i• 

..

~ ;.,. 

~,it."•`:~:~~'..; ;f;• ,. =...~..; _ _ „s.,:••::= .,~ 

. 

. ~• ° i~~ ,..•'.,

~f 

' ~~ _. _ =._ • . : 

~ ..1.?:r i y 4 

~ A +L~ 1 - _ 

- ,:.= 

~,;,::i7-• _..,: ',.:--::}' 

- i.li`

~.•~ - - _ - :::i:i'4~'~'~.~ .... 

. ... .{v . 

j•~~~ _ _ `~-~-.rT-Y. ._ 

~~~~,:i•' h, 

-~,~.u+~u:.+ •~~ 

.~J~.~ '.:

~

.i: w •. 

':•W 

:

~-.ii. ~~!~~~,. 

i''=+a •.,~,~

... - 

/ :' •'. ~ r 

;;: '~ •+';~'~'~~ - "'~~ s 

EPA-600/8-84-020dF 

August 1986 

Air Quality Criteria 

for Ozone and Other, 

Photochemical Oxiclanits 

Vol u me IV of V 

Environmental Criteria and Assessment Office 

Office of Health and Environmental Assessment 

Office of Research and Development 

U.S. Environmental Protection Agency 

Research Triangle Park, N.C. 27711 
DISCLAIMER 

This document has been reviewed in accordance with U.S. Environmental 

Protection Agency policy and approved for publication. Mention of trade 

names or commercial products does not cbnstitute.endorsement or 

recommendation. 

ii 
ABSTRACT 

Scientific information is presented and evaluated relative to the health 

and welfare effects associated w.ith exposure to ozone and other photochemical 

oxidants. Although it is not intended as a complete and detailed literature 

review, the document covers pertinent literature through early 1985. 

Data on heaith and welfare effects.are emphasized, but additional information is provided for understanding the nature of the oxidant pollution problem and for evaluating the reliability of effects data as well as their 

relevance to potential exposures to ozone and other oxidants a -t concentrations 

occurr.ing in ambient air. Information is p-resented on the following exposurereiated topics: nature, source, measurement, and concentrations of precursors 

to ozone and other photochemical oxidants; the formation of ozone and other 

photochemical oxidants and their tra'nsport once formed; the properties, chemistry, and measurement of ozone and other photochemical oxidan -ts; and the 

concentrations of ozone and other photochemical oxidants that are typically 

found in ambient air. 

The specific areas addressed by chapters on health and welfare effects 

are the toxicological appraisal of effects of ozone and other oxidants; effects 

observed in controlled human exposures; effects observed in field and epidemiological 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. I 

iii 
AIR QUALITY CRITERIA FOR OZONE 

AND OTHER PHOTOCHEMICAL OXIDANTS 

Pa e 

VOLUME I 

Chapter 1. Summary and Conclusions .............................. 1-1 

VOLUME II 

Chapter 2. Introduction ............ .... .. ............. .. 

~ 

2-1 

Chapter 3. Properties, Chemistry, andTransport of Ozone and 

Other Photochemical Oxidants and Their Precursors .... 3-1 

Chapter 4. Sampling and Measurement of Ozone and Other 

Photochemical Oxidants and Their Precursors .......... 4-1 

Chapter 5. ° Concentrations of Ozone and Other Photochemical 

Oxidants in Ambient Air ...............,............... 5-1 

VOLUME III 

Chapter 6. Effects of Ozone and Other Photochemical Oxidants 

on Vegetation ........................................ 6-1 

Chapter 7. Effects of Ozone on Natural Ecosystems and Their 

Components........... ............ ..... . ... ... 7-1 

Chapter 8. Effects of Ozone and Other Photochemical Oxidants 

on Nonbiological Materials ........................... 8-1 

VOLUME IV 

Chapter 9. Toxicological Effects of Ozone and Other 

Photochemical Oxidants ............................... 9-1 

VOLUME V 

Chapter 10. Controlled Human Studies of the Effects of Ozone 

and Other Photochemical Oxidants .... .. ........... 10-1 

Chapter 11. Field and Epidemiological Studies of the Effects 

of Ozone and Other Photochemical Oxidants ............ 11-1 

Chapter 12. Evaluation of Health Effects Data for Ozone and 

Other'Photochemical Oxidants ......................... 12-1 

iv 
TABLE OF COiiTENTS 

Page 

LIST OF TABLES ........................................................ viii 

LIST OF FIGURES ....................................................... i'x 

