexposure. Immediately after exposure,
the desaturase activity was quite depressed, but this was attributed to anorexia
in the rats, and not to 0^. 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 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 03 exposure are involved in the activa-
tion, which makes full interpretation of the effect of 0- on this metabolism
0190Z4/A 10-86 5/1/84
-------
impossible. The impact of the decrease in cytochrome P-450 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 10.3.3.2; 10.3.3.3),
3
acute exposure to a high 0_ level (3920 ug/m , 2 ppm; 8 hr) reduced NADPH
O
cytochrome c reductase activity (DeLucia et al., 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. , 1977). Continuous and intermittent exposures
were not different. The time course of the response to 1568 ug/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
3
exposure for 8 hr/day for 7 days to 1568 |jg/m (0.8 ppm) of 0, (DeLucia et al. ,
1975a). However, monkey data were not reported in any detail.
10.3.3.5 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 on Table 10-7. 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
10.3.1), such an increase in type 2 cells and alveolar macrophages and polymor-
phonuclear 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 10.4.3.
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
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 07 continuously for
3
7 days. Lower levels (196 ug/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
0190Z4/A 10-87 5/1/84
-------
TABLE 10-7. LACTATE DEHYDROGENASE AND LYSOSOMAL ENZYMES
Ozone
concentration
|jg/m3
196
' 392
980
1568
980
1568
i—"
o
i
Co 1568
CO
1372
1568
1372-
1568
i
i 1568
1568
ppm
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
a h
Measurement '
method
NBKI
MAST,
NBKI
NBKI
NBKI
MAST,
NBKI
MAST,
NBKI
MAST,
NBKI
Exposure
duration and
protocol
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 in Rat
diet group receiving 0 ppm
vitamin E. Groups with 11 or
110 ppm of vitamin E had no effect.
Increased lung lysozyme activity only Rat
after continuous exposure to 0.8 ppm.
Increased LOH activity in lungs. Rat
Change in LDH isoenzyme distri-
bution at 0.8 ppm.
No change in total LDH activity or Monkey
isoenzyme 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 HH activity not Rat
affected by vitamin E (0 or
45 mg/kg diet).
Reference
Chow et al. ,
Chow et al . ,
Chow et al . ,
Oil lard et al
1972
Castleman et
1973a
Castleman et
1973b
1981
1974
1977
.,
al.,
al.,
Chow and Tappel ,
1973
Measurement method: MAST = Kl-coulometric (Mast meter); NBKI = neutral buffered potassium iodide.
bCalibration method: NBKI = neutral buffered potassium iodide.
-------
LDH activity and isoenzyme pattern (relative ratios of different LDH isoenzymes)
3
in the lungs, plasma, and erythrocytes of 0--exposed rats (980 or 1,568 (jg/tn ,
3
0.5 or 0.8 ppm) and monkeys (1568 ug/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
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
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 03- 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 03 at concentrations of 1,372 ± 294 ug/m (0.70 ± 0.15 ppm) for 5
days and 1,548 ± 274 pg/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 03 exposure. Similarly, Castleman et al. (1973a,b) found that activity
of lung acid phosphatase was increased in young rats that had been exposed to
1,372 to 1,568 ug/m (0.7 to 0.8 ppm) of 0- continuously for 7 days. Increases
in p-glucuronidase activity were not observed. The histochemical and cytochem-
ical localization suggested that 0., exposure results in damage to the lung's
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
0190Z4/A 10-89 5/1/84
-------
represent an enzymatic protective mechanism operating locally in the centri-
acinar regions of 0~-exposed lungs. Chow et al. (1974) also observed an
increase in lysozyme activity (lung homogenate) in rats exposed continuously
3
to 1568 ug/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 ug/m (0.2 or 0.5 ppm) of 03.
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.
10.3.3.6 Protein Synthesis. The effects of 03 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 10-8.
Hesterberg and Last (1981) found that increased collagen synthesis caused
3
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 03 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 OT for 30 days, the augmentation of activity seen earlier at
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
o J
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
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
0190Z4/A 10-90 5/1/84
-------
TABLE 10-8. EFFECTS OF OZONE ON LUNG PROTEIN SYNTHESIS
Ozone .
concentration Measurement '
ug/mj
392
784
1176
ppm method
0.2 NO
0.4
0.6
Exposure
duration and
protocol
8 hr/day for
3 days
Observed
Decreased rate of
tracheal explants
effect(s)
glycoprotein secretion by
at 0.6 ppm.
Species Reference
Rat Last and Kaizu,
1980; Last and
Cross, 1978
1568
980
o
i
0.8
0.5
Continuous for
1 through 90 days
Continuous for
3 or 14 days and
combined with
H,S04
Decreased rate of glycoprotein secretion.
Increased rate of glycoprotein secretion.
392
980
1568
392
784
1176
1568
392
1568
3920
980-
3920
0.2 MAST
0.5
0.8
0.2 UV
0.4
0.6
0.8
0.2 UV,
0.8 NBKI
2.0
0.5- UV
2
Continuous for
7 days
8 hr/day for
1 to 90 days
6 hr/day, 5 days/
wk, 12.4 wk (62
days of exposure)
1, 2, or 3 wk
Concentration-dependent increase in lung prolyl Rat
hydroxylase activity. No effect at 0.2 ppm. Meta-
bolic adaptation suggested at 980 MS/™3 (0-5 PPm)
At 0.8 ppm, collagen and noncollagenous protein
synthesis increased; effect on proyl 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 rate of collagen synthesis; fibrosis Rat
of alveolar duct walls; linear concencentration
response.
Hussain et al . ,
1976a,b
Last et al. , 1977
Costa et al. , 1983
Last et al. , 1979
-------
TABLE 10-8. EFFECTS OF OZONE ON LUNG PROTEIN SYNTHESIS (continued)
o
i
Ozone . Exposure
concentration Measurement ' duration and
ug/m3
882
1568
980
1568
1568
1568
2352
2940
ppm method
0.45 ND
0.8 ND
0.5 UV
0.8 NO
0.8 NBKI
0.8 UV
1.2
1.5
protocol
Continuous
for 7 days
Continuous
for 90 days
Continuous for
up to 180 days
Continuous
for 7 days
Continuous
for 3 days
Continuous
for 7 days
Observed effect(s)
Increased collagen synthesis at 5 and 7, but
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
7 days. No effect 20 days and beyond.
Increase in protein and hydroxyprol ine content of
lungs. No change 2 mo postexposure.
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
Mouse Bhatnagar et al . ,
1983
Rat
Rat Last and Greenberg,
1980
Rat Mustafa et al . ,
1977
Rat Chow et al. , 1976b
Rat Hesterberg and Last,
1981
Measurement method: MAST = Kl-coulometric (Mast meter); UV = UV photometry; NBKI = neutral buffered potassium iodide; ND = not described.
Calibration method: NBKI = neutral buffered potassium iodide.
-------
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
exposure to ozone for I, 2, or 3 weeks at levels ranging from 980 to 3920 ug/m
(0.5 to 2 ppm). A general correlation was found between fibrosis detected
histologically and the quantitative changes in collagen synthesis in minces of
0«-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 0, concentration to which the rats were
exposed.
Continuous exposure of mice for 7 days to 882 jjg/m (0.45 ppm) of 0.,
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 2, 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.
Bhatnagar et al. (1983) also studied rats exposed continuously for 90
days to 1568 ng/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.
Effects of subchronic 0- exposure have been studied by Last and Greenberg
3
(1980). Rats were exposed to 980 ug/m (0.5 ppm) continuously for up to 180
days and examined at various times during exposure and 2 months after exposure
ceased. The total protein content of the lungs increased during exposure,
with the greatest increase after 88 days of exposure. By 53 days postexposure,
values had returned to control levels. Hydroxyproline content of the lungs
0190Z4/A 10-93 5/1/84
-------
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 hydroxypro-
line 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 discussing their data, the authors referred
to a concurrent morphological study (Moore and Schwartz, 1981) that showed an
increase in lung volume, mild thickening of the interalveolar septa and alveo-
lar interstitium, and an increase in collagen (histochemistry) in these areas.
Different results for collagen levels were observed after another longer term
exposure (6 hr/day, 5 days/wk, 12.4 wk) to 392, 1568, or 3920 pg/m (0.2, 0.8,
or 2.0 ppm) of 0., (Costa et al., 1983). At the two lower concentrations, rats
exhibited an equivalent decrease in hydroxyproline. At the highest concentra-
tion, an increase was observed. Similar findings were made for elastin levels.
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 0,
(Chow at al. , 1976b). Recovery had occurred by 6 days postexposure. No
adaptation was observed, because when animals were re-exposed to the same 0«
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 in vivo incorporation of radioactive ami no acids. Rats were exposed
3
continuously for 7 days to 1568 pg/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 mucus glycoproteins and their secretion by tracheal
explants have been reviewed by Last and Kaizu (1980). Mucus glycoprotein syn-
3
thesis and secretion were measured by the rate of incorporation of H-glucos-
amine into mucus glycoproteins and their subsequent secretion into supernatant
fluid of the tracheal exp'lant culture medium. This method has been found to
be a reproducible index of mucus production, and these authors maintain that
0190Z4/A 10-94 5/1/84
-------
this measurement ex vivo following exposure i_n vivo is representative of
injuries occurring Jjn vivo. When rats were exposed to 1568 pg/m (0.8 ppm) of
CL 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,
3
and 392 ug/m (0.6, 0.4, 0.2 ppm) of 0~. Glycoprotein secretion decreased
only at the higher concentration. Trachea! explants from Bonnet monkeys
3
exposed to 0, 980, or 1568 ug/m (0, 0.5 or 0.8 ppm) 03 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.
3
A combination exposure to 0, (980 ug/m ; 0.5 ppm) and sulfuric acid
3
(HLSO.) aerosol (1.1 mg/m ) caused complex effects on mucus 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 mucus secretion rates, but H^SO. had no
effect. In rats exposed to the combination of H^SO. aerosol and 03, mucus
secretion significantly increased. After 14 days of continuous exposure, the
rats receiving a combined exposure to both HUSO, aerosol and 0« had elevated
values (132 percent) over the control group of animals. Because mucus secretion
and synthesis are intimately involved in diminishing the exposure of underlying
cells to 0« and removing adventitiously inhaled particles, alterations in the
mucus 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.
10.3.3.7 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 10-9. Generally, the unsaturated fatty
3
acid content decreased in rats exposed to 980 ug/m (0.5 ppm) of 03 for up to
6 weeks (Roehm et al., 1972).
0190Z4/A 10-95 5/1/84
-------
TABLE 10-9. EFFECTS OF OZONE EXPOSURE ON LIPID METABOLISM AND CONTENT OF THE LUNG
Ozone
concentration
jjgTni3 ppm
980
H- 980
*p 1568
<-D
01 :
1960
1960
0.5
0.5
0.8
1.0
1.0
a 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
NO 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 ID vitamin £/kg diet, decreased
ethane and pentane production.
Decreased incorporation of fatty Rabbit
acids into lecithin.
Reference
Roehm et al . , 1972
Dumel in et al . ,
1978a
Dumel in et al . ,
1978b
Kyei-Aboagye
et al. , 1973
Measurement method: NBKI = neutral buffered potassium iodide; UV = UV photometry; I = iodometric; ND = not described.
-------
PRELIMINARY DRAFT
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
1960 (jg/m (1 ppm) of 0~ for 60 min. The provision of 11 (minimum vitamin
requirement) or 40 ID (supplemented level) of vitamin E acetate per kg of diet
resulted in a decrease in expired ethane and pentane after 0, exposure.
Dumelin et al. (1978a) also measured breath ethane and pentane in Bonnet
3
monkeys exposed to 0, 980, or 1568 ug/m (0, 0.5, or 0.8 ppm) of 0_ for 7, 28,
o
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.
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 (jg/m (1 ppm) of 0~ 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 0~ 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.
(1971) and Huber et al. (1971) when using high levels of 0., (> 9800 |jg/m ;
5 ppm).
10.3.3.8 Lung Permeability. Table 10-10 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. (1989) studied the effect of 0~ on active transport of
3
phenol red in the lungs of rats exposed to 1176 to 4116 ug/m (0.6 to 2.1 ppm)
of 0- continuously for 24 hr. Ozone inhibited the carrier-mediated transport
of intratracheally instilled phenol red from the lung to the circulation and
0190Z4/A 10-97 5/1/84
-------
TABLE 10-10. EFFECTS OF OZONE ON LUNG PERMEABILITY
o
i
<-o
oo
Ozone
concentration
ug/m-3
196
510
1000
1960
353
510
980
1000
490
980
1960
4900
1176
2156
3136
4116
1960-
2940
ppm
0.1
0.26
0.51
1.0
0.18
. Exposure
Measurement ' duration and
method protocol
CHEM, 3 hr or 72 hr
UV
3 hr; 8 hr/day
! for 5 or 10 days
0.26 3, 24, or 72 hr
0.5
|
0.51
0.25
0.5
1.0
3 hr
MAST, 6 hr
NBKI
2.5
0.6 NBKI 24 hr
1.1
1.6
2.1
1.0- MAST 2 hr
1.5
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
0.5 ppm, 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:
MAST = Kl-coulometric (Mast meter); CHEM = gas phase chemiluminescence; UV = UV photometry; NBKI = neutral buffered
potassium iodide.
Calibration method: NBKI = neutral buffered potassium iodide; UV = UV photometry.
-------
I
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 03 exposure,
the appearance of albumin and immunoglobins in
examined. Reasor et al. (1979) found that dogs
(1.0 to 1.5 ppm) of 0~ had increased albumin a
their airway secretions. Alpert et al. (1971a),
airway secretions has been
breathing 1960 to 2940 ug/m3
rid immunoglobin G content of
using rats exposed for 6 hr to
1.0 ppm) of 03 for 72 hr and
490 to 4900 ug/m (0.25 to 2.5 ppm) 0~, also fdund 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. (198J2) exposed guinea pigs to 196,
510, 1000, or 1960 ug/m3 (0.1, 0.26, 0.51, or
found increased lavage fluid protein content sampled immediately after exposure
3
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
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 ei concentration-related manner
3 3
from 500 to 1470 ug/m (0.256 to 0.75 ppm). Again 196 ug/m (0.1 ppm) had no
3
effects. The lavage fluid protein content of guinea pigs exposed to 353 ug/m
consecutive days was not dif-
C deficiency could be found on
i guinea pigs exposed to 196 to
et al., 1982). In contrast,
(0.18 ppm) of 03 for 8 hr per day for 5 or 10
ferent from air controls. No effect of vitamir
the accumulation of the lavage fluid protein i
1470 ug/m3 (0.10 to 0.75 ppm) 0, for 3 hr (Hi
-------
3
edema in rats from exposure to 7890 pg/m (4 ppm) of 0~ for 4 hr (Giri et al. ,
1975). Prostaglandins F? and E? were markedly increased in plasma and lung
lavage of rats exposed to 7840 ng/m (4 Ppro) for up to 8 hr (Giri et al. ,
1980). Ozonolysis of arachidonic acid i_n 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
(Pryor, 1976; Pryor et al. , 1976). Acute exposure to 5880 pg/m3 (3 ppm) 03
inhibits uncompetitively rat lung prostaglandin cyclooxygenase (Menzel et al.,
1976).
Earlier reports that prostaglandin synthesis inhibitors exacerbated
0~-produced edema (Dixon and Mountain, 1965; Matzen, 1957), as did methyl pred-
nisolone (Alpert et al., 1971a), tend to confuse the interpretation of the role
of prostaglandin in 0,,-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 0~-produced edema, their exact role is
still unexplained.
10.3.3.9 Proposed Molecular Mechanisms of Effects. Experts generally agree
that the toxicity of Q~ depends on its oxidative properties. The precise
mechanism of 0 '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 Association
(1970); North Atlantic Treaty Organization (1974); National Research Council
0190Z4/A 10-100 5/1/84
-------
(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.
10.3.3.9.1 Oxidation of polyunsaturated lipids. The first hypothesis is that
Og 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 peroxidation all can react
with proteins. In addition to the direct oxidation of amino acids by 0~,
secondary reaction products from 0,-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
changes of the membrane-embedded protein. At present, methods are not availa-
ble to differentiate effects on membrane proteins from effects on membrane
lipids.
Some of the strongest evidence that the toxic reaction of 0., 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 0.,-induced increase
in enzyme activities of the glutathione peroxidase system (Section 10.3.3.2)
and those involved in oxygen consumption (Section 10.3.3.3). More details are
provided in Table 10-5. Morphological effects due to 0- exposure are also
lessened by dietary vitamin E (Section 10.3.1.4.1.1). Although this is not
the strongest evidence, vitamin E supplementation also prevented 0--induced
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
0190Z4/A 10-101 5/1/84
-------
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 ug/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 pg
of vitamin E/g of tissue), but morphological responses were very similar.
Stephens et al. concluded that 0--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-
10.3.3.9.2 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 0~ (Mudd et al., 1969; Mudd and Freeman, 1977; Previero et al., 1964). In
the following descending order of rate, 03 oxidizes the ami no acids cysteine >
methionine > tryptophan > tyrosine > histidine > cystine > phenylalanine. The
remaining common amino 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 i_n vitro by 03
as shown by studies of avidin, the biotin-binding protein. Oxidation of
tyrosine in egg albumin by 03 occurs i_n vitro, converting the Deoxidized egg
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 03, alpha-1-protease inhibitor lost its ability to
inhibit trypsin, chymotrypsin, and elastase.
0190Z4/A 10-102 5/1/84
-------
Meiners et al. (1977) found that 03 reacted i_n vitro with tryptophan,
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 0- also generates hydrogen peroxide. Hydrogen peroxide is a
toxic substance in itself and initiates peroxidation of lipids (McCord and
Fridovich, 1978). Other active Q? 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
03 (viz. , 1960 to 7840 ug/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 |jg/ro , 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,
and mitochondrial 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 glyce ides. Mudd and Freeman contend,
however, that proteins are far more easily oxidized by 0^ than are lipids.
Indirect evidence in support of the idea that amines in particular are oxidized
preferentially by 0- is the protective effect of p-aminobenzoic acid (Goldstein,
B., et al. , 1972). Rats injected with p-aminobenzoic acid were partially
protected from the mortality due to high concentrations of 0.,. Presumably,
the added p-aminobenzoic acid is oxidized by 0- in place of proteins. Goldstein
and Balchum (1974) later suggested that the protection of p-aminobenzoic acid,
allylisopropylacetamide, 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
0190Z4/A 10-103 5/1/84
-------
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 in vitro
3
exposure to 78,400 ug/m (40 ppm) 03 through p-aminobenzoic acid treatment
(Goldstein, B., et al., 1972). Amines are also efficient lipid antioxidants,
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
well.
10.3.3.9.3 Formation of toxic compounds through reaction with polyunsaturated
lipids. The effects of 0- 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 composition of the serum and lung lipids were similar to
those occurring after Q~ exposure (Roehm et al., 1971a,b, 1972; Menzel et al.,
1976).
Peroxidation of lung lipids could lead to cytotoxic products. Phosphati-
dyl choline liposomes (spheres formed by emulsifying phosphatidyl choline in
water) were lysed on exposure to 0, (Teige et al., 1974). Liposomes exposed
to 03 were more active than 03 alone in lysing red blood cells. The products
of ozonolysis of phosphatidyl choline could be stable, yet toxic, intermediates.
10.3.3.9.4 Formation of free radicals and injury mediated by pharmacologic
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 ROOR) rather than oxidation of alkenes to higher oxidation
states (e.g., ROM, 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.
0190Z4/A 10-104 5/1/84
-------
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 03 (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,
3
at levels of 03 below 1960 ug/m (1 ppm), increases in glutathione have been
observed (Plopper et al. , 1979; Fukase et al. , 1975; Moore et al. , 1980;
Mustafa et al., 1982). The rat lung is sensitive to the increase of glutathione
peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase
3
activities at levels as low as 196 ug/m (0.1 ppm) 03 continuously for 7 days
(Plopper et al., 1979; Chow et al., 1981; Mustafa, 1975; Mustafa and Lee,
1976) in vitamin E deficient rats. With vitamin E-supplemented rats, 392 pg/m
3
(0.2 ppm) caused similar effects. After acute exposures to >1960 ug/m (1 ppm)
0-, decreases are observed in these enzyme activities and glutathione levels.
Other species (mice and monkeys) exhibit similar effects at different concentra-
tions (Table 10-5). These changes at the lower levels of 03 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 al., 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
lung. This was substantiated by Mustafa (1975) and DeLucia et al. (1975a),
who found that the 0? consumption per mitochondrion was not increased in
0,,-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 0,,-exposed rats were due to the demands of the glutathione peroxidase
O i
system for reducing equivalents in the form of NADPH. Cross et al. (1976) and
DeLucia (1975a,b) reported that 0- 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 03 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
0190Z4/A 10-105 5/1/84
-------
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 0Q in vitro.
*5 —— _—__—_
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 0, and nitrogen dioxide. Fairchild and
Graham ascribed the mortality following 0~ to pulmonary edema. Wong and
Hochstein (1981) suggested that 0, 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 0, sensitivity.
10.3.3.9.5 Summary. The actual toxic mechanism of 0- may involve a mixture
of all of these chemical mechanisms because of the interrelationships between
the peroxide detoxification mechanisms and glutathione (See Section 10.3.3.2)
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
0190Z4/A 10-106 5/1/84
-------
free radical reactions (vitamins E and C), and differences in membrane composi-
tion of PUFA (relative ease of attack of 03), for example.
10.3.4 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 0- 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 CL 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 CL on its integrity (Gardner,
1981; Ehrlich, 1980; Gardner and Ehrlich, 1983).
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
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 * Sections. The immune system,
which defends the overall host against both infectious and neoplastic diseases,
is also discussed.
10.3.4.1 Mucociliary Clearance. The mucociliary system is one of the lung's
primary defense mechanisms. It protects the conducting airways by trapping
and quickly removing material that has been deposited or is being cleared from
the alveolar region. The effectiveness of mucociliary clearance can be deter-
mined iDy 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 distribution of mucus-secreting
cells. Once this defense mechanism has been altered, a buildup of both viable
0190Z4/A 10-107 5/1/84
-------
and nonviable inhaled substances can occur on the epithelium and may jeopardize
the health of the hose, depending on the nature of the uncleared substance.
A number of studies v/ith various animal species have reported morpho-
logical damage to the cells of the tracheobronchial tree from acute and sub-
chronic exposure to 490 to 1960 ug/m (0.20 to 1.0 ppm) of 0 . (See Section
O
10.3.1.) 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 tracheal 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 al., 1980). In the
same study, when the animals were subsequently exposed for 2 hr to 1090 ug of
H?SO. per cubic meter (0.30 urn volume median diameter), a significant reduction
in ciliary beating frequency occurred. The magnitude of this effect was,
however, significantly less than that observed due to the effect of H^SO^
exposure 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
3
slight decrease in beating frequency with a simultaneous exposure to 196 |jg/m
(0.1 ppm) of 03 and 847 ug/m H2S04 (Grose et al., 1982). These data indicate
that 03 appears to partially protect against the effects of H,,S04 on ciliary
beating frequency. Grose et al. (1982) proposed that the ciliary cells may be
partially protected due to the increase in mucus tracheal secretion of glyco-
proteins (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 03 on the host's
ability to physically remove deposited particles (Table 10-11). The slowing
of mucus transport in both rat and rabbit trachea as a result of 03 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
O
bactericidal activity but not to significantly affect physical removal of the
deposited bacteria. In these studies, mice were exposed to an aerosol of
32P-labeled Staphylococcus aureus either after a 17-hr exposure to 03 or
0190ZS/A 10-108 5/1/84
-------
TABLE 10-11. EFFECTS OF OZONE ON HOST DEFENSE MECHANSIMS: DEPOSITION AND CLEARANCE
VQl
Ozone
concentration
ug/'m3
196
784
784
785, 1568,
1960
0.
0.
0.
0,
1,
ppm
1
,4
.4
.4, 0
.0
Measurement
method
CHEM
NBKI
ND
.8, UV
Exposure
duration and protocol
3
3
4
4
hr
hr
hr
hr
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
did not enhance 03 effect.
Delay in mucociliary clearance,
in alveolar clearance.
Silicosis
acceleration
Species Reference
Hamster Grose et al., 1980
Mouse Coffin and Gardner, 1972b
Mouse Goldstein, E. , et al., 1972
Rat Kenoyer et al . , 1981
1764
0.5
MAST
1.4 hr
Increases nasal deposition and growth of
virus; no effect in the lungs.
Mouse Fairchild, 1974, 1977
980
980
0.
0
5
5
NBKI
NBKI
16
7
2
hr/day, No effect on clearance of polystyrene and
months iron particles.
months
Reduced clearance of viable bacteria.
Rabbit
Guinea
I Friberg
pig(Friberg
1
et
et
al.,
al.,
1972
1972
980, 1960 0.5, 1.0 CHEM
2 hr
Reduced tracheal mucus velocity at 1.0 ppm.
No effect at 0.5 ppm.
Sheep Abraham et al., 1980
784
0.57-2.03 M
17 hr before Physical clearance not affected, but bactericidal
bacteria activity affected at 0.99 ppm. Decrease in
deposition of inhaled organisms at 0.57 ppm.
Mouse Goldstein et al. , 1971a
-------
TABLE 10-11. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: DEPOSITION AND CLEARANCE (continued)
o
i
Ozone
concentration Measurement
ug/m3 ppm method <
1176 0.62-4.25 M
1372 0.7 M
1372 0.7 G
1568 0.8 UV
1960 1.0 NBKI
2352 1. 2 UV
Exposure
duration and protocol
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
Mouse Coffin and
Rat Frager et
et al . , 1971b
et al . , 1974
al . , 1982
al . , 1980
Gardner, 1972b
al., 1979
Measurement method: ND = not described; CHEM = gas phase chemiluminescence; UV = UV photometry; NBKI = neutral buffered potassium iodide;
MAST = Kl-coulometric (Mast meter); M = microcoulomb sensor; G = galvanic meter.
-------
before a 4-hr exposure. Concentrations of 0~ were 1180, 1370, 1570, or
o J
1960 ug/m (0.6, 0.7, 0.8, or 1.0 ppm). The mechanical 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
3
bactericidal activity beginning at 1960 ug/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 03 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.
Friberg et al. (1972) studied the effect of a 16-hr/day exposure to 980
3
ug/m (0.5 ppm) of 03 on the lung clearance rate of radiolabeled monodispersed
polystyrene and iron particles in the rabbit and of bacteria in the guinea
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 0^ 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 radiolabeled monodis-
perse polystyrene latex spheres were followed after 0, exposure. A 4-hr
3
exposure to 1568 ug/m (0.8 ppm) of 0, 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(S04)2, H2$04, (NH4)2$04]
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 02 free radicals, which have the capability for tissue destruction.
Essentially no data are available on the effects of prolonged exposure to 0,
0190ZS/A 10-111 5/1/84
-------
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 pptn) of 03. 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 0- 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 0- 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.
These results were confirmed when Kenoyer et al. (1981) repeated these
studies, with three different concentrations of 0~, 784, 1568, and 1960 ug/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 GO-
The velocity of the tracheal 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 0- (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
tracheal mucus velocity.
10.3.4.2 Alveolar Macrophages. Within the gaseous exchange region of the
lung, the first line of defense against microovjanisms 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 10-12 illustrates the
effect of 0- on the alveolar macrophage (AM).
0190ZS/A 10-112 5/1/84
-------
TABLE 10-12. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: MACROPHAGE ALTERATIONS
Ozone
concentration
ug/n
196
1960
392
490
980
980
980
980
980
1313
980
1960
1058
n3 ppm
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
method
NBKI
MAST
NBKI
NBKI
NBKI
NBKI
CHEM
NBKI
NBKI
UV
1 Exposure
duration and protocol
2.5 hr or
30 min 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 effects
Lung protective factor partially inactivated,
increasing fragility of macrophages (concen-
tration-related).
Increased number of macrophages in lungs
(morphology).
Decreased activity of the lysosomal
enzymes lysozyme, acid phosphatase,
and p-glucuronidase.
Increased osmotic fragility.
Decreased enzyme activity and increased influx
of PMNs.
Decreased red blood cell rosette binding to
macrophages.
Decreased ability to ingest bacteria.
Decreased agglutination in the presence of
concanavalin A.
Increased number of macrophages (morphological).
Species
Rabbit
Monkey
Rat
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Rat
Mouse
Reference
Gardner et al. ,
Castleman et al
Dungworth, 1976
Stephens et al.
1971
. , 1977
, 1976
Hurst et al., 1970, 1971
Dowel 1 et al . ,
Alpert et al. ,
Hadley et al. ,
1970
1971b
1977
Coffin et al., 1968b
Coffin and Gardner, 1972b
Goldstein et al
Zitnik et al. ,
. , 1977
1978
-------
TABLE 10-12. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: MACROPHAGE ALTERATIONS (continued)
Ozone I
concentration Measurement '
ug/m3 ppm I method
1568 0.8 NO
1568 0.8 NO
1568 0.8 UV
1568 0.8 MAST
1960 1 CHEM
9800 5
1960 1 UV
3136 1.6 to NBKI
6860 3.5
4900 2.5 M
4900 2.5 M
Exposure
duration and protocol Observed effects Species Reference
11 and 24 days No effect on in vitro interferon production. Mouse
90 days Eightfold increase in number of macrophages at Rat
7 days, reducing to fourfold after 90 days.
7 days Decreased number of migrating macrophages Monkey
and total distance migrated.
3, 7, 20 days Increased phagocytosis. Rat
3 hr Decreased ability to produce interferon Rabbit
in vitro.
4 hr Decreased jji vitro migrational ability, as Rat
evidenced by decreased number of macrophages able
to migrate.
2 hr to 3 hr Decreased superoxide anion radical production. Rat
5 hr Loss of B-glucuronidase and acid phosphatase in Rat
RAM with ingested bacteria; decreased rate of
bacterial ingestion.
5 hr Diminished rate of bacterial killing, increased Rat
numbers of intracellular staphylococcal clumps;
lack of lysozyme in macrophages with staphylococcal
clumps.
I Ibrahim et al. , 1976
Boorman et al. , 1977
Schwartz and Christman, 1979
Christnian and Schwartz, 1982
Shingu et al., 1980
McAllen et al., 1981
Amoruso et al . , 1981
Witz et al., 1982
I
Goldstein et al., 1978b
Kimura and Goldstein, 1981
Measurement method: NO = not described; CHEM = gas phase cht'mi luminescence; UV = UV photometry; NBKI = neutral buffered potassium iodide;
MAST = Kl-coulometric (Mast meter); M = microcoulomb sensor.
Calibration method: NBKI = neutral buffered potassium iodide.
cAbbreviations used: PMN = polymorphonuclear leukocytes; RAM = pulmonary alveolar macrophage.
-------
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, CL, 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 reserve 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
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 0-
can result in a macrophage response in several animal species. Exposure of
3
mice to 1058 ug/m (0.54 ppm) of 0, 23 hr/day for a maximum of 34 days resulted
in an increased number of macrophages within the proximal alveoli of the
alveolar ducts (Zitnik et al., 1978). These cells were highly vacuolated and
contained many secondary 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 in the number of pulmonary free cells after exposure
3
to 1568 ug/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 both monkeys and rats exposed to concentra-
3
tions as low 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 approxi-
mately equal in susceptibility to the short-term effects of 0.,.
Thus, the total available data would indicate that, after short periods
of 0~ insult, there is a significant reduction in the number of free macrophages
available for pulmonary defense, and that these macrophages are more fragile,
0190ZS/A 10-115 5/1/84
-------
are less phagocytic, and have decreased enzymatic activity (Dowell et al.,
1970; Coffin et al., 1968b; 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 decrease in the ability of macrophages to phagocytize bacteria after
3
exposure to concentrations as low as 980 ug/m (0.5 ppm) of 0- for 3 hr was
demonstrated by Coffin et al. (1968b). 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 pg/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, 1, 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 enhance-
ment correlated well with a significant increase in cell spreading of AMs from
exposed rats as compared to controls. If 03 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 va1 d, 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 collec-
ted cells adhered after 3 days of 0- 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 0, 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 0., exposure might never have been tested, because they were
O
discarded.
0190ZS/A 10-116 5/1/84
-------
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
membrane organization. A decrease in agglutination of rat AMs was found after
3
exposure to 980 or 1960 (jg/m (0.5 or 1.0 ppm) of 0~. A decrease in concanavalin-
A agglutinabi1ity of trypsinized red blood cells obtained from rats exposed
for 2 hr to 1960 ug/m (1 ppm) was also noted. Hadley et al. (1977) investigated
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 0.,-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 NO™ mixtures on the
activity of AMs isolated from the lungs of mice exposed for 1, 2, and 3 months.
Only after a 3-month exposure to the mixture of 196 ug/m (0.1 ppm) of 0~ and
0.5 ppm of NO^ (3 hr/day, 5 days/week) did viability in macrophages decrease
significantly, lr\ 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.
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 H9/m3 (1-0
ppm) of 03 for 4 hr on the migration rate of AMs. Migration was measured by
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 o^ 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.
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
3
can be inactivated by a 2.5-hr exposure to 196 ug/m (0.1 ppm) of 0». When
normal AM's are placed in fluid lavaged from 0.,-exposed animals, they showed
an increase in lysis (10 percent over control). A similar effect was seen
0190ZS/A 10-117 5/1/84
-------
when normal AM's were placed in protective fluid that had been exposed J_n
vitro to 00. The data indicate that some of the effects of 00 on lung cells
j o
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 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 (L-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, any perturbation of the metabolic
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 rabbit macrophages after a 3-hr exposure of rabbits to
3
concentrations as low as 490 |jg/m (0.25 ppm) of 03 (Hurst et al. , 1970).
Such enzymatic reductions were also observed in AMs exposed i_n vitro (Hurst et
al. , 1971). The ability of 0-, to alter macrophages1 enzyme activity was also
studied by means of unilateral lung exposure of rabbits (Alpert et al., 1971b).
A significant reduction in these same three intracellular enzymes was found to
be specific to the lung that breathed 0- rather than a generalized systemic
3
response. These effects v/ere concentration-related, beginning at 980 ug/m
(0.5 ppm) of 0,,. The extracellular release o. such enzymes may occur either
as a result of direct cytctoxic damage and leakage of intracellular contents,
or they may be selectively released without any cell injury. Hurst et al.
(1971) showed that the reduction in intracellular lysosomal enzyme activity
observed after jm vitro exposure coincides with the release of the enzyme into
the surrounding medium. In these studies, the sum of the intra- plus extracel-
lular enzyme activity did not equal the total activity, indicating that the
pollutant itself can inactivate the hydrolytic enzyme as well as alter the
cell membrane. Recently, Witz et al. (1982) and Amoruso et al. (1981) reported
that i_n vivo 03 exposure affected the production of superoxide anion radicals
(Op) by rat AMs. This oxygen radical is important in antibacterial activity.
0190ZS/A 10-118 5/1/84
-------
Exposure to concentrations above 3136 ug/m (1.6 ppm) of 0~ for 2 hr appears
to result in a progressive decrease in 0? 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 09 production arises from the
*3 * ^
direct oxidative damage of the membrane enzyme involved in the metabolism of
0- to 0~, or whether it is a result of oxi dative degradation of membrane
lipids that may serve as a cofactor function.
Shingu et al. (1980) reported the effects of 0_ on the ability of two
O
cell types, macrophages and tonsillar lymphocytes, to produce interferon, a
substance that aids in defending the host organism against viral infections.
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
3
tonsillar lymphocytes was not significantly depressed by 9800 ug/m (5.0 ppm)
of 03. The authors suggested that an impairment of interferon production
might play an important role in the ability of the host to combat respiratory
viral infections. 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.
10.3.4.3 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? Measurement of the
competency of the host's antimicrobial mechanisms can best be tested by chal-
lenging 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., mechanical clearance via the mucociliary escalator,
biological clearance mediated through macrophages, and associated cellular and
humoral immunological events) that would normally function in defending the
host against this microbe, the microbe, in its attempt to survive, would take
advantage of these weaknesses. A detailed description of the infectivity
0190ZS/A 10-119 5/1/84
-------
model commonly used for ()„ 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 CL. 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 concentra-
tions of 0- used do not cause any mortality. The mortality in the control
O
group (clean air plus exposure to the microorganism) is approximately 10 to
20 percent and reflects the natural resistance of the host to the infectious
agent. The difference in mortality between the 0_-exposure group and the
O
controls is concentration-related (Gardner, 1982a).
If the test agent does not prevent the host's defense mechanisms from
functioning normally, there is a rapid inactivation of inhaled microorganisms
that have been deposited in the respiratory system. However, if the gaseous
exposure alters the ability of these defense cells to function, the number of
microbes in the lungs increases rapidly (Coffin and Gardner, 1972b; Miller et
al., 1978; Gardner, 1982a). This acceleration in bacterial growth is 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 could be predicted from a positive blood
culture (Coffin and Gardner, 1972b).
Coffin et al. (1968a) treated mice with 03 (0.08 ppm for 3 hr) and subse-
quently exposed them to an aerosol of infect', jus Streptococcus sp. In this
study, 0- increased the animals' susceptibility to infection, resulting in a
O
significant increase in mortality rate in the 0.,-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 to 03 at
196 ug/m (0.1 ppm) in which the bacterial aerosol was administered either
immediately or 2, 4, or 6 hr after cessation of the 0~ exposure. In each
replicate experiment, 20 mice per group were used, and 0_ was monitored contin-
uously by chemiluminescence. For these postexposure challenges, only the 2-hr
time resulted in a significant increase in mortality (6.7 percent) over controls.
0190ZS/A 10-120 5/1/84
-------
However, when the animals were infected with Streptococci during the actual CL
exposure, a significant increase in mortality of 21 percent was observed.
When this latter experimental regimen was used, exposure to 157 ug/m (0.08 ppm)
of 0- resulted in a significant increase in mortality of 5.4 percent.
The differences in results among these studies may have been due to a
variation in the sensitivity of the method of 0~ 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 0,-treatment group were significantly less
effective in combating the infectious agent to the extent that, even at this
low concentration, there was a significant increase in mortality rate in this
group over controls. As the 0- concentration increased, mortality increased.
The effective concentration of 0- that increases the lung's susceptibility to
infection is lowered when another pollutant, such as N0? or H~SO., is combined
with 0.,. 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 tha 0~ 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 10-13).
3
When mice were exposed 4 hr to 392 to 1372 ug/m (0.2 to 0.7 ppm) of 03
and than challenged with virulent Klebsiella pneumoniae, a significant in-
crease in mortality was noted at 785 ug/m (0.4 ppm) 0, (Bergers et al.,
1982). 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
0190ZS/A 10-121 5/1/84
-------
TABLE 10-13. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: INTERACTIONS WITH INFECTIOUS AGENTS
Ozone
concentration
ug/m3
157
157-196
196
196
196, 588
392-1372
588
1372-1764
1960
2940
ppm
0.08
0.08, 0.
0.1
0.1
0.1, 0.3
0.2-0.7
0.3
0.7-0.9
1.0
1.5
Measurement3
method
ND
1 CHEM
CHEM
UV
CHEM
G
ND
ND
CHEM
ND
Exposure
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,
Observed effects
j Increase in mortality to Streptococcus pyogenes.
Significant increase in mortality during 02
exposure (Streptococcus pyogenes).
Increased mortality to Streptococcus pyogenes.
Increased susceptibility to bacterial infection
(Streptococcus pyogenes).
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
(Pasturella haemolytica).
Increased susceptibility to infection
(Streptococcus pyogenes).
Increase in Mycobacterium tuberculosis lung titers.
No effect on resistance to Mycobacterium
Species
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Sheep
Mouse
Mouse
Mouse
Reference
Coffin et al. ,
Miller et al . ,
Ehrlich et al . ,
Aranyi et al . ,
Illing et al. ,
Bergers et al. ,
Abraham et al. ,
1968a
1978
1977
1983
1980
1982
1982
Coffin and Blommer, 1970
Thomas et al . ,
Thienes et al . ,
1981b
1965
-------
(0.4 ppm) of 0., a nearly threefold lowering of the bacterial LD™ value computed
from the three challenge doses of bacteria was found for the CL-exposed group,
indicating a significant increase in mortality. In the same study, the authors
3
also found that. 1372 pg/m (0.7 ppm) of 0, for 3 hr resulted in a decreased
ability of the lung to clear (bactericidal) inhaled staphylococci. This
finding is consistent with that of Goldstein et al. (1971b) and confirms
previous studies reported by Miller and Ehrlich (1958) and Ehrlich (1963).
Chiappino and Vigiani (1982) also reported that 0, (1.0 ppm) potentiated
pulmonary infection in rats. In this study, the investigators wanted to know
in what way 0., modified the reactions to silica in specific pathogen-free
(SPF) rats. Silica-treated animals were exposed to 0~ (8 hrs/day, 5 days a
week for up to 1 year) and housed in either an SPF environment or in a conven-
tional animal house, thus being exposed to the bacterial flora normally present
there. The SPF-maintained, 0~-exposed rats showed a complete absence of
saprophytes and pathogens in the lungs, whereas the microbial 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 0, 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 0^ exposure have
also been tested in sheep. In these studies, sheep were infected by an inocu-
lation of Pasturella haemolytica either 2 days before being exposed to 588
3
ug/m (0.3 ppm) of 0., or 2 days after the 0., exposure. In both cases, the 0,,
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 al. ,
1982). Unfortunately, only a small number of animals were used in each 0-,
treatment (n = 3).
Thomas et al. (1981b) 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 03 3 hr/day, 5 days/week for up
to 8 weeks, initiated 7 or 14 days after the infectious challenge with
Mycobacterium tuberculosis H37RV, resulted in significantly increased bacterial
lung titers, as compared with controls. Exposure to lower concentrations of
0190ZS/A 10-123 5/1/84
-------
0, did not produce any significant effects. In an earlier study, Thienes et
o
al. (1965) reported that exposure to 2940 ug/m (1.5 ppm) of (k 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 10-14 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 03 and NO,, mixtures. In the earlier studies (Ehrlich et al. ,
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/m (1.5 to 5 ppm) of NO 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 5 months to mixtures of 196 ug/m (0.1 ppm) of 0, and 940 ug/m (0.5
«3
ppm) of N02 and challenged with bacterial aerosol. The 1- or 2-month exposure
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 0, and N09 mixture
O £.
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
o
superimposed 3-hr daily peak (5 days/week) containing a mixture of 196 \jg/m
(0.1 ppm) of 03 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 not statistically 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
mixture of 03, SO,,, and (NH.^SO.. The concentrations were 200 ug/m of 0.,,
13.2 mg/m of SO,,, and 1.04 mg/m of (NhOpSO,. 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 OT was the major constituent of the mixture affecting the host's suscepti-
bility to infection. These investigators also measured a number of specific
0190ZS/A 10-124 5/1/84
-------
TABLE 10-14. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: MIXTURES
Ozone
concentration Measurement Exposure ^
ug/m3 ppm Pollutant
98 0.05 03 +
3760 N02
98-196 0.05-0.1 03 +
100-400 N02 +
1500 ZnS04
196 0.1 03 +
241-483 : H2S04
196 0.1 03 +
S 900 H2S04
i
rn j
196 0.1 ' 03 +
1090 H2S04
196 0.1 03 +
980 N02
196 0.1 03 +
980 N02
196 0.1 03 +
13200 S02 +
1040 (NH4)2S04
method duration and protocol
CHEM 3 hr
CHEM 3 hr
CHEM 3 hr
CHEM 3 hr +
2 hr
CHEM 3 hr +
2 hr
CHEM 3 months
CHEM 1-6 months
UV 5 hr/day,
5 days/wk
for 103 days
Observed effects Species
Exposure to mixtures caused synergistic Mouse
effect after multiple exposures.
Additive effect of pollutant mixtures Mouse
with infectivity model.
Increased susceptibility to Streptococcus Mouse
pyogenes.
Sequential exposure resulted in signifi- Mouse
cant increase in respiratory infection.
Neither alone produced a significant
effect.
Reference
Ehrlich et al. , 1977
Ehrlich, 1983
Grose et al. , 1982
Gardner et al. , 1977
Sequential exposure resulted in signifi- Hamster 1 Grose et al . , 1980
cant reduction in ciliary beating
activity.
Significant decrease in viability of Mouse
alveolar macrophages.
At 3 and 6 months, susceptibility to Mouse
pulmonary infection increased sig-
nificantly. Delayed clearance rate.
Highly significant increase in Mouse
susceptibility to infection. Effects
attributed to 03. Increased bactericidal
rate. Mixture showed greater growth
inhibition in leukemia target cells and an
increase in blastogenic response to PHA,
Con-A, and al loantigens.
Ehrlich, 1980
Ehrlich, 1980
Aranyi et al. , 1983
-------
TABLE 10-14. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: MIXTURES (continued)
o
I
CT>
Ozone
concentration
flgTifr3 ppm
216-784 0.11-0.4
3760-13720 2-7.3
980 0.5
11-3000
1568 0.8
3500
3500
3500
Pollutant
03 +
NO 2
03 +
H2S04
0, H-
Fe2(S04)2 +
H2S04 +
(NH4)2S04
Measurement 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 et al., 1978
secretion; synergism reported;
effect reversible in clean air.
Exposure to mixtures produced same Rat i Phalen et al., 1980
effect as exposure to 03 alone.
j Measurement method:
Abbreviations used:
CHEM = gas phase chemi luminescence; UV = UV photometry; A = amperometric.
PHA = phytohemagglutinin; Con-A = concanavalin-A
-------
host defenses to determine if host response to the mixtures was significantly
different from that of the controls or of the O^-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 0--alone and the control animals.