LIST OF ABBREVIATIONS . . ...................:.:.............. xiii 

AUTHORS, CONTRIBUTORS, ~ AND REVIEWERS ......................... ....... xiv 

9. TOXICOLOGICAL EFFECTS OF OZONE AND OTHER PHOTOCHEMICAL OXIDANTS ... 9-1 

9.1 INTRODUCTION ................................................. 9-1 

9.2 REGIONAL DOSIMETRY IN THE RESPIRATORY TRACT ... .:' .. 9-3 

9.2.1 Absorption in Experimental Animals ..:............:. 9-4 

9.2.1.1- Nasopharyngeal Absorption ..... .......... 9-4 

9.2.1.2 Lower Respiratory Tract Absorption ........ 9-5 

9.2.2 Ozone Dosimetry Models . .. . .. . ....................... . 9-6 

9.2.2.1 Modeling Nasal Uptake . ........:.... 9-6 

9.2.2.2 Lower Respiratory Tract Dosimetry 

Models ........ ....... .... ...........: 9-6 

9.2.3 Predictions of Lower Respiratory Tract Ozone 

Dosimetry Modeling ..... .. ........ .. .....:.. 9-10 

9.2.3.1 Illustration of Dosimetry"Simulations ..... 9-11 

9.2.3.2 Comparison of Simulations to Experimentai 

Data . .. .. . ..................... 9-14 

9.2.3.3 Uses of Predicted Dose .......:......."..:.. 9-15 

9.3 EFFECTS OF OZONE ON THE RESPIRATORY TRACT .................. 9-16 

9.3.1 Morphological Effects ........ .............. ... 9-16 

9.3.1.1 Sites Affected ... ... . .... . .:..:.. 9-16 

9.3.1.2 Sequence in which Sites are Affected 

as a Function of Concentration and 

Duration of Exposure . ..:.....:.... 9-41 

9:3.1.3 Structural Elements Affected .... ..... 9-42 

9.3.1.4 Considerations of Degree of Susceptibility to Morphological Changes .......... 9-46 

9.3.2 Pulmonary Function Effects ......................... 9-52 

9.3.2.1 Short-Term Exposure ...................... 9-52 

9.3.2.2 Long-Term Exposure ....................... 9-57 

9.3.2.3 Airway Reactivity ........................ 9-62 

9.3.3 Biochemically Detected.Effects ..................... 9-73 

9.3.3.1 Introduction ...... ..................... 9-73 

9.3.3.2 Antioxidant Metabolism ... . ... ....... 9-73 

9.3.3.3 Oxidative and Energy Metabolism .......... 9-88 

9.3.3.4 Monooxygenases ........ ....... ..... . 9-92 

9.3.3.5 Lactate Dehydrogenase andLyso somal 

Enzymes .... .. .......................:. 9-95 

9.3.3.6 Protein Synthesis ............... ........... 9-98 

9.3.3.7 Lipid Metabolism and Content ofthe 

Lung ..................................... 9-108 

v 
TABLE OF CONTENTS (continued) 

Page 

9.3.3.8 Lung Permeability ........................ 9-110 

9.3.3.9 Proposed Molecular Mechanisms of 

Effects ..... ... .. .................... 9-113 

9.3.4 Effects on Host Defense Mechanisms ................. 9-119 

9.3.4.1 Mucociliary Clearance .................... 9-120 

9.3.4.2 Alveolar Macrophages .............:....... 9-125 

9.3.4.3 Interaction with Infectious Agents ....... 9-132 

9.3.4.4 Immunology . ............................... 9-141 

9.3.5 Tolerance .......................................... 9-144 

9.4 EXTRAPULMONARY EFFECTS OF OZONE ............................ 9-153 

9.4.1 Central Nervous System and Behavioral Effects ...... 9-153 

9.4.2 Cardiovascular Effects .. .. .... ................. 9-158 

9.4.3 Hematological and Serum Chemistry Effects .......... 9-159 

9.4.3.1 Animal Studies - In Vivo Exposures ....... 9-159 

9.4.3.2 In Vitro Studies ....................... 9-166 

9.4.3.3 Changes in Serum .......................... 9-170 

9.4.3.4 Interspecies Variations .................. 9-172 

9.4.4 Reproductive and Teratogenic Effects ............... 9-173 

9.4.5 Chromosomal and Mutational Effects ................: 9-176 

9.4.5.1 Chromosomal Effects of Ozone ............. 9-176 

9.4.5.2 Mutational Effects of Ozone .............. 9-189 

9.4.6 Other Extrapulmonary Effects ....................... 9-190 

9.4.6.1 Liver ..................................... 9-190 

9.4.6.2 The Endocrine System ..................... 9-197 

9.4.6.3 Other Effects ............................ 9-204 

9.5 EFFECTS OF OTHER PHOTOCHEMICAL OXIDANTS .................... 9-205 

9.5.1 Peroxyacetyl Nitrate ............................... 9-205 