Previous studies (Gardner et al., 1977) indicated that a sequential expo-
sure to Oo followed by H^SO^ significantly increased streptococcal pneumonia-
induced mortality rates in mice. Ozone and H2SO. had an additive effect when
exposed in this sequence. However, the reverse sequence did not affect inci-
dence of mortality. Grose et al. (1982) expanded these studies and showed
3
that a 3-hr exposure to the combined aerosol of 196 ug/m (0.1 ppm) of 0- and
3
483 or 241 ug/m H^SO^ also significantly increased the percent mortality, as
compared to control.
The effects of exercise on the response to low levels of 0., were also
3
studied by using the infectivity model. Mice were exposed to 196 ug/m (0.1 ppm)
of 03 and 588 ug/m (0.3 ppm) of 0, for 3 hr while exercising. Each exposure
level yielded mortality rates that were significantly higher than those observed
in the 0~ group that was not exercised (Illing et al., 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.
10.3.4.4 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
10-15).
o
The effects of 2900 \jg/m (1.48 ppm) of 0, 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
0190ZS/A 10-127 5/1/84
-------
TABLE 10-15. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: IMMUNOLOGY
Ozone
concentration Measurement Exposure
pg/m3
196
H- 980, 2940
o
t
C»;980, 1568
i
980-2900
I
1150
ppm method duration and protocol
0.1 UV 5 hr/day,
5 days/wk for
90 days
0.5, 1.5 NO 4 hr
0.5, 0.8 Mast Continuous
3-4 days
0.5-1.48 CHEM 3 hr
0.59 NO 36 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
Levamisole 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 ppm for 5 days. Hemagglutination antibody
titers increased over control.
Impaired resistance to toxin stress.
Immunosuppression.
Species Reference
Mouse Aranyi et al., 1983
Mouse Goldstein et al., 1978a
Mouse Osebold et al . , 1979, 1980
Gershwin et al . , 1981
Guinea pig Thomas et al . , 1981b
Mouse Campbell and Hilsenroth,
1976
Measurement method: ND = not described; CHEM = gas phase chemiluminescence; UV = UV photometry; Mast = Mast meter.
Abbreviations used: PHA = phytohemagglutinin; con-A = concanavalin-A; LPS = lipopolysaccharide; IgE = immunoglobulin-E; IgA = immunoglobulin-A.
-------
with Mycobacteriurn tuberculosis, the cutaneous sensitivity to PPD was signifi-
cantly affected by 0». The diameters of the erythemas from the 03-exposed
animals were significantly smaller during the 4 to 7 weeks after the infectious
challenge, indicating depressed cell-mediated immune response. Exposure to
980 ug/m3 (0.5 ppm) of 0_ 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 (200 ug/m of 0- 5 hr/day, 5 days/week for 90 days). No
alteration in the response to alloantigens or the B-cell mitogen lipopoly-
saccharide (IPS) was noted, but a statistically significant suppression in
blastogenesis to the T-cell mitogens (PHA and con-A) was detected. The authors
suggested this indicated that the cellular mechanisms for recognition and
proliferation to LPS and alloantigens were intact, but 0, might have been in-
O
terfering 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).
Campbell and Hilsenroth (1976) used a toxoid immunization-toxin challenge
approach to determine if continuous exposure to 1150 ug/m (0.59 ppm) of 0.,
O
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 03-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 stephylococci 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
tested, 2940 and 980 ug/m3 (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.
0190ZS/A 10-129 5/1/84
-------
The possibility that 03 may be responsible for the enhancement of allergic
sensitization has important implications for human health effects. Gershwin
et al. (1981) reported that 0- (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. In general, the number of IgE-containing cells correlated positively
with levels of anaphylactic 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). These authors showed that a signi-
ficant increase in anaphylactic sensitivity occurred when antigen-stimulated
and Og-exposed animals were compared to controls (Osebold et al., 1980).
Significantly greater numbers of animals were allergic in experimental groups
which 0- exposure ranged from 0.8 ppm to 0.5 ppm for 3 days. Further studies
are needed to determine the threshold level of these effects.
10.3.5 Tolerance
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 11, 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
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 forms,
tolerance and adaptation, are not yet fully understood in any concise manner.
0190ZS/A 10-130 5/1/84
-------
Further research is needed to focus on the specific structural/functional
response being addressed in each specific experimental situation and to relate
this to the exposure concentration and profiles of 03- An attempt to relate
the modified responses seen in animals to what has been described in human
subjects will be made in Chapter 13.
In animal oxidant toxicity studies, the term tolerance classically is
defined as the phenomenon wherein a previous exposure to a nonlethal dose of
0« will provide some protection against a subsequent exposure to a dose of CL
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 morpho-
logical changes. This protective phenomenon seen with oxidants was originally
described by Gildemeister (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
O
early as 1956, Stokinger et al. presented data clearly indicating that an
animal could also become tolerant to the lethal effects of 03- Such tolerance
has also been reported by many investigators, including Matzen (1957a), Menden-
hall and Stokinger (1959), Henschler (1960), and Fairchild (1967). The obser-
vation of this tolerance phenomenon in experimentally exposed animals has led
to the speculation that it may also be a mechanism for protecting environmental-
ly exposed humans.
The previous criteria document for 0_ and other photochemical oxidants
cited various studies (Table 10-16) that examined 03 tolerance and presented
some evidence indicating possible mechanisms of action. Review of these
earlier data reveals that pre-exposure to a certain concentration of 03 can
protect test animals from the acute lethal effects of a second exposure to 03-
This protection has been attributed to a significant reduction in pulmonary
edema in the pre-exposed animals. Table 10-16 lists the key studies on 03-
Because 0- has a marked proclivity to reduce the ability of alveolar
<3
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 10.3.4.3), the pre-exposed (tolerant)
animals were only partially protected from the aerosol infectious challenge
0190ZS/A 10-131 5/1/84
-------
TABLE 10-16. TOLERANCE TO OZONE
Ozone
(ug/m3)
pre-
exposure
196-1960
196
490
i— '
0
1
t-1 980
OJ
ro
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
(ug/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
1, 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.
lolerance 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 al., 1971a
with methylprednisolone.
Edema as measured by recovery of 1321
in pulmonary lavage fluid.
Tolerance to edema effects of 0., did not Mouse Gregory et al., 1967
develop in thymectomized animals but
developed in sham-operated animals, in- i
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 0, 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 a local effect and seen Alpert and Lewis, 1971
only in the pre-exposed lung.
-------
TABLE 10-16. TOLERANCE TO OZONE (continued)
Ozone
(pg/rn3)
pre-
exposure
980
t— '
V 1470
i—1
OJ
CO
1600
1960
1960
Ozone
Ozone (ug/m3)
(ppm) Length of after
pre- pre- latent
exposure exposure period
0.5 3 hr 5880 or
0.75 3 days 7840
0.8 4 hr 2352
1 1 hr ND
1 1 hr 3920
Ozone Length of
(ppm) exposure
after after
latent latent
period period
3 and 22 3 hr
4.0 8 hr
1.2 4 hr
ND ND
2 1 hr
Observed effect(s) Species Reference
With unilateral lung exposure technique, Rabbit Gardner et al., 1972
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 Chow, 1976
peroxidase, glutathione reductase, glucose-6- Chow et al., 1976b
phosphate dehydrogenase and levels of reduced
glutathione in lungs of tolerant animals, as
compared to nontolerant animals.
Pre-exposure to 03 caused complete tolerance to Rat Frager et al., 1979
delay in mucociliary clearance at 3 days, but
not 13 days.
All animals X-irradiated to 800 R. 60% of Mouse Hattori et al . , 1963
03-pre-exposed mice survived. 100% of
controls died.
Tolerance to allergic response to inhaled Guinea Matsumura et al . , 1972
acetylcholine. pig
ND = not described.
-------
(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 ug/m (0.10 ppm) of 03, 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 al., 1972), studies were conducted to evaluate the effects of tolerance at
the cellular level. These studies indicated that the initial Q3 exposure did
induce tolerance against pulmonary edema in the exposed lung; however, there
was no protection afforded against the cytotoxic effects of 03 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 polymorphonuclear leukocytes—PMN)
showed that there was no protection against these cellular defense mechanisms.
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
00 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 0., 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 10.3.1.2, and they indicate
that with continuous exposure to 03, the lung attempts to initiate the repair
of the 03 lesion, resulting in somewhat reduced or less than expected total
0190ZS/A 10-134 5/1/84
-------
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 03 (Evans et al., 1976a; Dungworth et al., 1975a; Schwartz et al., 1976).
Thus, there is also the possibility that this reparative-proliferative response
relines the airway epithelium with cells that have a biochemical armamentarium
more resistant to oxidative stress (Mustafa et al., 1977; Mustafa and Lee,
1976).
Another suggestion is that with 03, 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 al.,
1978). As the airways become more obstructed, the 03 molecules are less likely
to penetrate to lumen. This may result in a filtering system that removes the
0, from the inhaled air before it reaches the alveoli.
More recently, there have been additional studies examining the protective
effects of this tolerance phenomenon in animals. Frager et al. (1979) studied
the possibility of tolerance to 0- in mucociliary clearance. Exposure of rats
to 1.2 ppm of 03 following particle deposition caused a substantial delay in
mucociliary clearance. This 0- effect could be eliminated by a pre-exposure
3
to 1600 [jg/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 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 0^. 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 (Nilding and Hi!ding, 1966) or the occurrence of some other temporary
change in the regenerating ciliated cell.
0190ZS/A 10-135 5/1/84
-------
Tolerance to 0_ has also been studied by using a variety of biochemical
indicators to measure the extent to which a pre-exposure to 0~ protects or
O
reduces the host response to a subsequent exposure. Chow et al. (1976a,b)
compared a variety of metabolic activities of the lung immediately after an
initial 3-day continuous exposure to 1600 ug/m (0.80 ppm) of 0 with the
O
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 rou-
tine. 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.
A number of biochemical effects of 0- gradually subside in intensity
despite continued exposure to the gas. An example is prolyl hydroxylase, a
key enzyme in collagen synthesis that serves as a sensitive indicator of
disturbances in collagen metabolism. The increase in superoxide anion caused
by 0- stimulates collagen synthesis. This is reflected in an increase in
prolyl hydroxylase activity. Bhatnagar et al. (1983) have shown that there is
also an increase in superoxide dismutase, which quenches free radicals, thereby
restricting hydroxylase activity and collagen formation.
Gertner et al. (1983b) presented data showing that the development of
adaptation and tolerance is rapid and mediated through the vagus nerve. These
investigators used a bronchoscope to study the response of the periphery of
the lung to 0~ exposure. Measurements of collateral resistance (Rcoll) were
used to monitor the response to 0~. 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
0_ exposure ceased, the Rcoll returned to normal. Subsequent exposure to 0.1
ppm of 0_ did not increase Rcoll, indicating that some protection existed.
These investigators 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.
0190ZS/A 10-136 5/1/84
-------
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 0« 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.
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 in the face of effective tolerance for
edema production. This suggests that the chemotactic effect of 0., may be
«J
separable from the edemagenic effect. This may explain why chronic morpholo-
gical 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 al. , 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 0~ exposure but did after
challenging with hi stamina, indicating that the parasympathetic system may
play a role in response to 03 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
0190ZS/A 10-137 5/1/84
-------
changes, such as an increase in mucus thickness, that may, in effect, prevent
03 from 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 with great potential for possible
future harm.
10.4 EXTRAPULMONARY EFFECTS OF OZONE
10.4.1 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 11), and the possible
implications of these and other symptoms as indications of low-level 0., effects,
few recent reports were found on behavioral and central nervous system (CNS)
effects of 03 exposure in animals. Table 10-17 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 03 by measuring their reaction to a 30-min exposure on one
side of an annular plastic mouse chamber. A concentration-related avoidance
of the 03 side was reported at 1176 to 16,660 ug/m (0.60 to 8.50 ppm) of Og.
However, the study had serious shortcomings, including a lack of position-
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 0.,. Mice were individually exposed to 0^ 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 ug/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 ug/m (10 ppm) of CL, all animals turned off
100 percent of the trials with an average latency of approximately 10 sec.
0190ZS/A 10-138 5/1/84
-------
TABLE 10-17. CENTRAL NERVOUS SYSTEM AND BEHAVIORAL EFFECTS OF OZONE
Ozone
concentration
ug/m3
110
98-1960
196-3920
o 235-1960
^ !
GO
392-980
588-1372
ppm
0.056
0.05-1.0
0.1-2.0
0.12-1.0
0.2-0.5
0.3, 0.7
Measurement3' Exposure
method duration and protocol
d 93 days, continuous
MAST 45 min
CHEM 6 hr
CHEM 6 hr
NBKI 6 hr
MAST 7 days, continuous
Observed effects(s)c Species Reference
No overt behavioral changes. Cholines- Rat Eglite, 1968
terase activity inhibited at 75 days of
exposure, returning to control levels
12 days after termination of exposure.
Motor activity progressively decreased Rat Konigsberg and
with increasing 03 concentrations up to Bachman, 1970
0.5 ppm. Slight increase in frequency
of 3-min intervals without motor activity.
Linear and/or monotonic decreases in Rat Weiss et al . ,
operant behavior during exposure. 1981
Wheel running activity decreased Rat Tepper et al.,
monotonically with increasing 03 con- 1982
centration. Components of running
were differentially affected at low
vs. high 03 concentrations.
Wheel running activity decreased 50%. Mouse Murphy et al . ,
1964
Running activity decreased 60% during
first 2 days, returning to control
levels during the next 5 days of expo-
sure; running activity decreased 20%
when 0.3 ppm exposure was followed
immediately by 0.7 ppm 03 exposure.
Adaptation with continued exposure
was apparent.
980-19600 0.5-10.0
CHEM
60 s
Exposure terminated by nose pokes with
increasing frequency .TS Q~ concentra-
tion increased.
Mouse Tepper et al.
1983
980 0.5
NU
mm
Elevation of simplp and choice reactive
time.
Nonhuman Reynolds and
primate Chaffee, 1970
-------
TABLE 10-17. CENTRAL NERVOUS SYSTEM AND BEHAVIORAL EFFECTS OF OZONE (continued)
c
I
Ozone
concentration
ug/m3
980-1960
1176-
16,660
1960
1960-
5880
1960
1960-
5880
ppm
0.5, 1.0
0.6-8.5
1.0
1-3
1
1-3
Measurement ' Exposure
method duration and protocol
NBKI 1 hr
e 30 min
MAST 7 days, continuous
NBKI
ND 18 months,
8 hr/day
18 months;
8, 6, 24 hr/day
ND 18 months,
8 hr/day
Observed eftects(s)
Evoked response to light flashes in the
visual cortex and superior collicus
decreased after exposure.
Avoidance behavior increased with
increasing 03 concentration.
Reduction in wheel running activity; no
effect of Vitamin E deficiency or
supplementation.
COMT activity decreased at 2 ppm, MAO
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 EEC patterns after
9 months, but not after 18 months of
exposure.
Species Reference
Rat ' Xintaras et al . ,
1966
Mouse Peterson and
Andrews, 1963
Rat Fletcher and
Tappel, 1973
Dog Trams et al . ,
1972
Dog Johnson et al.,
1974
Measurement method: MAST = Kl-coulometric (Mast meter); CHEM = gas phase chemiluminescence; NBKI = neutral buffered potassium iodide; ND = not
described.
Calibration method: NBKI = neutral buffered potassium iodide.
Abbreviations used: COMT = catechol-o-methyltransferase; MAO = monamine oxidase; EEC = electroencephalogram
Spectrophotometric method with dihydroacridine.
KI titration with sodium thiosulfate.
-------
Studies by Murphy et a "I. (1964) demonstrated that wheel-running activity
3
decreased by approximately 50 percent when mice were exposed to 392 to 980 ug/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 pg/m (0.30 ppm)
of 00. Running activity gradually returned to pre-exposure values during the
3
next 5 days of continuous exposure to 588 pg/m (0.30 ppm) of Ov If the same
3
mice were subsequently exposed to 1372 pg/m (0.70 ppm) of 0- for an additional
7 days, running activity v/as depressed to 20 percent of pre-exposure values.
3
Partial recovery was described during the final days of exposure to 1372 pg/m
(0.70 ppm), and complete recovery occurred several days after exposure was
terminated. However, partial tolerance was seen when the air-control mice
were subsequently exposed to 392 pg/m (0.20 ppm) of 0- for 7 days. Konigsberg
and Bachman (1970) used a capacitance-sensing device to record the motor
activity of rats during a 45~min exposure to 98, 196, 392, 980, and 1960 pg/m
(0.05, 0.10, 0.20, 0.50, and 1.0 ppm) of 0~. Compared with control rats,
motor activity following 0, exposure progressively decreased with increasing
3
0, concentrations up to 980 pg/m (0.50 ppm). No greater reduction was obtained
o
at 1960 pg/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 0, concentration.
A detailed microanalysis 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 OT- 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 pg/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 Q~. An increase in
the time interval between running bursts primarily accounted for the decreased
3
motor activity at the low (235 pg/m , 0.12 ppm) 0, concentration. Postexposure
increases in wheel running were seen following this low 0,, concentration. At
3
higher 0- 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.
0190ZS/A 10-141 5/1/84
-------
Effects of 03 on behavior were further investigated by Weiss et al.
(1981) in their studies on the operant behavior of rats during CL 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
3
rats were exposed for 6 hr to CL 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
3
afternoon. Ozone-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
3
monotonic from 196 to 3920 ug/m (0.10 to 2.0 ppm). Analysis of the distribu-
tion of responses during 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 contrast to other types of toxicants, 0, did not disrupt
the temporal pattern that characterized response during each fixed-interval
presentation. Based on tha 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 0.,. One-hour air perfusion following
the 0,, 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, includ-
ing O.,, 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
1960, 3920, or 5880 ug/m (1, 2, or 3 ppm) of 03- In 8 hr/day exposures,
reported decreases (35 percent) in the catecholamines norepinephrine and
0190ZS/A 10-142 5/1/84
-------
epinephrine were not statistically significant, although 0- exposure at
3
3920 ug/m (2 ppm) caused a statistically significant decrease in catechol-o-
methyl-transferase (COMT) activity. In contrast, monamine oxidase (MAO)
activity was significantly elevated at 1960 ug/m (1 ppm) of 0,, but not at
3 3
3920 or 5880 ug/m (2 or 3 ppm) of 0~. Increasing daily exposures to 1960 ug/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. (1974) measured electroencephalographic (EEC)
patterns in the same dogs and noted alterations in EEC patterns after 9 months
3
of exposure to 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
EEC 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 about chronic 0, exposure.
10.4.2 Cardiovascular Effects
Very few reports on the cardiovascular effects of 0., 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 0.20 ppm
of 0.3 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 03 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 03 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
combined, in rats. The rats were exposed to 0.60 ppm ozone of 0., 5 hr/day for
3
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.
0190ZS/A 10-143 5/1/84
-------
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
3
exposed to 392, 1568, or 3920 ug/m (0.2, 0.8, or 2 ppm) of 0., 6 hr/day, 5
days/week for 62 exposure days as part of a more extensive evaluation of lung
function (Section 10.3.2). 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
and 1960 ug/m (0.3 and 1.0 ppm) of 0- on the pulmonary gas-exchange region of
dogs ventilated through an endotracheal tube. Pulmonary capillary blood flow
and arterial 0? pressure (Pa02) were decreased 30 min following exposure to
both 0., concentrations, and arterial pH (pH ) was decreased following exposure
o o a
to 1960 ug/m (1.0 ppm) of Ov Decreases in pulmonary capillary blood flow
3
persisted 24 hr following exposure to 588 and 1960 ug/m (0.3 and 1.0 ppm) of
03 and as long as 48 hr following exposure to 1960 ug/m (1.0 ppm) of 0.,.
Persistent decreases in pH and Pa00 were observed 24 hr following exposure to
3
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 0., exposure
can cause both acute and delayed changes in cardiopulmonary function.
10.4.3 Hematological and Serum 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
0- or some reaction product of 03 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 i_n vitro and i_n vivo
0., exposure. Hematological 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.
0190ZS/A 10-144 5/1/84
-------
10.4.3.1 Animal Studies - In Vivo Exposures. The effects of jm vivo 03
exposure in animals, including studies reviewed in the previous 0^ criteria
document (U.S. Environmental Protection Agency, 1978), are summarized in Table
10-18.
Effects of 0- on the blood were first reported by Christiansen and Giese
(1954) after they detected an increased resistance to hemolysis of RBCs from
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 03 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 03 concentration 8 hr/day for 7 days.
In more recent studies, Clark et al. (1978) investigated the biochemical
changes in RBCs of squirrel monkeys exposed to 1410 ug/m (0.75 ppm) of 0,
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 ug/m (1
or 3 ppm) of 0~ 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 0.,.
Alterations in RBC morphology have been previously observed in 0,,-exposed
laboratory animals and man (Brinkman et al. , 1964; Larken et al. , 1978).
Similar observations have recently been made in monkeys exposed to 1254 ug/m
(0.64 ppm) of 03 for 8 hr/day over a 1-yr period (Larkin et al. , 1983).
Ultrastructural SEM studies of RBC's following exposure to 03 demonstrated
0190ZS/A 10-145 5/1/84
-------
TABLE 10-18. HEMATOLOGY: ANIMAL — IN VIVO EXPOSURE
o
I
CTV
Ozone
concentration Measurement
ug/m3
110
118
235
470
941
392
392
392
392-
1960
490
980
1372
627
784
ppm method
0.056 c
0.6 UV
0.12
0.24
0.48
0.2 ND
0.2 UV
0.2 UV
0.2- UV
1.0
0.25 UV
0.50
0.70
0.32 UV
0.4 ND
Exposure
duration and
protocol
93 days
2.75 hr
4 hr
8 hours/day,
5 days/week,
3 weeks
60 min
1-4 hr
2.75 hr
6 hr
± dietary
vitamin E
G hr/day,
5 days/week,
6 months
Observed effect(s) Species
Decreased whole blood chol inesterase, Rat
which returned to normal 12 days after
exposure ceased.
RBC survival decreased at 0.06, Rabbit
0.12, and 0.48 ppm; no concentra-
tion-response relationship.
Increased osmotic fragility and Rabbit
spherocytosis of RBC's.
Increased serum glutamic pyruvic Mouse
transaminase and hepatic ascorbic
acid. No change in blood catalase.
Small decrease in total blood sero- Rabbit
tinin.
Plasma creatine phosphokinase Mouse
activity altered immediately and
15 min postexposure; no effect
30 min postexposure. No change
in plasma histamine or plasma
lactic acid dehydrogenase.
RBC survival decreased at 0.25 ppm Sheep
only.
Increased erythrocyte G-6-PD and Mouse
decreased AChE (both diets).
Increased plasma vitamin E
(both diets).
No change serum trypsin inhibitor Rabbit
capacity.
Reference
Eglite, 1968
Calabrese et al . ,
1983a
Brinkman et al . ,
1964
Veninga, 1970
Veninga, 1967
Veninga et al . ,
1981
Moore et al. ,
1981a
Moore et al . , 1980
P' an and Jegier ,
1971
-------
TABLE 10-18. HEMATOLOGY: ANIMAL — IN VIVO EXPOSURE (continued)
o
I
Ozone
concentration
ug/m3
784
784
784
980
980
980
1254
1470
ppm
0.4
0.4
0.4
0.5
0.5
0.5
0.64
0.75
Exposure
Measurement duration and
method protocol
NO 6 hr/day,
5 days/week,
10 months
ND 10 months
ND 6 hr/day,
5 days/week,
10 months
UV 2.75 hr
MAST Continuous,
23 days
NBKI 8 hr/day,
7 days
i
UV 8 hr/day,
1 yr
ND 4 hr/day,
4 days
Observed effect(s)
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
Rabbit
Rabbit
Rabbit
Sheep
Rat
Monkey,
rat
Monkey
Monkey
Reference
Jegier, 1973
P'an and
1972
P'an and
1976
Moore et
1981b
Menzel et
Jegier,
Jegier,
al.,
al., 1972
Chow et al. , 1975
Larkin et
Clark et
al., 1983
al., 1978
-------
TABLE 10-18. HEMATOLOGY: ANIMAL — IN VIVO EXPOSURE (continued)
o
i
CO
Ozone
concentration
ug/m3 ppm
1568 0.8
1568 0.8
1568 0.8
1666 0.85
1686 0.86
1960 1.0
1960 1.0
Exposure
Measurement duration and
method protocol
NBKI 7 days
NBKI 8 hr/day,
7 days
NBKI Continuous,
29 days
MAST 4 hr
NO 8 hr/day,
5 days/week,
6 months
UV 4 hr ± vitamin E
ND 30 min
Observed effect(s) Species
Increased activity of GSH pero- Rat
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 G-6-OP,
catalase, and superoxide dismutase
and in levels of thiobarbituric acid
reactants, methemoglobin, hemoglobin,
and reticulocytes.
No change in total lactate dehydro- Monkey
genase activity or isoenzyme pattern
in plasma or erythrocytes.
Increased lysozyme activity by Rat
day 3.
Increased Heinz bodies in RBC's Mouse
(decreased with continual exposure).
Increased infestation and mor- Mouse
tality after infection with
Plasmodium berghei. Increased
acid resistance of erythrocytes.
Decreased filterability. No pro- Mouse
tection by vitamin E. No lipid
peroxidation.
Increased resistance to erythrocyte Mouse
hemolysi s.
Reference
Chow and Kaneko,
1979
Chow et al . ,
Chow et al . ,
Menzel et al
Schlipkoter
Bruch, 1973
Dorsey et al
Mizoguchi et
1973;
Christiansen
Giese, 1954
1977
1974
. , 1975a
and
. , 1983
al. ,
and
-------
TABLE 10-18. HEMATOLOGY: ANIMAL — IN VIVO EXPOSURE (continued)
Ozone
concentration
ug/ni3
1960-
3920
1960
5880
2940
11,760
15,680
7840
9800
19,600
ppm
1.0
2
1.0
3.0
1.5
6.0
8.0
4.0
5.0
10.0
Exposure
Measurement duration and
method protocol
CHEM 2 or 7 days
CHEM 4 hr
UV 3 days
4 days
4 days
UV 2, 4, or 8 hr
CHEM 3 hr
I 1 hr/week,
6 weeks
Observed effect(s)
No changes.
No effects on oxyhemoglobin affinity,
2,3-DPG concentrations, heme-02
binding.
No effect on SOD, GPx, K+ influx
ratios (all levels). Increased Hb,
Hct, echinocytes II & III (6 & 8 ppm);
echinocytes correlated with petechia
in lungs, indicative ot vascular
endothelial damage.
Increased plasma concentrations
of PGF2a and PGE2.
Inhibition of serum monoamine oxi-
dase.
Production of serum antibodies that
reacted with ozonized egg albumin
but not native ovalbumin.
Species Reference
Rat, Cavender et al . ,
guinea pig 1977
Rabbit Ross et al. , 1979
Rat Larkin et al . , 1978
Rat Giri et al. , 1980
Rat Suzuki , 1976
Rabbit Scheel et al., 1959
Measurement method: NO = not described; CHEM = gas phase chemiluminescence; UV - UV photometry; NBKI = neutral buffered potassium iodide;
MAST = KI - coulometric (Mast meter); I = iodometric.
Abbreviations used: RBC = red blood cell; G-6-PO = gl ucose-6-phosphate dehydrogenase; AChE = acetylcholinesterase; GSH = reduced
glutathione; GSH peroxidase = glutathione peroxidase; GSH reductase = glutathione reductase; FA = fatty acid; LDH = lactic dehydrogenase;
2,3-DPG = 2,3-diphosphoglycerate; SOD = superoxide dismutase; GPx = glutathione peroxidase; K = potassium, Hb = hemoglobin; Hct = hemato-
crit; PGF2a = prostaglandin F2a; PGE2 = prostaglandin E2.
Spectrophotometric method using dihydroacridine
-------
reduced numbers of normal discocytes and increased numbers of knizocytes,
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 Oo~exposed animals.
3
Moore et al. (1981a) reported reduced RBC survival in sheep exposed to 490 ug/m
(0.25 ppm) 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 ug/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
vitamin E-deficient rats to 1568 ug/m (0.8 ppm) of 0., 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
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
blood of 9-month-old C57L/J mice exposed to 627 ug/m (0.32 ppm) of 0~ 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 0~ 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 ug/m (0.3 ppm), 1372 pg/m (0.7 ppm),
or 1960 ug/m (1.0 ppm) of 0- 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-pm 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 0,. and incubation
of RBCs for 6 hr resulted in a significant increase in filtration time of RBCs
0190ZS/A 10-150 5/1/84
-------
from 0,-exposed mice, and a lack of protection by dietary vitamin E. The
3
hematocrit of vitamin E-deficient mice exposed to 1960 ug/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.
10.4.3.2 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 10-19.
The effects of in vitro 0- exposure on human RBCs have been evaluated by
.. ^
using a number of different end points, such as increases in complement-mediated
cell damage (Goldstein, B., et al., 1974), formation of Heinz bodies (Menzel et
al. , 1975b), decreases in RBC native protein fluorescence (Goldstein and
McDonaugh, 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 0~ exposure. (See Table 10-20 for a summary
of the human J_n vitro studies.)
Kesner et al. (1979) demonstrated that 0.,-treated phosphol ipids inhibited
RBC membrane ATPase. . Addition of semicarbazide to O^-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 0? through the solution at 4 ml/min (2.5 pM/min of 0~)
for 1 or 2 min. Semicarbazide (40 uM) or PABA (40 uM) inhibited the cross-
linking of 03-exposed spectrin. The inhibition of AChE and hexokinase activities
of RBC ghosts exposed to CL was also partially prevented by these two agents,
+ J
as was K influx into whole RBCs. The authors attributed the inhibition of
ATPase to oxidation of phospholipids with subsequent cross-linking of membrane
protein by lipid peroxidation products. Because the reaction of ozonolysis
0190ZS/A 10-151 5/1/84
-------
TABLE 10-19. HEMATOLOGY: ANIMAL — IN VITRO EXPOSURE
en
ro
Exposure
Ozone Measurement duration and
concentration method protocol
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 aminotriazole.
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 10-20. HEMATOLOGY: HUMAN - IN VITRO EXPOSURE
o
i
en
OJ
Ozone3
concentration
980 ug/m3 (0.5 ppm)
1960 pg/m3 (1.0 ppm)
03-treated phospholipids
4 pM/min
Methyl ozonide
10-4-2xlO-3 M
750 nM/min
106 nM/min
300 nM/min
0-9.8 uM/g of Hb
0.84 pM/min
78400 pg/m3 (40 ppm)
Measurement
method
CHEM
NO
I
NO
NBKI
NBKI
NBKI
NBKI
NBKI
Exposure
duration and
protocol
0.5-2 hr
5, 10, 15, and
20 min
1 min
30 min
14.3 or 43.0
nMol of 03 per 106
cell equivalent
5, 10, 20, 30,
40 and 50 min
NO
0-2 hr
2 hr
Observed effect(s)
Decreased agglutination of RBCs by
concanavalin A.
Decreased ATPase 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
a1-proteinase inhibitor.
Decreased glyceraldehyde-3-PD.
Decreased ATPase.
No statistics.
Decreased GSH. No effect on
Hb or on glucose uptake.
Increased complement-mediated cell
damage.
Species
Human
Human
(RBC ghosts)
Human
(RBC ghosts)
Human
Human
Human
Human
(RBC ghosts)
Human
(RBCs,
RBC ghosts)
Human
Reference
Hamburger et al. , 1979
Kesner et al. , 1979
Kindya and Chan, 1976
Menzel et al. , 1975b
Koontz and Heath,
1979
Johnson, 1980
Freeman et al . ,
1979
Freeman and Mudd,
1981
Goldstein, B. , et al. ,
1974
1,960 pg/m3 (1.0 ppm) NBKI
20 and 60 min Decreased native protein fluore-
scence. No statistics.
Human Goldstein et al.,
(RBC ghosts) 1975
-------
TABLE 10-20. HEMATOLOGY: HUMAN - IN VITRO EXPOSURE (continued)
Ozone3
concentration
40 nM/rain
t — i
0
i — »
en
-p>
2.5 uM/min
2.5 uM/min
L Exposure
Measurement duration and
method protocol
I 4 min
I 20, 40, and 60
rain
I 20, 40, and
60 min
Observed effect(s)
Decreased ATPase activity; lost 40%
membrane sulfhydryls. Lipid per-
oxidation and protein crosslinking
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 benzoic acid prevented
these 03 effects.
Species Reference
Human Chan et al., 1977
(RBC ghosts)
Human Verweij and
Steveninck, 1980
Human Verweij and Steveninck,
(RBC ghosts) 1981
bNot ranked by concentration; listed by reported values.
Measurement method: NO = not described; CHEM = gas phase chemiluminescence; NBKI = neutral buffered potassium iodide; I = iodometric.
-------
products with semicarbazide and PABA during 03 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 i_n vitro
reaction of 03 with sulfhydryl groups of human RBC membrane, proteins, and
cytoplasmic contents. After exposure of RBCs to 03 in 0^ at 20 ml/min (0.84
nMol/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 [jg/m (0.5 ppm) of 03 continuously for 23 days.
Although jjn 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 i_n vivo 03 toxicity in man is diffi-
cult. In most J_n vitro studies, RBCs were exposed by bubbling high 0., concen-
trations (> 1 ppm) through cell suspensions. Not only were the 0~ concentra-
O
tions unrealistic and the method of exposure nonphysiological, but the toxic
species causing RBC injury may be different during i_n vitro and i_n vivo 0.,
O
exposures. Because of its reactivity, it is uncertain that 03 per s_e 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.
10.4.3.3 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)
o
investigated the effects of 784 (jg/m (0.4 ppm) of 0., 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
10.3.1).
0190ZS/A 10-155 5/1/84
-------
P'an and Jegier (1976) also reported changes in serum proteins after
3 3
exposure of rabbits to 784 ug/m (0.4 ppm) and 1960 |jg/m (1.0 ppm) of Ov
3
Following exposure to 784 ug/m (0.4 ppm) of 0» for 105 days, the albumin
concentrations began to decrease, and or and 6-globulin concentrations began
to increase. At the end of 210 days of exposure, the mean albumin level fell
16 percent, the a-globulin 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-
3
tent (8 hr/day) exposure to 1568 ug/m (0.8 ppm) of 0~ for 7 days. The
increased release of lysozyme into the plasma was suggested to be a result of
0- damage to alveolar macrophages.
3
After exposure of rats to 7840 ug/m (4 ppm) of 0- for 2, 4, and 8 hr,
Giri et al. (1980) observed increases in plasma prostaglandin (PG) F- of 186,
109, arid 25 percent, respectively. Plasma concentrations of PGE» were approxi-
mately twice the control levels after exposure for 2 or 8 hr, but only a
slight increase was observed after exposure for 4 hr.
Veninga et al. (1981) reported that short-term exposures of mice to low
0~ concentrations induced changes in serum creatine phosphokinase (CPK) activity.
3
Ozone doses were expressed as the product of concentration and time; the
maximum 0- concentration was 1600 ug/m (0.8 ppm), and the maximum exposure
•3
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 0- doses employed. The authors concluded that these
responses may represent adaptation of the animals to 0_ toxicity by enhanced
metabolic processes.
10.4.3.4 Interspecies Variations. The use of animal models to investigate
the effects of 0~ on the blood is complicated, because few species respond
O
like humans. The rodent model has been most commonly used to predict the
effects of 0- on human RBCs. The reliability of this model was recently
challenged by Calabrese and Moore (1980) on the following grounds: (1) ascorbic
acid synthesis was significantly increased in mice following 0., exposure
(Veninga and Lemstra, 1975), (2) ascorbic acid protected human G-6-PD-deficient
RBCs in vitro from the oxidant stress of acety1pheny1hydrazine (Winterbourn,
0190ZS/A 10-156 5/1/84
-------
1979), and (3) humans lack the ability to synthesize ascorbic acid. Although
Calabrese and Moore (1980) stressed that this hypothesis 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 model, because these mice
responded to 0, exposure (627 ng/m > 0.32 ppm for 6 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 Academy of Sciences, 1977). However, Dorset sheep were
found to be no more sensitive than normal humans with respect to 0.,-induced
changes in GSH and also differed from humans in the formation of methemoglobin.
Further studies (Calabrese et al., 1982, 1983b; 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 0, intermedi-
ates, but G-6-PD-deficient human erythrocytes were considerably more suscep-
tible. Consequently, the authors also questioned the value of the sheep
erythrocyte as a quantitatively accurate predictive model.
10.4.4 Reproductive and Teratogenic Effects
Pregnant animals and developing fetuses may be at greater ris,k to effects
from photochemical oxidants, because the volume of air inspired by females
generally increases from 15 to 50 percent during pregnancy (Allman 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 10.5), or they used oxidant concentra-
tions greater than those typically found in ambient air. Brinkman et al.
(1964) exposed pregnant mice to lower concentrations of 03, but the results of
their experiments are difficult to interpret, because the time of 03 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 pg/m (0.1 ppm) of 0~
(Table 10-21). Unfortunately, without more details on the period of exposure,
0190ZS/A 10-157 5/1/84
-------
PRELIMINARY DRAFT
TABLE 10-21. REPRODUCTIVE AND TERATOGENIC EFFECTS OF OZONE
o
en
Oo
Ozone
concentration
Mg/mJ
196
392
862
ppm
0.1
0.2
0.44
Measurement3
method
NO
ND
I
Exposure
duration and protocol
7 hr/day, 5 days/week
for 3 weeks
7 hr/day, 5 days/week
for 3 weeks
8 hr/day over entire
Observed effect(s)
Increased neonatal mortality £4.9 to 6.8%
vs. 1.6 to 1.9% for controls)0.
Unlimited growth of incisors (5.4% incidence
vs. 0.9% in controls) .
Decreased average maternal weight gain.
Species
Mouse
Mouse
Rat
Reference
Brinkman et al .
Veninga, 1967
Kavlock et al . ,
, 1964
1979
2920
1.49
period of organogenesis
(days 6 to 15)
Continuous during mid-
gestation
Increased fetal resorption rate (50% vs. 9%
for controls).
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 = iodometric (Saltzman and Gilbert, 1959).
No statistical evaluation.
-------
it is impossible to ascertain whether the decreased infant survival rate was
due to development interference ij} 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,
3
Veninga (1967) found that mice exposed to 392 ug/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
O
effects were found in rats exposed 8 hr/day to concentrations of 03 varying
from 863 to 3861 ug/m3 (0.44 to 1.97 ppm) during early (days 6 to 9), mid
(days 9 to 12), of late (days 17 to 20) gestation, or the entire period of
3
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.
3
A single dose of 150 mg/kg sodium salicylate followed by 1960 ug/m (1.0 ppm)
of OT during midterm produced a significant synergistic increase in the resorp-
tion rate, a decrease in maternal weight change, and an average fetal weight.
3
Exposure of pregnant rats 8 hr/day to 862 ug/m (0.44 ppm) of 0.. 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 ug/m (1.5 ppm) of 0, for periods of 4 days
3
during gestation. Pups from litters of females exposed to 1960 ug/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
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 ug/m (1.5 ppm) of 0~ (1.0 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.
0190ZS/A 10-159 5/1/84
-------
10.4.5 Chromosomal and Mutational Effects
10.4.5.1 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 |jg/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 conduc-
3
ted at or below 1,960 ug/m (1 ppm) of 0~. Data derived predominantly from j_n
3
vitro experiments conducted at 0, concentrations in excess of 1,960 ug/m (1
o
ppm) of 0^ will be discussed first (Table 10-22), followed by a discussion of
the genotoxicity data from both i_n vitro and in v"ivo research conducted at. or
below I ppm of 03 (Table 10-23).
The potential for genotoxic effects relating to 0~ exposure was predicted
from the radiomimetic properties of GO- The decomposition of 0^ in water
produces OH and H0« 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
human KB cells exposed to 15,680 ug/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, slightly more efficient in the production of
chromatid breaks than 50 rad of x-rays. Significant mitotic delay was measured
in neuroblasts from the grasshopper Chortophaga viridifaciata exposed to 3500
to 4500 ug/L of 0~ 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 when
cells were exposed to 1 ug/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 03-
The molecular mechanism for the clastogenic and lethal effects resulting
from 0~ exposure are not precisely known. Bubbling 8 percent 0., through a
phosphate buffer solution (0.05M, 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 pyrim-
idine bases, nucleosides, and nucleotides. In a more recent report, Shiniki
et al. (1981) examined the degradation of a mixture of 5' nucleotides, yeast
0190ZS/A 10-160 5/1/84
-------
TABLE 10-22. CHROMOSOMAL EFFECTS FROM IN VITRO EXPOSURE TO HIGH OZONE CONCENTRATIONS
03 Measurement
concentration method
Exposure
duration and protocol
Observed effect(s)
Species
Reference
15,680 pg/m3 (8ppm)
98,000 |jg/m3 (50 ppm)
UKI 5-25 min Chromatid deletions.
MAST 30 min lex mutants deficient in
repair of x-ray- induced
Humans KB
cells
Escherichia
col i
Fetner, 1962
Hamelin and
Chung, 1974
DNA strand breaks were more
sensitive to lethal effects
of 03 than were the wild-type
repair-proficient parental strains
98,000 pg/m3 (50 ppm) MASTQ
30 min
o
i
CT>
DNA Polymerase I mutant strains
(KMBL 1787, 1789, 1791) were more
sensitive to the cytotoxic effects
of 03, and DNA 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 ug/m3 (50 ppm) ! MAST
30 min
Mucoid mutapt 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 03 and
degraded DNA to a greater extent
than the Ion + (MQ259) strain.
E. coli
Hamelin et al. ,
1977b
98,000 pg/m3 (50 ppm) MAST0
up to 3 hrs 15 different DNA repair-deficient
strains were tested for sensitivity
to the cytotoxic effects of 03; DNA
Polymerase 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 10-22. CHROMOSOMAL EFFECTS FROM IN VITRO EXPOSURE TO HIGH OZONE CONCENTRATIONS (continued)
03 Measurement
concentration method
0.1 ug/ml [51 ppm] UV
0.5 ug/ml [255 ppm] UV
0.18 ug/ml [92 ppm] UKI
1.0 ug/ml [510 ppm]
0.5-6 ug/ml [255- NBKI
3061 ppm]
Si- 10 ug/ml [510- NBKI
^ 5100 ppm]
CTi
(V)
5% [50,000 ppm] NBKI
3.5-4.5 ug/ml [1786- UKI
2296 ppm]
2% [20,000 ppm] e
8% [80,000 ppm] GPT
Exposure
duration and protocol Observed effect(s) Species Reference
60 min (70 ml/min) Preferential degradation of yeast Yeast Shiniki et al.,
RNA at the N-glycosyl linkage; 1981
sugar-phosphate linkage was
03 stable. ;
30 min (330 ml/min) 5-ribonucleotide guanosine
monophosphate was degraded
most rapidly.
ND Release of nucleic acids; E. coli Scott and Lesher,
cell lethality. • 1963
0-5 min 03 reacts with pyrimidine E. coli Prat et al . , 1968
bases from nucleic acids
(thymidine > cytosine >
uracil ).
30 min Cell death and nonspecffic Chick , Sachsenmaier
chromosomal aberrations: embryo et al., 1965
shrunken and fragmented nuclei, fibroblasts
clumped metaphase chromosomes
and chromosome bridges.
0-40 min Rapid loss of glycolytic and Mouse !
respiratory capacity; loss of ascites
tumorigenicity after 20 min. cells
exposure.
ND Mitotic delay. Chortophaga Fetner, 1963
viridifaciata
3 min Abnormal nuclei; fragmentation. Am. oyster MacLean et al.,
1973
5, 15, 60 s Rapid degradation of nucleic ND Christensen and
acid bases, nucleosides or Giese, 1954
nuc"?eotides in 0.05M phosphate
buffer, pH 7.2.
aNot 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 flow rate given in (ml/min), when available. ND = not described.
Concentrations of 03 were not measured in the cell suspensions.
e03 analyzer (Fisher and Porter, Warminster, PA).
-------
3920 2.0
TABLE 10-23. CHROMOSOMAL EFFECTS FROM OZONE CONCENTRATIONS AT OR BELOW 1960 pg/m3 (1 ppm)
Concentration Measurement3 >b
Mg/mJ
294
412
1940
451
2548-
14,700
3234-
27,832
ppm method
0.15 NBKI
0.21
0.99
0.23 NBKI
1.3- NBKI
7.5
1.65- NBKI
14.2
Exposure0
duration and protocol
5 hr
5 hr
2 hr
5 hr
(in vitro)
ND
(in vitro)
NO
(in vitro)
Observed effect(s)
No effect induced by 03 treatment
on the frequency of chromosome or
chromatid aberrations in Chinese
hamster or mouse peripheral
blood lymphocytes stimulated with
PHA; no effect on spermatocytes
in mice 8 wk following exposure.
Peripheral blood lymphocytes exposed
to 03 in culture 12 hr after stim-
ulation with PKA showed na increase
in chromosome or chromatid aberrations.
Peripheral blood lymphocytes exposed
to 03 in culture, 36 hr after PHA
Species Reference
Mouse Gooch et al . ,
1976
Hamster
'
Human
lymphocytes
Human
lymphocytes
NBKI
5-90 min
(in vitro)
stimulation, showed no change in the
frequency of chromosome or chromatid
aberrations at any concentration
except 7.23 ppm of 03.
No apparent increase in the fre-
quency of chromosome or chromatid
aberrations 12 or 36 hr after
PHA stimulation.
Human
lymphocytes
392 0.2
MAST
UKI
5 hr
(in vivo)
Combined exposure to 03 and radi-
ation (227-233 rad) produced an
additive effect on the number
of chromosome breaks measured in
peripheral blood lymphocytes.
Hamster Zelac et al.
1971b
470- 0.24-
588 0.3
MAST
UKI
5 hr
(in vivo)
A significant increase in chro-
mosome aberrations (deletions,
ring dicentrics) in the peri-
pheral blood lymphocytes; in-
creased break frequency was
still apparent at 6 and 16 days
following exposure.
Hamster Zelac et al .
1971a
-------
TABLE 10-23. CHROMOSOMAL EFFECTS FROM OZONE CONCENTRATIONS AT OR BELOW 1960 pg/m3 (1 ppra) (continued)
o
I—«
en
03
Concentration,
ug/m3 ppm
490- 0.25-
1960 1.0
588- 0.3-
1568 0.8
843 0.43
3920 2.0
1960- 1.0-
9800 5.0
Measurement * Fxpncnrp
_. p i —
method duration and protocol
UV 1 hr
(in vitro)
UV 8 days,
continuous
(in vitro)
UV, 5 hr
NBKI (j_n vivo)
6 hr
(in vivo)
CHEM 24 hr
NBKI (in vivo)
Observed effect(s)d Species Reference
Dose-related increase in SCE fre- Human Guerrero et
quency in WI-38 diploid fibroblasts fibroblasts al., 1979
exposed in culture.
Growth of cells from lung, breast, Human ! Sweet et al.,
and uterine tumors were inhibited tumor 1980
to a greater degree than IMR-90, cells
a nontumor diploid fibroblast.
Increase in chromatid-type Hamster Tice et al.,
aberrations in peripheral blood 1073
lymphocytes of 03-exposed hamsters;
Increase in deletions at 7 days
and increase in achromatic lesions
at 14 days after exposure; chromosome- !
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: MAST = Kl-coulometric (Mast meter); NBKI = neutral buffered potassium iodide; UV = UV photometry
Calibration method: UKI = unbuffered potassium iodide; NBKI = neutral buffered potassium iodide.
ND = not described.
Abbreviations used: PHA = phytohernagglutinin; SCE = sister chromatid exchange.
-------
t-RNA or tobacco mosaic virus RNA with 0., (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 0_ 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 extensive
ozonolysis of yeast t-RNA (0.5 ug/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 0_-labile,
the sugar-phosphate backbone was intact and extremely stable against 0^. Prat
et al. (1968) investigated the reactivity of the pyrimidines in £_._ coli DNA
with 0~ (0.5 to 6 mg/L, 0 to 5 min) and radiation. Ozone preferentially
reacted with thymidine, then with cytosirie and uracil, in decreasing order of
reactivity. The results are slightly different from those reported by Shiniki
et al. (1981) 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_. 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 significa'ntly more sensitive to the cytotoxic effects of
OT 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 coworkers investigated the survival of bacterial strains with
known defects in DNA repair. Closely related strains of E^ coli K-12 with
mutations in DNA polymerass I were shown to be more sensitive to the cytotoxic
effects of 03 than the DNA polymerase proficient (pol +) strain (Hamelin et
al. , 1977a). Polymerase I-deficient strains also exhibited an extensive
degradation of DNA in response to 0., 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
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
0190ZR/A 10-165 5/1/84
-------
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 thsy were exposed to 0,.
Although DNA polymerase I was shown to be involved in the repair of
O^-induced DNA damage (Hamelin et al. , 1977a), £_._ coli cell strains with
mutations in DNA polymerase II or III were not found to be more sensitive to
CL 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 E^
coli with defects in DNA synthesis (DNA A, B, C, D, and G) showed no enhanced
sensitivity to 0~. Therefore, the DNA gene products are probably not involved
in the repair of 0~ damage. Recombinational repair mutants, rec A and rec B,
only showed a slightly increased sensitivity to CL than the wild-type, suggest-
ing that the rec gene products are only partially involved in the repair of
(k-induced DNA lesions (Hanelin and Chung, 1978).
Other effects have been observed than those described above on bacteria.
In the commercial American oyster exposed to CL-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 CL (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 i_n vi tro effects of extremely high 0., concentrations on
biolog4cal systems or biologically important cellular components. Although
these investigations may be important for the elucidation of the types of
damage or responses that might be expected to occur at lower 0_ concentrations,
the most relevant data or. the genotoxicity of Oq should be obtained from
3
investigations where the 0., concentration did not exceed 1960 ng/m (1 ppm).
Research conducted at or below 1 ppm of 0,. will be presented below (See Table
10-24).
Several investigators have examined the i_n vivo cytogenetic effects of 0,,
in rodents and human subjects. Until the reports of Zelac et al. (1971a,b),
0190ZR/A 10-166 5/1/84
-------
PRELIMINARY DRAFT
TABLE 10-24. MUTATIONAL EFFECTS OF OZONE
°3 a
concentration Measurement Exposure
ug/m-* ppm method duration and protocol Observed effect(s)
196 0.1 MAST 60 min Various mutated, growth factor auto-
(2.1 ml/min) trophic states, were recovered; mutant
strains differed from parental strains
in sensitivity to UV light and excessive
production of capsular polysaccharide.
58,800 30 NBKI 3 hr Induction of a dominant lethal muta-
tion during stages of spermatogenesis;
sperm were found to be twice as sen-
sitive as earlier stages.
-------
the toxic effects of CL 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 0,, 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 (jg/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 0,-,-exposed animals
when compared with nonexposed control animals. Zelac et al. (1971b) reported
that 0- was additive with radiation in the production of chromosome breaks.
Both 03 and radiation produced chromosome breaks independently of each other.
Simultaneous exposure to 3.2 ppm of CL 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 0~ 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 pg/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 (jg/m (5 ppm) of 0- 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 0--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 0^
treatment, and the induction of alkaline-labile lesions in DNA by 0,, which
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 0, effect on DNA at 9800 pg/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
0190ZR/A 10-168 5/1/84
-------
inhalation, Tice et al. (1978) tried to repeat the experiments of Zelac as
3
closely as possible. Chinese hamsters were exposed to 843 jjg/m (0.43 ppm) of
0~ by inhalation for 5 hr. The authors investigated chromatid and chromosome
aberrations in peripheral blood lymphocytes and bone marrow of control and
(L-exposed animals immediately after exposure and at 7 and 14 days after 0.,
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 0- for 6 hr were examined
in peripheral blood cells collected from the animals immediately after 0~
exposure and at 7 and 14 days after 0- exposure.
The authors reported no significant increase in the SCE frequency of the
0_-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 0,-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 less 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 |jg/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
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 0~.
Zelac et al. (1971a) administered 470 to 590 (jg/m3 (0.24 to 0.3
ppm) of 03 to Chinese hamsters for 5 hr, whereas Tice et al.
(1976
of 0,
(1978) exposed animals to an atmosphere of 840 pg/rn (0.43 ppm)
3'
0190ZR/A 10-169 5/1/84
-------
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 i_n 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 0,
O
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 v/ith the results with the use of polkweed mitogen.
There is evidence thet the B-lymphocyte may be more sensitive to 0~ than
the T-lymphocyte. Savino et al. (1978) measured the effects of CL on human
cellular and humoral immunity by measuring rosette formation with human lympho-
cytes (See Chapter 11). Rosette formation measures the reaction of antigenic
red cells with surface membrane sites on lymphocytes. Different antigenic
RBCs are used to distinguish T-lymphocytes from B-lymphocytes. Rosette forma-
tion with B-lymphocytes was significantly depressed in eight human subjects
exposed to 784 ug/m (0.4 ppm) of 0- by inhalation for 4 hr. A similar inhibi-
tion of rosette formation was not observed with T-lymphocytes from the same
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 0.,
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 &1. (1978). There are sufficient differences in the
experimental protocols of the two reports so that the results need not be con-
sidered directly contradictory.
0190ZR/A 10-170 5/1/84
-------
An assumption that is made in all of the reports in which lymphocytes are
stimulated with mitogens is that the lymphocytes from the O^-exposed animals
and the control animals are 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 11.7). 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 GQ or G, cells into the cycling state, cells exposed to sufficient concen-
trations of 0^ would not be stimulated to divide, and hence no 0.,-induced cy-
togenic effects would be observed in activated cells. In their report, Tice
et al. (1978) stated that the lymphocytes of the 03~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 0,-damaged lymphocytes were selected
against in the cytogenetic investigations.
In other investigations with rodents, Gooch et al. (1976) analyzed bone
3
marrow samples from Chines? 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 C-H mice were exposed to 294 or 412 ug/m (0.15
o
or 0.21 ppm) of 03 for 5 hr, or to 1940 ug/m (0.99 ppm) of 03 for 2 hr.
Blood samples were drawn from these animals at various times for up to 2 weeks
following 03 exposure. The mice were killed 8 weeks following 0, exposure,
and spermatocyte preparations were made and analyzed for reciprocal transloca-
tions. Data from the Chinese hamster bone marrow samples and the mouse leuko-
cytes indicated that there was no effect induced by 03 treatment on the fre-
quency of chromatid or chromosome aberrations, nor were there any recognizable
reciprocal translocations in the primary spermatocytes.
Several investigators have examined the effects of 0,, on htiman cells i_n
vitro. Fetner (1962) observed the induction of chromatid deletions in human
KB cells exposed to 15,68C ug/m (8 ppm) of 03 for 5 to 25 min. Sweet et al.
(1980) reported that the growth of human cells from breast, lung, and uterine
3
tumors was inhibited by exposure to 588 to 1568 ug/m (0.3 to 0.8 ppm) of 03
for 8 days in culture.
0190ZR/A 10-171 5/1/84
-------
Guerrero et al. (1979) performed SCE analysis on diploid human fetal lung
cells (WI-38) exposed to 0, 490, 980, 1470, or 1960 ug/m3 (0, 0.25, 0.5, 0.75,
or 1.0 ppm) 0., for 1 hour in vitro. A dose-related increase in the SCE fre-
^
quency was observed in the WI-38 human fibroblasts exposed to 0^. 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 0_ by inhalation (Chapter 11). Unless the lymphocyte is intrinsi-
cally less sensitive to the induction of SCE by 0, than the WI-38 human fetal
lung fibroblast, the results indicate that exposure of human subjects to 980
ug/m (0.5 ppm) of 0_ for 2 hr did npt result in a sufficiently high con-
centration of 03 or 03 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 ug/m (0.25 ppm) for 1 hour.
Gooch et al. (1976) also investigated the effects of 0^ exposure on human
cells HI vitro. In these experiments, lymphocytes were stimulated with PHA
for 12 or 36 hr before the 0~ exposure to obviate the potential problems of
OT inactivation of the PHA receptor. Human leukocyte cultures were exposed to
3
3920 M9/m (2 ppm) of 07 for various lengths of time to accumulate total 0.,
•3 o ^
exposure doses of 3234 to 27,832 ug/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
aberrations at a total exposure of 14,170 ug/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
T j
on cells and cellular components including the genetic material. Cytogenetic
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.
10.4.5.2 Mutational Effects of Ozone. The mutagenic effects of 0., have been
investigated in surprisingly few instances (Table 10-24). No publication to
date has investigated the mutagenic effects of 0, in mammalian cells.
0190ZR/A 10-172 5/1/84
-------
Sparrow and Schairn (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 CL concentration was reported in this publics-
O
tion.
E^ coli, strain MQ 259, were mutated to various growth factor auxotrophic
states, including requirenents for most common amino acids, vitamins, and
3
purines and pyrimidines (Hamelin and Chung, 1975a). Ozonated air 196 ug/m ,
0.1 ppm) was passed through the bacterial suspensions at a rate of 2.1 L/min
for 30 min. Many of the 0 - 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
3
mutations in Drosophila virilis exposed to 58,800 ug/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 Saccharomyces cerevisae. Several different strains
*j
were utilized to investigate forward, reverse, and recombinational mutations.
ozone (98,000 ng/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 (jQ/m » 50 ppm; 30 to 90 min) was compared with other known mutagens.
13 2
The positive controls were UV light (1.54 J/m per second, 1-tnin exposure),
N-methyl-N1-nitrosoguanidine (MNNG) (50 ug/mL, 15 min), or x-rays (2 kR/ min,
40 min). By comparing the induced mutation frequency at similar cellular
survival levels for 03, MNNG, UV light, and x-rays, it was shown that 03 was a
very weak mutagen. Induced mutation frequencies were generally 20 to 200
times lower for 0 than for the other three mutagens.
0190ZR/A 10-173 5/1/84
-------
In summary, the mutagenic properties of 0., have been demonstrated in
procaryotic and eucaryotic cells. Only one study, however, (Hamelin and
Chung, 1975a, with E^ coli) investigated the mutagenic effect of CL at concen-
trations of less than 1 pom. The results clearly indicate that if cells in
culture are exposed to sufficiently high concentrations of (L 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 CL toward these species can be determined.
10.4.6 Other Extrapulmonary Effects
10.4.6.1 Liver. A series of studies reviewed by Graham et al. (1983a) have
shown that 0., increases drug-induced sleeping time in animals (Table 10-25).
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 sleeping time, because liver xenobiotic metabolism transforms the drug
into an inactive form. Therefore, this response is interpreted as an extrapul-
monary effect.
Gardner et al. (1974) were the first to observe that 0., increases pentobar-
bital-induced sleeping time. Female CD-I mice were exposed for 3 hr/day for
2
up to 7 days to 1960 ug/m (1.0 ppm) of CU and the increase was found on
days 2 and 3 of exposure, with the greatest response occurring on day 2. Com-
plete tolerance did not occur; when mice were pre-exposed (1960 ug/m , 1.0 ppm;
2
3 hr/day for 7 days) and then challenged with a 3-hr exposure to 9800 [jg/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 ex, al., 1981).
3
Ozone (1960 ug/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 ug/m3 (1.0 ppm) of 03 (Graham et al., 1981). The females of all
0190ZR/A 10-174 5/1/84
-------
TABLE 10-25. EFFECTS OF OZONE ON THE LIVER
Ozone
concentration
ug/m3
1960
ppm
Measurement3'b
method
Exposure
duration and protocol
Observed effects(s)
Species Reference
196- 0.1- CHEM 3 hr/day,
9800 5 GPT 1-17/days
588 0.3 UV 3 hr
1470 0.75 NBKI 3 hr
i-- 5880 3
-------
TABLE 10-25. EFFECTS OF OZONE ON THE LIVER (continued)
Ozone
concentration Measurement
pgTm3" ppm method
a,b
Exposure
duration and protocol
Observed effects(s)
Species
Reference
1960
1960
1 CHEM
GPT
1 CHEM
GPT
5 hr/day,
1,2,3 or
4 days
5 hr
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 hexobarital-and thiopen-
tal-induced sleeping time and zoxazo-
Mouse
(female)
Mouse
(female)
Graham et al . ,
1981
Graham et al . ,
1982a
CTl
1 amine-induced paralysis time. Pre-
treatment with mixed function oxidase
ip.d'jcers (phenobarbetal, pregr.eolor.e
-16a-carbonitrile, and B-naphthofla-
vone, but not pentobarbital) decreased
phenobarbital-induced sleeping time in
CD-I mice, and 03 increased the sleeping
time in all groups. Pretreatment 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
9800
CHEM
GPT
5 hr
3 hr
No effect on hepatic cytochrome
P-450 concentration, aminopyrine
M-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.
Mouse
(female)
Graham et al.
1982b
-------
PRELIMINARY DRAFT
TABLE 10-25. EFFECTS OF OZONE ON THE LIVER (continued)
Ozone
concentration
ug/m3 ppm
Measurement
method
a,b
Exposure
duration and protocol
Observed effects(s)
Species Reference
1960
9800
CHEM
GPT
5 hr
At 1 ppm: 71% increase in plasma
half-life of pentobarbital, decrease
(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
brain
Mouse Graham 1979;
(female) Graham et al.
1983a,b
1960
NO
90 inin
No effect on hepatic cytochrome
P-450 concentration.
Rabbit Goldstein
! et al., 1975
3920
UV
8 hr/day
Supplementing or depriving rats of
vitamin E or selenium altered the
03 effect. 03 caused changes in
several iji vitro enzyme activities
in the liver and kidney (see text).
Rat
Reddy et al.
1983
Measurement method: CHEM = gas-phase chemiluminescence; NBKI = neutral buffered potassium iodide; UV = UV photometry; NO = not described
Calibration method: GPT = gas phase titration
-------
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.
3
Female CD-I mice were exposed to 196 to 9800 ug/m (0.1 to 5.0 ppm) of 0, for
3
3 hr/day for a varying number of days (Graham et al. , 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 ug/m (0.5 ppm), the greatest
increase in pentobarbital-induced sleeping time was observed on day 3, but at
3
490 ug/m (0.25 ppm), 6 days of exposure were required to cause an increase.
At the lowest concentration evaluated (196 ug/m3, 0.1 ppm), the increase was
only observed at days 15 and 16 of exposure. Thus, as the concentration of 0~
-------
and metabolism. Although there are different mechanisms involved in hexobarbi-
tal, 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 CL to differences in the actions pf 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 elucida-
ted.
The effect of 0., 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 0~ as high as 9800 ug/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-
lase). However, this exposure regimen and another (1960 ug/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
3-hr exposure to 9800 ug/rn (5.0 ppm) of 0- did not affect brain concentra-
tions of pentobarbital at time of awakening, even though sleeping time was
increased (Graham, 1979). Therefore, it appears that 0~ 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, 1979). Following this
exposure, first-order clearance kinetics of pentobarbital were observed in
both the air and 03 groups, and 03 increased t^e plasma half-life by 106 per-
cent (Graham, 1979). Mice exposed to 1960 ug/m3 (1.0 ppm) of 0., for 5 hr had
»3
a 71 percent increase in the plasma half-life of pentobarbital (Graham et al. ,
1983b). 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.
0190ZR/A 10-179 5/1/84
-------
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., 1983a) 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 10.2). 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 10.3.3).
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 0_ on liver xenobiotic metabolism by perform-
ing HI vitro enzyme assays. Although lower Og 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
cytochrome 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 0~ 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 activity in 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
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
0190ZR/A 10-180 5/1/84
-------
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 0- 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 we"e 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 0, and
examined at 5 postexposure periods up to 24 hr. Rats had significantly increased
ascorbic acid levels in both the Q~ and air groups, with the greater change
taking place in the. air group. Thus, there were no changes due to 0.,. Likewise,
there was no 0.. effect on reduced ascorbic acid content in the serum.
10.4.6.2 The Endocrine System. A summary of the effects of 0^ on the endo-
crine system, gastrointestinal tract, and urine is given in Table 10-26.
Fairchild and co-workers were the first to observe the involvement of the
3
endocrine system in 0~ toxicology. Mice exposed 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 Q- 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 0.,. Thyroid-blocking agents and thyroidectomy increased the
survival of mice and rats acutely exposed to 0~, and injections of the thyroid
hormones, thyroxine (T.), or triodothyronine (T,) decreased their survival.
This response in animals with altered thyroid function was not specific to a
0190ZR/A 10-181 5/1/84
-------
TABLE 10-26. EFFECTS OF OZONE ON THE ENDOCRINE SYSTEM, GASTROINTESTINAL TRACT, AND URINE
Ozone
concentration Measurement
ug/m3
5.4
21
110
490
2940
980
1960
ppm method
0.003 c
0.01
0.056
0.25 d
1.5
0.5 I
1
Exposure
duration and
protocol
93 days,
continuous
2 hr
30 min
5 hr/day,
4 days
Observed effect(s) Species Reference
From 6th wk to end of exposure, 0.056 ppm Rat Eglite, 1968
increased the urine concentration
of 17-ketosteroids. After 93 days of
exposure to 0.056 ppm, the ascorbic and
level of the adrenal glands was decreased.
No data were presented for these effects.
1.5 ppm of 03 (30 min) inhibited gastric Rat Roth and Tansy,
mortality; recovery was rapid. The 1972.
lower level caused no effects.
No effects on thyroid release of 131I, Rat Fairchild et al.,
96-384 hr post lMI injection. 1964.
o
I
00
ro
1470
0.75
ND
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
ND 48 hr Early postexposure parathyroid glands
postexposure enlarged and congested with focal
1-20 days vasculitis. After 7 days postexposure,
parenchyma! atrophy, leukocyte infiltra-
tion and capillary proliferation. Authors
suggest lesions may be due to autoimmune
reactions.
Rabbit
Atwal et al., 1975
1470
0.75
ND
48 hr Microvascular changes in the parathyroid
postexposure glands, including hemorrhage, endothelial
12-18 days proliferation, platelet aggregation, and
lymphocyte infiltration.
Dog
Atwal and Pemsingh, 1981
1568- 0.8-
2940 1.5
(range) (range)
NBKI
6 hr/day, Lower titratable acidity of urine,
4 days/wk with no changes in levels of creatine,
about 19 wk uric acid/creatinine, amino acid
nitrogen/creatinine, or excretion of
12 ami no acids.
Rat
1962
Hathaway and Terrill,
-------
TABLE 10-26. EFFECTS OF OZONE ON THE ENDOCRINE SYSTEM, GASTROINTESTINAL TRACT, AND URINE (continued)
o
i
00
GO
Ozone
concentration
ug/m3
1960
3920
7840
ppm
1
2
4
Exposure
Measurement duration and .
method protocol Observed effect(s) Species Reference
I 5 hr Decreased release of 131I from thyroid, Rat Fairchild et al.,
48-384 hr post 131Injection to all 03 1964.
levels above 1 ppm.
1960
ND
24 hr
Decreased serum level of thyroid-
stimulating hormone from anterior
pituitary, thyroid hormones (T3, 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 time up to a
potential maximum of 500 hr also caused
a decrease in TSH levels.
Rat
demons and
Garcia, 1980a,b.
79,800 > 5.0 I
> 3 hr
< 8 hr
Anti-thyroid agents, thyroidectomy,
hypophysectomy, and adrenal ectomy
protected against 03- induced mor-
tality. Injection of thyroid hormones
decreased survival after 03 exposure.
Mouse,
rat
Fairchild et al. ,
1959; Fairchild and
Graham, 1963;
Fairchild, 1963.
9800
CHEM
3 hr
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, 1956); ND = not described.
Abbreviations used: LM = light microscopy; TEM = transmission electron microscopy; RER = rough endoplasmic reticulunr
T3 = trnodothyronine; T4 = thyroxine.
Spectrophotometric technique (dihydroacridine).
Flow rates from ozonator.
-------
concomitant altered metabolic rate, because another drug (dinitrophenol),
which increases metabolism, had no effect on the (k response.
Hypophysectomy and adrenalectomy also protected against 0,-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 adrenocorti costeroi ds reduces 0.,
toxicity. Rats that have been adrenalectomized and treated with adrenergic
blocking agents are more resistant to 0~ than rats that have only been adrena-
lectomized, indicating that decreases in catecholamines reduce 0~ toxicity.
Potential tolerance to the effect of CL 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 ug/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 0,. 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 07 (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 |jg/m (4.0 ppm) on the third day. These animals exhibited a greater
3
effect than rats that received only the 7840-|jg/m (4.0 ppm) challenge. This
difference persisted for 43, 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 0~ challenge.
demons and Garcia (2.980a,b) extended this area of research by investi-
gating the effects of 0., on the hypothalamo-pitui tary-thyroi d axis of rats.
Generally, these three endocrine organs regulate the function of each other
through complex feedback mechanisms. Either stimulation or inhibition of the
hypothalamus regulates the release of thyrotropin-rel easing hormone (TRH).
The thyroid hormones (T~ and T.) can stimulate TRH. Thyrotropin-releasing
0190ZR/A 10-184 5/1/84
-------
hormone and circulating thyroid hormones (T., 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 ng/m (1.0
ppm) of 0~ caused decreases in the serum concentrations of TSH, T,, T., free
T,, and protein-bound iodine. There was no change in the uptake of T~, 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
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 exoosure 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 I960, 3920, 5880 or 7840 ug/m3 (1.0, 2.0, 3.0, or 4.0 ppm) of 03 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 of 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.
The thyroid gland itself was altered by the 0.. exposure (apparently 1960
3
Ug/m , 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
0190ZR/A 10-185 5/1/84
-------
tissue. There was no change in T. release per gland. demons and Garcia
(I980a,b) interpreted these findings as an (L-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.
The susceptibility of the parathyroid gland to CL 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 0_ exposure. Changes included hyperplasia of chief cells;
hypertrophy and proliferation of the rough endoplasmic reticulum, free ribo-
somes, mitochondria, Golgl 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.
Atwal et al. (1975) also investigated possible autoimmune involvement in
3
parathyroiditis of rabbits following a 48-hr exposure to 1470 M9/m (0.75 ppm)
of 03- 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 e'ectron 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. The microvascular changes were further
studied by Atwal and Pernsingh (1981) in dogs exposed to 1470 jjg/m3 (0.75 ppm)
of 0, for 48 hr. They reported focal hemorrhages, vascular endothelial pro-
«J
liferation, intravascular platelet aggregation, and lymphocytic infiltration.
A potential autoimmunity after 0_ exposure was also observed by Scheel et al.
(1959), who showed the presence of circulating antibodies against lung tissue.
0190ZR/A 10-186 5/1/84
-------
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
ug/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 live1" or kidney.
Adrenal cortex function after a 93-day (continuous) exposure to 0., was
3
investigated in rats (Eglite, 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
O
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.
10.4.6.3 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 relatively 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 0,,-exposed animals. Titratable acidity was also lower on day 98.
«J
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 differ-
ences were observed. The authors interpreted the results as a reflection of
respiratory alkalosis, assuming no kidney toxicity.
Gastric secreto-motor activities of the rat were investigated by Roth and
Tansy (1972). A 2-hr exposure to 490 pg/m (0.25 ppm) caused no effects.
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.
0190ZR/A 10-187 5/1/84
-------
10.5 EFFECTS OF OTHER PHOTOCHEMICAL OXIDANTS
10.5.1 Peroxyacetyl Nitrate
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 (LC5[)) (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. , 1977). The concentration of
3 3
PAN used in these studies ranged from 22.3 mg/m (4.5 ppm) to 750 mg/m (150
ppm). They are considerably higher than the 0.174 mg/m (0.037 ppm) daily
maximurr. concentration of PAN reported for ambient air samples in areas having
relatively high oxidant levels (Chapter 6) and are of questionable relevance
to the assessment of effects on human health.
Campbell et al. (1967) estimated that the LC,-n for mice ranged from 500
Q OU
to 750 mg/m (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 LC™ (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. 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 depres-
sed both the 6-hr and 24-hr activity when compared to similar pre-exposure
periods. Depression was irore 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)
O
(Section 10.4.1).
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 pro!iferative
peri bronchiolitis.
Thomas et al. (1977) 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
0190ZR/A 10-188 5/1/84
-------
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 et al. (1981a) published additional data that extend observations
of reduced resistance of mice to streptococcal pneumonia over a range of
3
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
39 percent. Mice exposed to 0~ at 0.98 mg/m (0.5 ppm) and challenged with
the S. pyogenes aerosol resulted in a mean excess mortality (38 percent) that
was almost equivalent to the excess mortality for the group exposed to 28.4
mg/m of PAN. The results agreed with earlier reports that PAN is less toxic
than 0- to mice exposed under ambient conditions. Exposure to 7.4 mg/m (1.5
ppm) of PAN 3 hr/day, 5 Cays/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 Mycobacterium tubercu-
losis (RIRv) was influenced by subsequent exposure to PAN. The exposure to
this oxidant (25 mg/m for 6 days) did not alter the pattern of bacterial
growth in the lungs of mice.
10.5.2 Hydrogen Peroxide
3
Although concentrations of hydrogen peroxide (HLO,,) as high as 0.14 mg/m
(0.10 ppm) have been reported to occur in urban air samples (Chapter 6), very
little is known about the effects of H»0? from inhalation exposure. Most of
the early work on H?0? toxicity involved exposure to very high concentrations.
Oberst et al. (1954) investigated the inhalation toxicity of 90 percent H 09
3
vapor in rats, dogs, and rabbits at concentrations ranging from 10 mg/m (7
ppm) daily for six months to an 8-hr exposure to 338 mg/m (243 ppm). After
autopsy, all animals showed abnormalities of the lung. In a recent experiment,
Last et al. (1982) exposed rats for 7 days to > 95-percent H?0« gas with a
3
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
0190ZR/A 10-189 5/1/84
-------
lobe weights, and protein or DNA content of lung homogenates. The authors
suggested that because HLO,, 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 the H?CL explore possible mechanisms for the
effects of H000. These include direct cellular effects associated with in
c- £-
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 Iff2 to IQ~1M of H202
increased the surface tension of saline extracts of dog lung homogenates. The
3
authors estimated that a clog breathing 1.4 mg/m (1 ppm) of H909 for 30 hr
-2
would build up a pollutant concentration of 10 M in the surfactant, assuming
that all the H^O^ was retained by the lungs. However, as stated above, this
estimate of tissue dose is not realistic, because most of the H?02 would be
absorbed in the upper airway.
Another mechanism by which H«0« may affect ventilation is by changing the
-4
tone of airway smooth muscle. Stewart et al. (1981) reported that 10 M of
H?0? caused significant constriction of strips of subpleural canine lung
parenchyma and of bovine trachea!is 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 idomethacin
markedly reduced the response to H^O,,. This suggested that the increase in
airway smooth muscle tone produced by H?02 involves prostaglandin-like sub-
stances.
The potential genotoxic effects from jm vitro H?0? exposure have been
evaluated in isolated cell systems. Bradley et al. (1979) reported that H202
produced both toxicity and single-strand DNA breaks but was not mutagenic at
concentrations up to 530 |jM. They observed a significant increase in the
frequency of reciprocal sister chromatid exchanges in V-79 Chinese hamster
cells at a concentration of 353 pM of H^CL. 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
H?0? for 3 hr at 37°C. Increases in the frequency of sister chromatid exchanges
in Chinese hamster cells fiave also been found by MacRae and Stich (1979),
Speit and Vogel (1982), and Speit et al. (1982). Wilmer and Natarajan (1981)
0190ZR/A 10-190 5/1/84
-------
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
H?CL. In comparison, cells were killed with a concentration of 10 M of H^O,,.
Similarly, H?0? (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 al., 1978; Kawachi et al., 1980).
Johnson et al. (1981) reported that the intrapulmonary instillation of
glucose oxidase, a generator of H»0?, 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-
HpOp-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
l-LOp 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
halide-dependent pathway described for cell injury produced by H?0p and lac-
toperoxidase/myeloperoxidase (MPO) (Klebanoff and Clark, 1975). Any source of
HLOp 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 H^O,, 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 j_n vitro experiments, Suttorp and Simon (1982) demonstrated that
HpOp 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 jjn situ type 2 pneumo-
cytes from which they were derived.
0190ZR/A 10-191 5/1/84
-------
10.5.3 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
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 03 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 0,,
but most of them could have been produced by CL. In most cases, however, the
biological effects presented would be difficult to associate with any one
pol1utant.
Research on ambient air and UV-irradiated or nonirradiated exhaust mix-
tures is summarized in Table 10-27. 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 infarr: 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
0190ZR/A 10-192 5/1/84
-------
TABLE 10-27. EFFECTS OF COMPLEX POLLUTANT MIXTURES
o
i
GO
Concentration,3
(ppro)
Pollutantb
Exposure
duration and
protocol
Observed effect(s)
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)
0.
c5
NOj,
NO
ox
COX
HC
NO;,
NO
PAN
ox
NO*
HC
ox
COX
HC
NO
X
0
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 ppm or at com-
bined concentrations of > 40 ppm of CO,
16 ppm of HC, and 1.2 ppm of N0x- Indi-
dividual sensitivity demonstrated. Tem-
perature was an important variable.
No consistent effects on conception rate,
litter rate, or newborn survival.
Mouse, Wayne and Chambers,
rat, 1968
hamster,
guinea pig,
rabbit
Mouse, Esnik ct al. , 1971
rat,
rabbit
Mouse Emik and Plata, 1969.
Guinea pig Swann and Balchum, 1966
Mouse Kotin and Thomas, 1957
-------
TABLE 10-27. EFFECTS OF COMPLEX POLLUTANT MIXTURES (continued)
O
i
Concentration,3 .
(ppm) Pollutant
Exposure
duration and
protocol Observed effect(s)
Species
Reference
B, Automobile exhaust
0.012-
3.0
0.04 -
0.06 -
0.06 -
0.04 -
0.15 -
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
1.8
36
100
0.5
0.6
B
60
0.4
30
1.0
2.0
0.49
0.03
Os (max)
HC (propylene)
N02
NO
SO,
03
N02
NO
HC (CH4)
ox
N0x
HC*
CO
03
CO
HC (CH4)
NOj.
NO
SOj,
H2S04
0.5-6 hr
(diesel )
1.5-23 mo
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- irradiation of propylene, SOj, , NO, and
NOj, produced Oa and a routagenic moiety
when collected particles were tested by
the plate-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.
Salmonel la
typhimurium
Mouse,
rat,
hamster,
guinea pig
Mouse
Dog
Dog
Claxton and Barnes, 1981
Hueter et al. , 1966
Lewis et al. , 19t>/
Nakajima et al. , 1972
Bloch et al. , 1972, 1973
Gillespie, 1980
Orthoefer et al. , 1976
Pulmonary function for groups receiving
oxidants [irradiated exhaust (I) + SO ]
18 months: no effects
36 months: no effects
Dog
Vaughan et al., 1969
Lewis et al., 1974
-------
TABLE 10-27. EFFECTS OF COMPLEX POLLUTANT MIXTURES (continued)
Concentration/
(ppm)
Pollutant
Exposure
duration and
protocol
Observed effect(s)c
Species
Reference
o
i
en
61 months:
RL increased (I, I+SOx).
washout increased (I);
P CO,
3 £.
increased (I+SO );
X
2 years recovery:
VD increased (I, I+SOx); DLcQ/TLC decreased
and V increased in all groups; lung com-
partment volumes increased (I+SO ).
Morphology (32-36 months recovery): air
space enlargement; nonci listed bronchi olar
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
ox
NOz
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
0.76
2.05
1.71
0.3
1.0
2.0
03
SO*
T-2 Butene
acetaldehyde
o,
NO.,
SO.
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
(AN;;) and increased diffusing capacity.
Voluntary activity (wheel running) Mouse Stinson and Loosli, 1979
decreased 75% after 1-3 days, returning
to 85% of pre-exposure levels by the end
of 14 days.
-------
O
I
en
TABLE 10-27. EFFECTS OF COMPLEX POLLUTANT MIXTURES (continued)
Exposure
Concentration,3 . duration and
(ppm) Pollutant protocol Observed effect(s) Species Reference
0.40 - 0.52
1.0 - 2.15
1.25
03 24 hr Decreased spontaneous wheel running Mouse Boche and Quilligan,
0^ (gas vapor) activity. I960
Ox (gas vapor) 19 weeks, Decreased conception rate, litter rate, Mouse Kotin and Thomas, 1957
continuous and newborn survival.
.Ranked by nonspecific oxidant concentration (03 or 0 ).
Abbreviations used: 03 = ozone; 0 = oxidant; CO = carbon monoxide; NO = nitrogen oxide, NO^ = nitrogen dioxide;
NO = nitrogen oxides; SO;, = sulfur* dioxide; HC = hydrocarbon; CH4 = methane; H2S04 = sulfuric acid; PAN = peroxyacetyl nitrate.
c x
See Glossary for the identification of pulmonary symbols.
-------
products that were biologically more active than nonirradiated mixtures. The
concentration of total oxidant as expressed by (L ranged from 588 to 1568
3
ug/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
(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 10-27 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
alone or in combination with SO .
/c
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
J\
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
X X
0190ZR/A 10-197 5/1/84
-------
groups. The diffusing capacity for carbon mom'xide (D. ) was similar in all
exposure groups, but when normalized for total lung capacity (TLC), the D. /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
/\
dogs exposed to I was too small (n = 5) to permit meaningful comparisons. In
general, pulmonary function changes were found to be similar in all groups
exposed to automobile exhaust alone or supplemented with SO . Exposure to
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 rconths 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+SO groups showed ramdom enlargement of alveolar airspaces centered in
A
respiratory bronchioles and alveolar ducts. Small hyperplastic lesions were
observed at the junction o~ the terminal bronchiole and the first-order respi-
ratory bronchiole. Foci cf ciliary loss associated with squamous metaplasia
were also observed in the intrapulmonary bronchi of the I+SO group. However,
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
)\
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 plate-incorporation test (Ames et al. , 1975).
The authors demonstrated that the irradiation of propylene, SO,,, NO, and N02
produced 0- and a mutagenic moiety. In baseline studies on diesel exhaust, in
which 0- 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.
0190ZR/A 10-198 5/1/84
-------
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 |jg/m
(0.3 ppm) of GO, 1 ppm of NCL, and 2 ppm of SO,,, 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 NO^ and SOp alone had only moderate effects, the authors
concluded that 0- had the major influence on depression of activity.
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, 0^, and $$2- Decreases in the nitrogen washout slope and
increases in the diffusing capacity indicated a significant compensatory
change in distrubution of ventilation in the lungs of exposed animals. Animals
compromised by the presence of emphysema were unable to respond to this pulmo-
nary insult in the same manner as animals without impaired lung function.
10.6 SUMMARY
10.6.1 Introduction
The biological effects of 0, have been studied extensively in animals and
a wide array of toxic effects have been ascribed to 03 inhalation. Although
much has been accomplished to improve the existing data base, refine the con-
centration-response relationships and interpret better the mechanisms of On
effects, many of the present data were not accumulated with the idea that
quantitative comparisons would be drawn. In many cases, only qualitative
comparisons can be made. To maximize the extent that animal toxicological
data can be used to estimate the human health risk of exposure to 0.,, the
qualitative as well as quantitative similarities between the toxicity of 0.,
to animals and man must be considered 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 scien-
tifically sound manner, the possible human responses to 0.
0190ZR/A 10-199 5/1/84
-------
10.6.2 Regional Dpsimetry in the Respiratory Tract
Experiments on the nasopharyngeal removal of 0 in animals demonstrate
that the fraction of 0~ uptake depends inversely on flow rate and 0~ concen-
tration, that uptake is greater for nose than for mouth breathing, and that
tracheal 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.
The model of Aharonson et al. (1974) is useful in analyzing nasopharyngeal
uptake data. Applied to 0~ data, the model indicates that the average mass
transfer coefficient in the nasopharyngeal region increases with increasing
air flow. Application of the LaBell et al. (1955) model to nasopharyngeal
uptake is inappropriate, because it does not include the effects of chemical
reactions.
The McJilton model (McJilton et al., 1972) and the Miller model (Miller
et al. , 1978) for lower respiratory tract 0., uptake are very similar in their
treatment of 0., in the airways (taking into account convection, diffusion,
wall losses, and ventilatcry patterns) and in their use of morphological data
to define the dimensions of the airways and liquid lining. However, the
Miller model is more realistic, because it accounts for chemical reactions of
0- with constituents of the mucous-serous layer. Tissue dose is predicted by
the Miller model to be relatively low in the trachea and to increase to a
maximum around the junction of the conducting airways and the gas exchange
region and then to decrease distally. Comparison of the Miller model results
to the morphological effects of 0- indicates qualitative agreement in the
pulmonary region. However, comparison in the tracheobronchial region indicates
further research is needed to define the relevant toxic, chemical, and physical
mechanisms.
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 0~ and its role in toxicity, but also to
support and to lend confidence to the modeling efforts. With experimental
confirmation, models will become practical tools and further our understanding
of the role of 0~ in the respiratory tract.
Ozone dosimetry modeling is in its initial stages; refinements and experi-
mental information are needed. The models (nasopharyngeal and lower respira-
tory tract) raise many questions, not only about the model formulations and
0190JE/A 10-200 5/1/84
-------
assumptions, but more importantly, they bring to the forefront questions about
what is needed to understand the absorption of 0_ in the respiratory tract.
10.6.3 Effects of Ozone on the Respiratory Tract
10.6.3.1 Morphological Effects. Morphological studies of the effects of 0
have indicated that the pattern and distribution of the tissue lesions are
similar in the species studied and depend on the locations of the sensitive
cells and the junction between the conducting airways and the gas exchange
regions of the lung. Damage to all parts of the respiratory tract can occur
in animals, depending on the 0_ concentration. At low concentrations of 0_
3
(< 1960 ug/m , 1 ppm) damage is principally confined to the junction between
the alveoli and the conducting airways. Dogs, monkeys, and man have respira-
tory and nonrespiratory bronchioles, but the common experimental animals
(mice, rats, rabbits, and guinea pigs) have only nonrespiratory bronchioles.
The location of the 0~ lesion thus differs according to the species examined
O
(Plopper et al., 1979; Castleman et al., 1977, 1980; Dungworth et al., 1975a;
Eustis et al. , 1981). In both types of lungs, the effects of 0_ have been
3
found at concentrations as low as 392 ug/m (0.2 ppm) and for exposure times
as short as 2 hr (Stephens et al., 1974a).
In the upper and lower conducting airways, ciliated cells appear to be
the most sensitive cell type; they are damaged by exposures as low as 392 to
1568 ug/m (0.2 to 0.8 ppm) 8 or 24 hr for 7 days in rats (Schwartz et al.,
1976), bonnet monkeys (Castleman et al., 1977), Rhesus monkeys (Dungworth et
al., 1975b; Mellick et al. , 1977), and mice (Ibrahim et al. , 1980). When
Moore and Schwartz (1981) and Boorman et al. (1980) extended these exposure
levels to 180 days, similar changes in these ciliated cells were observed.
Uniform damage is not always noted; most often reports are of shortened and
less dense cilia occurring in random patches. Electron microscopy revealed
severe cytoplasmic changes and condensed nuclei. Damage is present in both
trachae and bronchi (Eustis et al. , 1981; Mellick et al. , 1977; Castleman et
al., 1977). The damaged cilia are replaced by nonciliated clara cells that
become hyperplasic (Evans et al., 1976a; Lum et al. , 1978).
3
In mice, 0 levels of 980 and 1568 ug/m (0.5 and 0.8 ppm) elicited a
pronounced hyperplasia of these nonciliated cells that persisted for 10 days
after cessation of the exposure (Zitnik et al., 1978; Ibrahim et al., 1980).
In a number of species, 0» damage is clearly evident at the centriacinar
0190JE/A 10-201 5/1/84
-------
region, which includes the terminal bronchiole region, portions of the respira-
tory bronchioles, and possibly the alveolar ducts, depending on the species
(Stephens et al., 1973, 1974a,b; Schwartz et al., 1976; Mellick et al. , 1977).
The type 1 epithelial cells are significantly affected (Stephens et al. ,
1974a; Evans et al., 1976a; Castleman et al., 1980; Eustis et al. , 1981; Barry
et al., 1983; Boorman et al. , 1980). In monkeys, for example, at 1764 pg/m
(0.9 ppm) the type 1 cell death reaches a maximum at 12 hr after continuous
exposure (Castleman et al., 1980). With the destruction of these cells, there
is a hyperplasia of type 2 alveolar epithelial cells, which recovers the
denuded basal lamina (Stephens et al., 1974a,b; Sherwin et al., 1983; Eustis
et al. , 1981). The type 2 cells are relatively resistant to 0,. exposure.
O
Following and during continued exposures, these type 2 cells begin to
proliferate, which is a hallmark of Q» injury regardless of species. In rats,
DNA synthesis in type 2 cells was reported as early as 4 hr after exposure to
3
392 ng/m (0.2 ppm) (Stephens et al. , 1974a) and reached a maximum at 2 days
3
following continuous exposure to 686 or 980 ug/m (0.35 or 0.5 ppm) (Evans et
3
al., 1976a,b), or 1568 (jg/m (0.8 ppm) (Boorman et al. , 1980). Although type
2 cells proliferated in the 0_-exposed lung, complete maturation of type 2
cells to type 1 cells did not occur, even as late as 180 days of exposure
(Moore and Schwartz, 1981). In the normal progression, type 2 cells would
have matured into type 1 cells. Continued 0_ exposure inhibits both cilia-
•5
genesis and type 2 cell maturation.
Inflammation occurs in all species so far examined. The inflammatory
response is seen as early as 4 hr after exposure to 1568 M9/m (0.8 ppm) in
monkeys (Castleman et al. , 1980). In rats and monkeys, inflammation persists
with continued exposure, although at reduced levels. The inflammatory exudate
includes both fibrin and various leukocytes in the initial phase. In the
later phase the inflammatory cells are predominantly macrophages (Castleman et
al., 1980; Brummer et al. , 1977; Boorman et al. , 1980; Moore and Schwartz,
1981). Quantitative estimates of the degree of inflammation are, however,
lacking at present. The contribution of the inflammatory response to sub-
sequent long-term features of 0_ toxicity has not been studied in detail, even
though techniques are available. A number of studies have reported that
3
exposure to 980 (jg/m (0.5 ppm) and above thickens the interalveolar septa of
centriacinar alveoli (Schwartz et al. , 1976; Castleman et al., 1980; Boorman
et al., 1980). Thickness could be due to interstitial fibrosis.
0190JE/A 10-202 5/1/84
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Although some delay has been observed between the exposure to 0_ and the
O
maximum manifestation of morphological changes, the shortest time required for
0_ exposure to result in significant morphological changes is 2 hr of exposure
to 392 or 980 ng/m3 (0.2 or 0.5 ppm) in rats (Stephens et al., 1973, 1974a,b)
or 4.7 or 6.6 hr of exposure by endotracheal tube in cats to 510, 980, or
3
1960 ug/m (0.26, 0.5, or 1 ppm) (Boatman et al., 1974). However, most of
these morphological studies were specifically designed to look at long-term
(i.e., days, months, years) exposures rather than acute effects. These data
based on acute studies are in agreement with molecular theories that ozone
acts rapidly to oxidize some critical tissue component. Little difference in
severity was noted by Schwartz et al. (1976) between exposure to 8 hr/day or
24 hr/c!ay when the exposure was continued for 7 days. Quantitative studies of
different exposure regimens are lacking, but technically feasible. The sequence
in which the anatomic sites are affected appears to be a function of concentra-
tion rather than of exposure duration. Increasing concentrations not only
results in more severe lesions, but also extends the lesion to higher genera-
tions cf the respiratory structure.
Morphological studies of vitamin E-deficient or supplemented rats have
been undertaken to correlate the biochemical findings with morphological
alterations (Plopper et al. , 1979; Stephens et al., 1983; Chow et al., 1981;
Schwartz et al. , 1976; Sato et al. , 1976a,b, 1978, 1980). Despite the pre-
sence of vitamin E in the diets of these animals, the morphological lesion of
0,. exposure was unchanged.
When comparisons are made at the analogous anatomical site, the morphol-
ogical effects of 0,. on the lungs of a number of species of animals are re-
markably similar. Despite the inherent differences in anatomy between most
experimental animals and man, the junction between the conducting airways and
the gas exchange region is most affected by 0_.
10.6.3.2 Lung Function. Changes in lung function following 0~ exposure have
been studied in mice, rats, guinea pigs, rabbits, cats, dogs, sheep, and
3
monkeys. Short-term exposure for 2 hr to concentrations of 431 to 980 yq/m
(0.22 to 0.5 ppm) produces rapid, shallow breathing and increased pulmonary
resistance measured 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 fol-
lowing short-term 0- exposures lasting 3 hr to 14 days are usually greatest
0190JE/A 10-203 5/1/84
-------
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 a!., 1979).
Long-term exposure of 4 to 6 weeks to 0 concentrations of 392 to
3
490 ug/m (0-2 to 0.25 ppm) increases lung distensibility at high lung volumes
in young rats (Bartlett et al., 1974; Raub et al. , 1983a). Similar increases
3
in lung distensibility were found in older rats exposed to 784 to 1568 ug/m
(0.4 to 0.8 ppm) for up to 180 days (Moore and Schwartz, 1981; Costa et al.,
1983; Martin et al., 1983). Three to twelve months of exposure to 0» concen-
3
trations of 1176 to 1568 ug/m (0.6 to 0.8 ppm) increased pulmonary resistance
and caused impaired stability of the small, peripheral airways in both rats
and monkeys (Costa et al. , 1983; Wegner, 1982). 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.
Studies of airway reactivity following 0,. exposure in experimental animals
show that 0~ increases the reactivity of the lung to mechanical and chemical
stimulation partly by increased sensory neural activity travelling in the
vagus nerve and partly by a local action of 03 on the tissues. Aerosolized
ovalbumin can reach immunologic receptors in the lungs of mice exposed to 980
3
or 1568 ug/m (0.5 or 0.8 ppm) continuously for 3 to 5 days (Osebold et al. ,
1980), resulting in an increased incidence of anaphylaxis. Increased sensiti-
vity to histamine or cholinomimetic drugs by aerosol or injection has been
noted in several species. Easton and Murphy (1967) showed that the lethal
dose of histamine in guinea pigs was reduced in those animals exposed to
concentrations as low as 980 or 1960 ug/m (0.5 or 1 ppm) of 0. for 2 hr. The
pulmonary resistance due to subcutaneous injection of histamine increased in
3
guinea pigs exposed to 1568 ug/m (0.8 ppm) of 0« for 1 hr (Gordon and Amdur,
3
1980). Similarly, dogs exposed to 1372 and 1960 ug/m (0.7 and 1.0 ppm) of 03
for 2 hr had greater changes in pulmonary resistance following histamine
aerosol inhalation (Lee et al., 1977; Holtzman et al., 1983a). Sheep exposed
to 980 ug/m (0.5 ppm) of 0_ for 2 hr experienced increased pulmonary resis-
•J
tance for the cholinomimetic drug, carbachol (Abraham et al., 1980). The time
course of 0_-induced airway hyperreactivity suggests a possible association
with inflammation (Holtzman et al. , 1983a,b; Sielczak et al. , 1983; Fabbri
et al., 1984), but responses are variable and not very well understood.
0190JE/A 10-204 5/1/84
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Additional studies that demonstrate increased collateral resistance following
30-min local exposure of Cu or histamine in sublobar bronchi of dogs (Gertner
et a!., 1983a,b,c) suggest that other mechanisms, along with amplification of
reflex pathways, may contribute to changes in airway reactivity.
10.6.3.3 Biochemically Detected Effects of Ozone. Increased activity of the
glutathione peroxidase system is one of the most sensitive indices of exposure
to <_ 1960 ug/m (1 ppm) of 0,. measured biochemically. The increases in the
glutathione peroxidase system are thought to be protective, because this
system is involved in antioxidant metabolism. Concurrent increases in 0?
consumption of mitochondria are also observed. Mustafa (1975) and Mustafa
et al. (1976, 1983) summarized a number of experiments with rats; they showed
that continuous exposure between 392 and 1568 ug/m (0.2 and 0.8 ppm) for 7
days caused a concentration-related increase in the activities of succinate
oxidase, succinate-cytochrome c reductase, NADPH-cytochrome c reductase,
lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glutathione reductase,
glutathione peroxidase, and the levels of reduced glutathione. Numerous
investigators have also found similar increases when studying rats, mice, and
monkeys (Chow, 1983; Plopper et al. , 1979; Schwartz et al. , 1976; Moore et
al., 1980; Fukase et al. , 1975; Chow et al. , 1974, 1975; Mustafa et al. ,
1982). Increases in the glutathione peroxidase system have been reported
after exposure of rats on a vitamin E-deficient diet to levels as low as
196 (jg/m (0.1 ppm) for 7 days (Chow et al., 1981; Mustafa, 1975; Mustafa and
Lee, 1976). The dietary vitamin E fed to the rats influenced the increase of
this system. For example, when the diet of rats had 66 ppm of vitamin E,
increased activities were observed at 392 ug/m (0.2 ppm) of 0 • with 11 ppm
3
of vitamin E, increases occurred at 196 pg/m (0.1 ppm) (Mustafa and Lee,
1976). Several other investigators have shown that vitamin E deficiency in
rats makes them more susceptible to these enzymatic changes (Chow et al. ,
1981; Plopper et al. , 1979; Chow and Tappel, 1972).
The influence of the pattern of exposure on these effects of 0 on anti-
«3
oxidant metabolism and 0- consumption have been investigated (DeLucia et al. ,
1975a,b; Fukase et al. , 1975; Chow and Tappel, 1973; Mustafa and Lee, 1976).
Generally, over a 7-day continuous exposure to about 1568 pg/m (0.8 ppm) of
03, there is a slight (usually nonsignificant) decrease in enzyme activities
on day 1 of exposure. The activities then increase by day 2, plateau by about
day 4, and are still elevated at day 7. When both continuous and intermittent
(8 hr/day) exposure regimens are compared for several 0 concentrations (392,
«3
0190JE/A 10-205 5/1/84
-------
3
980, or 1568 (jg/m ; 0.2, 0.5 or 0.8 ppm) , no differences between the regimens
are observed in rats (Chow et al . , 1974; Schwartz et al., 1976; Mustafa and
Lee, 1976; DeLucia et al . , 1975a).
Antioxidant metabolism and 0^ consumption have been examined in mice,
rats, and monkeys after 0,, exposure. Generally, similar effects were noted,
although different concentrations were required sometimes to elicit effects.
Q
For example, rats were affected by an 8-hr/day, 7-day exposure to 980
(0.5 ppm) of 0-, but rhesus monkeys were not affected (Chow et al., 1975).
Rhesus monkeys were affected after 7 days of intermittent exposure to
3
1568 (jg/m (0.8 ppm) (Mustafa and Lee, 1976). Rats and bonnet monkeys have
been compared by Mustafa and Lee (1976). After 7 days of intermittent expo-
sure to 980 |jg/m (0.5 ppm), the rats and monkeys had equivalent increases in
succinate oxidase activity, Direct comparison of mice and rats shows that for
several of the biochemical endpoints, the increases in mice were significantly
greater than the increases in rats (Mustafa et al . , 1982). Quantitative
evaluation of species sensitivity from these studies is not possible. For
example, in the rat and monkey studies, some 03 concentrations were not equal,
and statistical design and analyses were inadequate. In addition, differences
in the deposition of 0., between rats and monkeys is not known. Thus, it may
O
only be generally stated that apparently rats were more responsive than monkeys.
Age-dependent responsiveness to 0, has been described in rats exposed to
3
1568 or 1764 ng/m (0.8 or 0.9 ppm) of 0~. Generally, the youngest rats (7 to
18 days old) exhibited decreases in enzyme activities; 20- to 24-day-old rats
had marginal increases (if at all); adult, 35- to 90-day-old rats had an
increase in enzyme activities (Tyson et al., 1982; Lunan et al . , 1977; Elsayed
et al . , 1982a). The reasons for this age dependence are unknown. It might be
due to a complex interrelationship between sensitivity (because basal levels
of enzymes studied differed by age) and dosimetry of 03_
The numerous studies of antioxidant metabolism and oxygen consumption are
summarized in Table 10-5. A few mechanisms for the increases in these metabo-
lic systems are possible: increased enzyme content within a given cell; same
enzyme content, but an increase in the number of cells with such enzymes; or a
combination of the two. The former potential mechanism has not been studied.
Mustafa et al. (1973), Mustafa (1975), and DeLucia et al . (1975a) found that
Op consumption of the whole lung increased, the number of mitochondria in the
lung increased, and the specific enzyme activity of mitochondria (activity per
0190JE/A 10-206 5/1/84
-------
milligram of mitochondria! protein, e.g., per mitochondrion) did not increase.
Several investigators have found close correlations between the pattern of
changes in these enzymes and increases in the number of type 2 cells, which
have more mitochondria than type 1 cells, in the lung (Plopper et al., 1979;
Chow et al., 1981; Schwartz et al., 1976; DeLucia et al. , 1975a). Thus, the
available experimental evidence suggests that the mechanism responsible for
these enzymatic changes is the hyperplasia of type 2 cells after 0_ exposure.
O
However, the presence of a concurrent increase in activities per cell that
makes a small contribution to the overall response cannot be ruled out.
The activities of other enzymes increase in the lung following 0 expo-
sure. The total lactate dehydrogenase activity was increased in lungs of rats
after exposure to 980 ug/irr (0.5 ppm) (Chow et al., 1977). Lysosomal enzymes
were increased in lungs of rats (Oil lard et al. , 1972; Castleman et al. ,
1973a) and may reflect part of the inflammatory process (e.g., the influx of
alveolar macrophages, which have high levels of these enzymes) seen throughout
periods of 0, exposure, as well as the breakdown of the exposed alveolar
macrophages.
Decreases in the activities of several enzymes in the lung have also been
reported. The cytochrome P-450 system has been studied because of its function
in drug and carcinogen metabolism. Montgomery and Niewoehner (1979) reported
a 50 percent decrease of benzphetamine N-demethylase activity, which is a
cytochrome P-450-dependent enzyme, in the rat following a 24-hr exposure to
3
1960 ug/m (1 ppm)- In other studies, a decrease in the activity of benzpyrene
hydroxylase in lung parenchyma of hamsters and in tracheobronchial mucosae of
3
the rabbit was observed at 1470 ug/m (0.75 ppm) (Palmer et al., 1971, 1972).
3
Acute exposure of rabbits to 1960 ug/m (1.0 ppm) also decreased levels of
lung micrososomal cytochrome P-450 (Goldstein, 1975).
Collagen, a major structural protein of the lungs, is also altered by 0-
exposure. Typically, the activities of prolyl hydroxylase (the rate-limiting
enzyme for the formation of hydroxyproline) and hydroxyproline (a major consti-
tuent of collagen) are measured. Last et al. (1979) observed a linear rela-
tionship between the increase in collagen synthesis and 0» concentrations
3
between 980 and 3920 ug/m (0.5 and 2 ppm). If exposure was prolonged, hydroxy-
3
proline decreased by 180 days of exposure to 980 ug/m (0.5 ppm) (Last and
Greenberg, 1980). Recovery from increases in collagen metabolism are not
3
immediate. After a 7-day exposure of rats to 1568 ug/m (0.8 ppm) of 0,. ceased,
0190JE/A 10-207 5/1/84
-------
about 10 days were required for recovery of prolyl hydroxylase activity;
hydroxyproline levels remained increased 28 days postexposure (Hussain, 1976a).
The minimal exposure regimen causing an increase in prolyl hydroxylase activity
3 3
in rats is a 7-day continuous exposure to 980 ug/m (0.5 ppm); 392
(0.2 ppm) caused no effects (Hussain et a!., 1976a,b). The increased collagen
synthesis has been correlated with fibrosis detected histologically in the same
experiment (Last et al . , 1979; Moore and Schwartz, 1981). Bhatnagar et al .
(1983) found that collagen synthesis also increases in mice exposed to
882 ug/m (0.45 ppm) of 0_. Thus, the response is not species-specific.
O
Although several investigators have found that in vitro exposure to 0_
" O
affects lipids, very few jjn vivo studies have been conducted. Continuous
exposure to 980 ug/m (0.5 ppm) for 2, 4, or 6 weeks alters fatty acid compo-
sition of the lungs in rats, and acute (4 hr) exposure to a higher level
3
(1960 pg/m ; 1 ppm) decreases incorporation of fatty acids into lecithin in
rabbits, (Roehm et al . , 1972; Kyei-Aboagye et al . , 1973).
3
Ozone exposure (1176 to 4116 ug/m ; 0.6 to 2.1 ppm) inhibits the phenol
red activity transport mecianism from the lung to the circulation in a concen-
tration-related manner (Williams et al., 1980). The noncarrier-mediated
diffusion of phenol red from the lung increased simutaneously with the inhibi-
tion of the carrier-mediated transport. An increase in lung permeability, as
evident, by the diffusion of plasma proteins into the airways following 0.,
exposure, has been reported in dogs (Reasor et al . , 1979), rats (Alpert et
al . , 1971a), and guinea pigs (Hu et al . , 1982). The protein content of lung
lavage fluid from vitamin C-deficient guinea pigs was not different from those
receiving adequate vitamin C. The influx of protein was proportional to the
0_ concentration to which the guinea pigs had been exposed in the concentra-
tion range of 510 to 1470 jjg/m3 (0.26 to 0.75 ppm) for 3 hr (Hu et al . , 1982).
It is generally agreed that the toxic effects of 0_ can be ascribed to
O
its oxidative capacity. As a powerful oxidant, 0_ can react with virtually
»J
every class of biological substance; this makes identification of the critical
or essential toxic lesion almost impossible.
Two general hypotheses have been put forth to explain the toxic effects
of 03 on a molecular basis. The first hypothesis is that the oxidation of
thiols or amino acids in tissue proteins or small molecular weight peptides is
responsible (Mudd and Freeman, 1977). The second is that the oxidation of
polyunsaturated fatty acids (PUFA) to fatty acid peroxides (FAR) results in
0190JE/A 10-208 5/1/84
-------
toxicity (Menzel, 1970). A careful study of the biochemical, physiological,
and morphological effects of 0, exposure points to the cellular membranes as
the site of toxicity. Both mechanisms could occur at the membrane. Because
membranes are composed of both proteins and lipids, damage to one is difficult
to separate from damage tc the other or to both. Because the metabolism of
FAR is linked to the glutathione peroxidase system (Chow and Tappel, 1972),
depletion of thiols such as glutathione (GSH) by 03 could enhance the toxicity
of FAPs. The two theories are thus not separate, but complementary, and most
likely oxidation of both PUFAs and proteins occurs simultaneously.
Although CL is a general reactant, the relative rates of reaction with
different biological substances is remarkably different. Ozone oxidizes
ethylene groups 1000 times faster than other compounds. Consequently, atten-
tion has been focused on PUFAs as targets for 0- reaction. The Criegee mecha-
nism for addition of 0^ across unsaturations is still a popular hypothesis. In
the presence of protom'c solvents such as water, peroxides can result directly
from the decomposition of the initial 03 adduct. Pryor et al. (1983) have
also suggested that free radicals are generated directly by other mechanisms
during the ozonation of PUFAs. Regardless of the chemical mechanism, once
free radicals are generated in membranes, the toxic effects are similar.
These effects are a loss of regulation of electrolytes and water (changes in
permeability), leakage of essential intracellular enzymes from the cell, and
inhibition of metabolic chains imbedded in mitochondria! or microsomal matri-
cies (Menzel, 1976). If damage is sufficiently severe, the cell cannot recover
and dies. Necrosis is commonly reported in the lungs of animals exposed to
high concentrations of 0^.
Perhaps the most compelling evidence for PUFA peroxidation jji vivo is the
protective effect of dietary vitamin E on 0- toxicity (Roehm et al., 1971a,b
and 1972; Chow and Tappel. 1972; Fletcher and Tappel, 1973; Donovan et al.,
1977; Sato et al., 1976a,b; Chow and Kaneko, 1979; Plopper et al., 1979; Chow
et al., 1981; Chow, 1983; Mustafa et al., 1982). Vitamin E as a phenolic
antioxidant easily inhibits the peroxidation of PUFAs initiated in vitro by 00
, . , j
(Roehm et al., 1971a). Indirect evidence that vitamin E protects against
03-initiated peroxidation of PUFAs u> vivo is presented by the studies of
Dumelir et al. (1978b), who found that the exhaled ethane and pentane in the
breath of rats was decreased when vitamin E was provided in their diets.
0190JE/A 10-209 5/1/84
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Ethane and pentane are thought to be produced by peroxidation of PUFAs initi-
ated by 0 inhalation. Changes in lung fatty acid composition due to peroxi-
dation are not easily interpreted because of the complexity of lung PUFA
composition and difficulties in quantitative analysis. Direct evidence of
peroxidation of PUFAs in human subjects has, however, been lacking.
Amino acids are oxidized by 0-3 (Mudd et al. , 1969; Mudd and Freeman,
1977; Previero et al. , 1964). Cysteine, methionine and tryptophan are the
three most easily oxidized amino acids. Human alpha-1-protease inhibitor is
destroyed by ozonolysis i_n vitro, presumably by oxidation of methionine,
tyrosine and tryptophan amino acid residues in the molecule (Johnson, 1980).
The activity of a large number of enzymes is decreased immediately after 0
3
exposure to 1960 to 7840 ug/m (1 to 4 ppm) for 1 hr or more (DeLucia et al.,
1972; Mountain, 1963; Kyei-Aboagye et al., 1973; Holzman et al., 1968; Goldstein
et al., 1975; Menzel et al., 1976). Lung mitochondria lose their respiratory
control following 0_ exposure (Mustafa et al., 1973). DeLucia et al. (1975b)
also reported the depletion of GSH and the formation of mixed disulfides in
the lungs of 0 -exposed rats.
O
In contrast to the decrease in enzyme activity by acute, high-level 0_
O
exposure, the glutathione peroxidase system consisting of glucose-6-phosphate
dehydrogenase, glutathione reductase and glutathione peroxidase is increased
3
in the lungs of rats exposed repeatedly to lower levels of 0_ (< 1960 yg/m , I
o
ppm) (Chow et al., 1981, 1974; Chow and Tappel, 1972, 1973; Mustafa and Lee,
1976; Moore et al. , 1980). These effects would favor the concept that 0
o
increases PUFA peroxidation, which induces the glutathione peroxidase system
as a protective mechanism. Thus, with the available data, it would appear
that both direct oxidation of low-molecular-weight compounds and proteins, as
well as lipid peroxidation, can occur with 0~ exposure.
10.6.3.4 Host Defense Mechanisms. To protect man and other mammals from
inhaled infectious agents, the mammalian lung possesses an array of defense
mechanisms. Considerable effort has been expended in studies of the effects
of 0- on such defense mechanisms in hopes of understanding the underlying
O
mechanisms and predicting concentration-response relationships.
Inhaled viable microorganisms as well as airborne particles are removed
from the lung by two mechanisms. Ciliated cells of the respiratory tract
propel mucus carrying entrapped particles and infectious agents upwards from
0190JE/A 10-210 5/1/84
-------
the distal regions of the lung to be swallowed and excreted. Alveolar macro-
phages engulf, kill, and remove infectious agents from the respiratory regions
of the lung either by migrating with the microbe to the mucus lining to be
propelled upwards or to the lymphatic system to be drained from the lung.
Ciliated cells are dcimaged 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. Ciliated cell
damage should result in decreased transport of viable and non-viable particles
from the lung. Rats exposed to 784, 1568, or 2352 ng/m (0.4, 0.8, or 1.2
ppm) for times as short as 4 hr have decreased short-term clearance of parti-
cles from the lung (Phalen et al., 1980; Frager et al. , 1979; Kenoyer et al.,
1981). Short-term clearance is mostly due to mucus removal of particles, and
the decreased short-term clearance is an anticipated functional result pre-
dicted from morphological observations. The mucous glycoprotein production of
the trachea is also altered by 0~ exposure, but not in a simple manner.
Mucous glycoprotein biosynthesis, as measured ex vivo in cultured tracheal
explants from exposed rats, was inhibited by short-term continuous exposure to
3
1568 ug/m (0.8 ppm) of 0 for 3 to 5 days (Last and Cross, 1978; Last and
*.!
Kaizu, 1980; Last et al., 1977). Glycoprotein synthesis and secretion re-
covered to control values after 5 to 10 days of exposure and increased to
greater than control values after 10 days of exposure.
The long-term clearance of particles from the lung is a function of the
alveolar macrophages (AM), which are damaged by 0~ inhalation. Most studies
3
have been with rabbits, in which a 3-hr exposure to 980 ug/m (0.5 ppm) reduces
phagocytosis (Coffin and Gardner, 1972b; Coffin et al. , 1968b), disrupts AM
membranes (Dowel! et al. , 1970; Goldstein et al., 1977; Hadley et al., 1977),
reduces AM lysosomal hydrolyase activity (Hurst et al., 1970, 1971), prevents
AM migration (Schwartz and Christman, 1979; McAllen et al., 1981), and reduces
AM numbers (Coffin et al.,, 1968b; Alpert et al. , 1971b). The bactericidal
capacity of AMs is also impaired; this has been attributed to the inhibition
of superoxide anion radical production capacity by 0~ exposure (Witz et al.,
«5
1982; Amoruso et al., 1981; Goldstein et al., 1971a,b, 1972; Warshauer et al.,
1974; Bergers et al., 1982).
An integrated measure of both of these mechanisms of viable organism
removal and the more general measures of host defense against airborne infec-
tions can be determined by the airborne infection of animals following 0
0190JE/A 10-211 5/1/84
-------
exposure. This method, known as the infectivity model, has recently been de-
scribed in detail by Gardner (1982a). A number of different animals and
bacterial infectious agents have been used to illustrate the increased suscep-
tibility of animals to airborne infections following very modest concentrations
and times of exposure. To date, it is one of the most sensitive measures of
ozone toxicity. Mice exposed to 157 to 196 ug/m (0.08 to 0.1 ppm) for 3 hr
are significantly more susceptible to bacterial infection than mice exposed to
clean air (Coffin et al. , 1968a; Ehrlich et al., 1977; Miller et al. , 1978).
This increase in susceptibility was also evident after an intermittent exposure
3
to 196 ug/m (0.1 ppm) fo<~ 103 days (Aranyi et al. , 1983). Adding SO and
(NH.)? SO. to 0- did not further enhance the mortality rate. However, combining
0- with either NO- or H?SC. had an additive effect in this infectivity system
(Gardner et al. , 1977; Grose et al. , 1982; Ehrlich et al., 1977; Ehrlich et
al., 1979; Ehrlich, 1980). The effects of 0» were more pronounced when the
test animals exercised during 0« exposure (Illing et al., 1980.) Such a study
o
demonstrates that the activity level of the test subject is an important
variable that can influence the host response.
Immunological effects of 0_ have only been scantily studied in mice and
guinea pigs. Depression of the cell-mediated immune response has been measured
3
at high 0., concentrations (2901 ug/m , 1.48 ppm) in the guinea pig (Thomas
et al., 1981b). The syste;nic immune response is depressed in mice, as measured
by the blastogenic response of splenic lymphocytes to the T-cell mitogens PHA
3
and Con-A (Aranyi et al. , 1983). Mice were exposed to 196 ug/m (0.1 ppm) 5
hr/day, 5 days/week for 90 days.
10.6.3.5 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 low levels of exposure to 0_ will certainly protect
against subsequent lethal doses and the development of edema (Stokinger et al.,
1956; Fairchild, 1967; Coffin and Gardner, 1972a). The delay in mucociliary
activity (i.e., clearance reported for 0_) can also be eliminated by pre-
exposure to a lower concentration. This effect is only 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 03, such as reduced functioning
activity of the pulmonary defense system (Gardner et al., 1972); hyperplasia
0190JE/A 10-212 5/1/84
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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 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 cells attempt to initiate early repair of the
O
specific lesion. This repair thus results in a reduction of the effect first
observed. 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). 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 routes, 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 nonreversible pulmonary changes.
10.6.4 Extrapulmonary Effects
It was formerly believed that 0-, on contact with respiratory system
tissue, immediately reacted and thus was not absorbed or transported to extra-
pulmonary sites. However, several studies suggest that either 0., or products
O
formed by the interaction of 0~ and respiratory system tissue produce effects
in lymphocytes, erythrocyt.es, and serum, as well as in the parathyroid gland,
the myocardium, the liver, and the CNS. Ozone exposure also produces effects
on animal behavior that may be caused by pulmonary consequences of 0 , or by
nonpulmonary (CNS) mechanisms. The mechanism by which 0_ causes extrapulmonary
O
changes is unknown. Mathematical models of 03 dosimetry predict that very little
0_ 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.
10.6.4.1 Central Nervous System and Behavioral Effects. Ozone significantly
affects the behavior of rats during exposure to concentrations as low as
3
235 ug/m (0.12 ppm) for 6 hr. With increasing concentrations of 0~, further
O
decreases in unspecified motor activity and in operant learned behaviors have
0190JE/A 10-213 5/1/84
-------
been observed (Konigsberg and Bachman, 1970; Tepper et al. , 1982; Murphy
et al. , 1964; and Weiss et al., 1981). Tolerance to the observed decrease in
motor activity may occur on repeated exposure. At low CL exposure concentra-
3
tions (490 ug/m , 0.25 ppm) an increase in activity is observed after exposure
3
ends. Higher CL concentrations (980 ug/m , 0.5 ppm) produce a decrease in
•3
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 0~, although this
•j
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 CL impairs the inclination to respond.
O
Two studies indicate that mice will respond to remove themselves from an
atmosphere containing greater than 980 ug/m (0.5 ppm) (Peterson and Andrews,
1963, Tepper et al. , 1983). These studies suggest that the aversive effects
of 0. may be due to lung "irritation. It is unknown whether lung irritation,
O
odor, or a direct effect on the CNS causes change in rodent behavior at lower
0- concentrations.
%J
10.6.4.2 Cardiovascular Effects. Studies on the effects of CL on the cardio-
vascular system are few, and to date there are no reports of attempts to con-
firm these studies. Structural changes in the cell membranes and nuclei of
the myocardium muscle fibers in mice were found after 3 weeks of exposure to
3
392 ug/m (0.2 ppm) (Brinkman et al., 1964), and these effects were reversible
in clean air. The exposure of rats to 0,, alone or in combination with cadmium
3
(1176 |-g/m , 0.6 ppm 0~) resulted in measurable increases in systolic pressure
•J
and heart rate (Revis et al., 1981). No additive or antagonistic response was
observed with the combined exposure. Pulmonary capillary blood flow and PaO_
3
decreased 30 min following exposure of dogs to 588 ug/m (0.3 ppm) of 0-
(Friedman et al. , 1983). The decrease in pulmonary capillary blood flow per-
sisted for as long as 24 hr following exposure.
10.6.4.3 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.
0190JE/A 10-214 5/1/84
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Effects of 0- 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
RBCs isolated from monkeys exposed to 1470 ug/m (0.75 ppm) of 0_ 4 hr/day for
3
4 days (Clark et al. , 1978). A single 4-hr exposure to 392 ug/m (0.2 ppm)
also caused increased fragility as well as sphering of RBCs of rabbits (Brinkman
et al., 1964). An increase in the number of RBCs with Heinz bodies was detected
3
following a 4-hr exposure to 1666 ug/m (0.85 ppm). The presence of such
inclusion bodies in RBCs is an indication of oxidant stress (Menzel et al.,
1975a).
These morphological changes are frequently accompanied by a wide range of
biochemical effects. RBCs of monkeys exposed to 1470 ug/m (0.75 ppm) of 0_
for 4 days also had a decreased level of glutathione (GSH) and decreased
acety1cholinesterase (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 uniquely sensitive to 0~. The RBCs
from these animals, after being exposed to 03, had a significant increase in
the activity of GSH peroxidase, pyruvate kinase, and lactic dehydrogenase, but
had a decrease in RBC GSH after exposure to 1568 ug/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, or catalase activities. At a lower level (980 ug/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 980 ug/m (0.5 ppm). These effects were not observed
in vitami;i E-supplemented rats. Mice on a vitamin E-supplemented diet and
those on a deficient diet showed an increase in G-6-PD activity after an
exposure of 627 ug/m (0.32 ppm) of 0 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
(Veninga, 1967). Six- and 10-month exposures of rabbits to 784 ug/m3 (0.4 ppm)
0190JE/A 10-215 5/1/84
-------
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; and Jegier, 1973). Chow et al. (1974) reported that
the se'^um lysozyme level of rats increased significantly after 3 days of
continuous exposure to 0, but was not affected when the exposure was interim" t-
6 3
tent (8 hr/day, 7 days). The CL concentration in both studies was 1568 ug/m
(0.8 ppm) of 0-.
Short-term exposure to low concentrations of CL induced an immediate
change in the serum creatine phosphokinase level in mice. In this study, the
CL 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
0. have also been seen following in vitro exposure of RBCs from humans (Freeman
3 ______
and Mudd, 1981; Menzel et al., 1975b; Verweij and Van Steveninck, 1981). A
common effect observed by a number of investigators is that 0~ 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 sphercytosis and increased fragility of RBCs seen in animal and human
cells.
Although these ij} 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 camot be determined with any accuracy.
10.6.4.4 Reproductive anc Teratogenic Effects. Pregnant animals may be at
greater risk to the effects of 0~ because the volume of air inspired generally
O
increases up to 50 percent during pregnancy. Experiments with mice suggest
3
that perinatal exposure to 392 ug/m (0.2 ppm) of 0_ can reduce infant survival
(Brinkman et al., 1964) and increase the incidence of unlimited incisor growth
3
in neonates (Veninga, 1967). Pregnant rats exposed to 2920 ug/m (1.49 ppm)
of 0_ during mid-gestation had a significantly higher embryo resorption rate
O
than control rats (Kavlock et al. , 1979). Progeny of dams exposed to
3
1960 ug/m (1.0 ppm) of 0., during late gestation showed significantly slower
0190JE/A 10-216 5/1/84
-------
neonatal growth rates, retarded early reflex development, and delayed onset of
grooming and rearing behavior (Kavlock et al., 1980).
10.6.4.5 Chromosomal and Mutational Effects. That 0_ may have the potential
O
for producing genotoxic effects has been postulated because of its radiomimetic
properties. The decomposition of 0- in water produces OH and HO radicals,
and it is these species that are considered to be the most biologically active
products of ionizing radiation. Cytogenetic toxicity has been reported both
in celTs in culture and in cells isolated from animals exposure to 0_. A
»J
large portion of this data base, however, has been derived from studies conduc-
ted at extremely high levels of 0 , and because of this, these data will not
O
be discussed further in this summary. The most relevant data concerning geno-
toxicity of 0 are obtained from investigations in which the 0 concentration
3
did not exceed 1,960 ug/m (1.0 ppm).
Zelac et al. (1971a,b) reported a significant increase in chromosome
aberrations in peripheral blood lymphocytes in Chinese hamsters exposed to
3
392 ug/m (0.2 ppm) for 5 hr. It was also reported that 0 was additive with
radiation. Tice et al. (1978) attempted to repeat the Zelac studies; however,
a number of significant differences existed between these two studies that
make direct comparison of the data difficult. Tice et al. did not find any
increase in sister chromatid exchange or any increase in number of chromosome
aberrations of phytohemagglutinin-stimulated peripheral blood, but they did
find an increase in the number of chromatid deletions and achromatic lesions.
Although both studies reported significant increases in chromosome aberrations
in peripheral blood lymphocytes, the specific type of lesions reported were
clearly different. Section 10.4.5 gives a detailed discussion of the importance
of these differences. Gooch et al. (1976) analyzed the bone marrow samples
3
from Chinese hamsters exposed to 451 \ig/m (0.23 ppm) of 0 for 5 hr and the
3
leukocytes and spermatocytes from mice exposed for up to 2 weeks to 412 |jg/m
(0.21 ppm) of 0_. No effect was found on either the frequency of chromatid or
chromosome aberrations, nor were there any reciprocal translocations in the
primary spermatocytes. Guerrero et al. (1979) reported a concentration-related
increase in the sister chromatid exchange in human fetal lung cells (WI-38)
exposed i_n vitro to concentrations ranging from 490 to 1960 ug/m (0.25 to
1.0 ppm) for 1 hr.
In summary, 0^ has been shown to produce a wide range of toxic effects on
cells and on cellular components, including genetic material. Cytogenetic
toxicity has been reported in cells in culture and in cells isolated from
0190JE/A 10-217 5/1/84
-------
animals if 03 exposure has occurred at sufficiently high levels and for suffi-
cient long periods. The irutagenic effects of 0- have been examined in only a
very few instances, and unfortunately none of these investigations have used
mammalian cells. The mutagenic properties of 0- have been demonstrated in
procaryotic and eucaryotic cells; however, the concentrations used were many
orders of magnitude highe1" than what is found in the ambient environment.
10.6.4.6 Other Extrapulmonary Effects. A series of studies was conducted to
show that 0- increases drug-induced sleeping time in a number of species of
animals (Gardner et al., 1974; Graham, 1979; Graham et al., 1981, 1982a,b,
1983a,b). At 1960 ug/m3 (1.0 ppm), effects were observed after 1, 2, and 3
days of exposure. As the concentration of 03 was reduced, increasing numbers
of dai'y 3-hr exposures were required to produce a significant effect. At the
3
lowest concentration studied (196 ug/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., 1983a).
Several investigators have attempted to elucidate the involvement of the
endocrine system in 03 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) investigated the effects of a 24-hr exposure to 1960
(1.0 ppm) of 03 on the hypothal amo-pi tui tary-thyroid system of rats. These
three organs regulate the function of each other through various hormonal
feedback mechanisms. Ozone caused decreases in serum concentration 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 03 on TSH. The thyroid
gland itself was altered (e.g., edema) by 0-. The authors interpreted these
findings as an 03-induced lowering of the hypothalamic set point for the
pituitary-thyroid axis and a simultaneous reduction of the activity of pro-
lactin inhibiting factor in the hypothalamus. The anterior pituitaries had
0190JE/A 10-218 5/1/84
-------
fewer cells but more TSH per cell. These cells also released a greater amount
of TSH into the culture medium.
By using LM and TEM, Atwal et al. (1975), Atwal and Wilson (1974), and
Atwal and Pernsingh (1981) described changes in parathyroid glands in dogs and
rabbits following short-term (up to 48 hr) exposure to 0.75 ppm of 0 . Changes
•5
identified include parenchyma! atrophy, congestion, abundant secretory granules,
leukocyte infiltration, and focal hemorrhages.
10.6.5 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. The few toxicological
studies on PAN indicate that it is much less acutely toxic than 0 . When the
O
effects seen after exposure to 0, 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 0« to produce a similar effect on lethality,
«J
behavior modification, morphology, or significant alterations in host pul-
monary defense system (Campbell et al., 1967; Dungworth et al. , 1969; Thomas
et al, , 1977, 1981a). The concentrations of PAN required to produce these
effects are many times greater than what has been measured in the atmosphere
(0.037 ppm).
Similarly, most of the investigations reporting H?CL toxicity have involved
concentrations much higher than those found in the ambient air (0.1 ppm), or
the investigations were conducted by using various j_n vitro techniques for
exposure. Very limited information is available on the health significance of
inhalation exposure to gaseous H_0_. Because H_0? is highly soluble, it is
generally assumed that it does not penetrate into the alveolar regions of the
lung but is instead deposited on the surface of the upper airways (Last et al.,
1982). Unfortunately, there have not been studies designed to look for pos-
sible effects in this region of the respiratory tract.
A few j_n vitro studies have reported cytotoxic, genotoxic, and biochemical
effects of H?0? 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.
0190JE/A 10-219 5/1/84
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Field and epidemiological studies have shown that human health effects
from exposure to ambient nixtures of oxidants and other airborne pollutants
can produce adverse human health effects (Chapter 12). 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 tc 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.,
1963; Murphy, 1964; Nakajima et al. , 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 end 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
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 S0v did not interact in any synergistic or additive
s\
manner.
0190JE/A 10-220 5/1/84
-------
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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
tissue^ 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 0,? 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 urn) 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 (PACQ?): Partial pressure of carbon dioxide
in the air contained in the lung alveoli.
Alveolar oxygen partial pressure (P/iOo)- 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.
019CC/C A-l May 1984
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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 (VQ .): Volume of the conducting airways down to the
level where, during enr Breathing, gas exchange with blood can occur, a
region probably situated at the entrance of the alveolar ducts.
Arterial oxygen saturation (SaCk): Percent saturation of dissolved oxygen in
arterial blood.
Arterial partial pressure of carbon dioxide (PaCO^): Partial pressure of
dissolved carbon dioxide in arterial blood.
Arterial partial pressure of oxygen (PaO?): 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.
Atropirie: A poisonous white crystalline alkaloid, C,7H,,~N03, from belladonna
arid related plants, used to relieve spasms of smootn 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.,
019CC/C A-2 May 1984
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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-
ogy, 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 tne 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 (carbamoylcholine chloride, CgH^ClN-O-)
that produces constriction of the bronchial smooth muscles.
Carbon dioxide production (VCO-): Rate of carbon dioxide production by organ-
isms, tissues, or cells. Common units: ml CO™ (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?.
019CC/C , A-3 May 1984
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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).
Closinc; capacity (CC): Closing volume plus residual volume, often expressed
as a ratio of TLC, i.e. (CC/TLC%).
Closing volume (CV): The volume exhaled after the expired gas concentration
is inflected from an alveolar plateau during a controlled breathing
maneuver. 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 VENTILATION 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 O'c 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 H^O or ml/cm H^O. 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, D|CO~, D.CO): Amount of gas (0,,,
CO, CO,,) commonly expressed as mi gas CSTPu) diffusing between alveofar
gas ana pulmonary capillary blood per torr mean gas pressure difference
per min, i.e., ml 02/(min-torr). Synonymous with transfer factor and
diffusion factor.
Dynamic compliance (C, ): 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.
019CC/C A-4 May 1984
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Elastance (E): The reciprocal of COMPLIANCE; expressed in cm H?0/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.
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 FEV./FVC. It is an index of airway obstruction.
t>
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.
FEF? _ nn = mean forced expiratory flow between 200 ml
U and 1200 ml of the FVC (formerly called the
maximum expiratory flow rate (MEFR).
FEF?C) 7C.% = mean forced expiratory flow during the middle
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 FEV0 _>
FEV_ 7t., FEV, „. These values are often expressed as a percent of the"
forced vital capacity, 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.
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.
019CC/C A-5 May 1984
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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.
Hematocrit (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 heme+ group. The heme is made of four pyrrole rings
and a divalent iron (Fe -protoporphyrin) which combines reversibly with
molecular oxygen.
Histamine: A depressor airiine derived from the ami no 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 P-? is low in the environment, whether because of
decreased barometric pressure or decreased fractional concentration of
0,,, the condition is termed environmental hypoxia. Hypoxia when referring
to the blood is termed hypoxemia. Tissues are said to be hypoxic when
their P._ 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.
Kilogram-meter/min (kgm/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.
019CC/C A-6 May 1984
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Maximal aerobic capacity (max V0~): The rate of oxygen uptake by the body
during repetitive maximal respiratory effort. Synonymous with maximal
oxygen consumption.
Maximum breathing capacity (MBC): Maximal volume cf 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 maximun voluntary ventilation (MVV).
»
Maximum expiratory flow (V ): Forced expiratory flow, related to the
total lung capacity o:" 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 -,.-0, = instantaneous forced expiratory flow when the
max 75% , . . -,..<* f • *. -riV
lung is at 75% of its TLC.
•
V o n = instantaneous forced expiratory flow when the
max i.u volume is 3 0 liters
Maximum expiratory flow rate (MEFR): Synonymous with FEF ~ .
Maximum mid-expiratory flow rate (MMFR or MMEF): Synonymous with FEF?c._7C.y.
Maximum ventilation (max Vc): 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 (VV): Volume of air breathed in one minute. It is a
product of tidal volume (VT) and breathing frequency (fD). See VENTILA-
TION. ' B
Minute volume: Synonymous with minute ventilation.
Mucociliary transport: The process by which mucus is transported, by ciliary
action, from the lungs.
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).
019CC/C . A-7 May 1984
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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 N0? 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 breatrnng ambient air to an inspired mixture of pure 0?. A
progressive decrease of N« concentration ensues which may be analyzed
into two or more exponential components. Normally, after 4 min of pure
()„ breathing the fractional N? concentration in expired alveolar gas is
down to less than 2%.
Normoxia: A state in which the ambient oxygen pressure is approximately 150 ±
1C 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 (V0_, Q0?): Rate of oxygen uptake of organisms, tissues,
or cells. Common unit?: 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 Qft_ = (jl 0?/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 (VO ): 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 02 stores
are constant.
Participates: 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.
Physiological dead space (Vn): Calculated volume which accounts for the
difference between the pressures of C0? 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.
019CC/C A-8 May 1984
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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 (SiIverman-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 enlargement of the air spaces distal to the
terminal nonrespiratory bronchiole, accompanied by destructive changes of
the alveolar walls. 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.
Resistance flow (R): The ratio of the flow-resistive components of pressure
to simultaneous flow, in cm HpO/liter per sec. Flow-resistive components
of pressure are obtained by subtracting any elastic or inertial components,
proportional respectively to volume and volume acceleration. Most flow
resistances in the respiratory system are nonlinear, varying with the
magnitude and direction of flow, with lung volume and lung volume history,
and possibly with volume acceleration. Accordingly, careful specification
of the conditions of measurement is necessary; see AIRWAY RESISTANCE,
TISSUE RESISTANCE, TOTAL PULMONARY RESISTANCE, COLLATERAL RESISTANCE.
019CC/C ; A-9 May 1984
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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 (fn): The number of breathing cycles per unit of time.
Synonymous with breathing frequency (fg)-
Respiratory quotient (RQ, R): Quotient of the volume of CO 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 C0? output to the 0» uptake by the lungs, "respiratory
quotient" being restricted to the actual metabolic CO- output and 0
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 0? and C0? stores.
Shunt: Vascular connection between circulatory pathways so that venous blood
is diverted into vessels containing arterialized blood (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 nalformations
are more important in respiratory physiology. Flow from ls?ft 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.
Static lung compliance (C ): 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., ^ TLC is static recoil pressure measured at
TLC (maximum recoil pressure).
Sulfur dioxide (S0?): Colorless gas with pungent odor, released primarily from
burning of fossil fuels, such as coal, containing sulfur.
019CC/C A-10 May 1984
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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..): 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
barometer 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.
Total pulmonary resistance (R.): Resistance measured by relating flow-dependent
transpulmonary pressure to airflow at the mouth. Represents the total
(frictional) resistance of the lung tissue (R..) and the airways (Raw).
R. = R + R+..
L aw ti
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.
019CC/C A-11 May 1984
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Transpulmonary pressure (P.): Pressure difference between airway opening
(mouth, nares, or cannula opening) and the visceral ploural surface, in
cm HLO. Transpulmonary in the sense used includes extrapulnonary s :ruc~
tures, e.g., trachea and extrathoracic airways. This usag? 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 thj lungs. The
word ventilation sometimes designates ventila~:ory flow rate (or ven\ila-
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
Vp = 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 vtntilatijn is
less than total ventilation because when a tidal volume of gas leaves the
alveolar spaces, the last part does not get expelled from 1 he body but
occupies the dead space, to be reinspired with the next ir spirati :>n.
Thus the volume of alveolar gas actually expelled completely is equal to
the tidal volume minus the volume of the dead space. This t^uly conplete
expiration volume times the ventilatory frequency constitutes the aiveolar
ventilation.
•
Ventilation, dead-space (Vn): Ventilation per minute of the physiologic dead
space (wasted ventilation), BTPS, defined by the following equation:
VD = V£(PaC02 - ?£C02)/(PaC02 - PjCOg)
Ventilation/perfusion ratio (V./Q): Ratio of the alveolar venti ation to th;
blood perfusion volume flow through the pulmonary parenchyma. This rat o
is a fundamental determinant of the Q~ and C0» pressure of the alveolar
gas and of the end-capillary blood. Throughout the lungs the looal
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 tha point of
maximum inspiration.
U.S. Environmental Protection Agency
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230 South Dearborn Street
Chicago, Illinois 60604
019CC/C A-12 ^lay 1984
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