9.5.2 Hydrogen Peroxide .................................. 9-206 

9.5.3 Formic Acid ........................................ 9-209 

9.5.4 Complex Pollutant Mixtures ......................... 9-209 

9 .6 SUMMARY .................................................... 9-217 

9.6.1 Introduction ...... ......... .................... 9-217 

9.6.2 Regional Dosimetry in the Respiratory Tract ........ 9-218 

9.6.3 Effects of Ozone on the Respiratory Tract .......... 9-221 

9.6.3.1 Morphological Effects .................... 9-221 

9.6.3.2 Pulmonary Function ....................... 9-224 

9.6.3.3 Biochemical Effects ..................... 9-228 

9.6.3.4 Host Defense Mechanisms .................. 9-235 

9.6.3.5 Tolerance ................................ 9-243 

9.6.4 Extrapulmonary Effects of Ozone .................... 9-244 

9.6.4.1 Central Nervous System and Behavioral 

Effects .................................. 9-245 

9.6.4.2 Cardiovascular Effects ................... 9-245 

9.6.4.3 Hematological and Serum Chemistry 

Effects .................................. 9-246 

vi 
TABLE OF CONTENTS (continued) 

9.6.4.4 Cytogenetic and Teratogenic Effects ...... 

9.6.4.5 Other Extrapulmonary Effects ............. 

9.6.5 Interaction of Ozone with Other Pollutants ......... 

9.6.6 Effects of Other Photochemical Oxidants ............ 

9.7 REFERENCES .................................................. 

APPENDIXA ................................................. 

Pa e 

9-247 

9-248 

9-249 

9-252 

9-257 

A-1 

vii 
LIST OF TABLES 

Table P age 

9-1 Morphological effects of ozone .................................. 9-17 

9-2 Effects of ozone on pulmonary function: short-term exposures ... 9-53 

9-3 Effects of ozone on pulmonary function: long-term exposures .... 9-58 

9-4 Effects of ozone on pulmonary funGtion: airway reactivity ...... 9-63 

9-5 Changes in the lung antioxidant metabolism and oxygen 

consumption by ozone ............................................ 9-75 

9-6 Monooxygenases .................................................. 9-93 

9-7 Lactate dehydrogenase and lysosomal enzymes ..................... 9-96 

9-8 Effects of ozone on lung protein synthesis ..................... 9-99 

9-9 Effects of ozone exposure on lipid metabolism and content of 

the lung.......... ........... .. .............:............... 9-109 

9-10 Effects of ozone onlung permeability ....... ... ............ 9-111 

9-11 Effects of ozone on host defense mechanisms: deposition and 

clearance ... ... ... . ....... ............................ 

~ 

9-122 

9-12 Effects of ~ ozone onho s t defense mechanisms: macrophage 

9-126 

9-13 

alterations ........ ............................................. 

Effects of ozone onhost defense mechanisms: interactions 

with infectious agents............. .,... . ....... . ................ 9-135 

9-14 Effects of ozone on host defense mechanisms: mixtures .......... 9-138 

9-15 Effects of ozone on host defense mechanisms: immunology ......... 9-142 

9-16 Tolerance to ozone ....... ... ... ............................ 9-147 

9-17 Central nervous system and behavioral effects of ozone .......... 9-154 

9-18 Hematology: animal--in vivo exposure ........................... 9-160 

9-19 Hematology: animal--in vitro exposure .......................... 9-167 

9-20 Hematology: human--in vitro exposure ........................... 9-168 

9-21 Reproductive and teratogen c effects of ozone ................... 9-175 

9-22 Chromosomal effects from in vitroexposure to high ozone 

concentrations .... .... .............. ............ ......... 9-177 

9-23 Chromosomal effects from ozone concentrations at or below 

1960 Ng/m3 (1 ppm) ............................................. 9-179 

9-24 Mutational effects of ozone ..................................... 9-184 

9-25 Effects of ozone on the liver ................ ....... ....... 9-191 

9-26 Effects of ozone on the endocrine system, gastrointestinal 

tract, and urine ................................................ 9-198 

9-27 Effects of complex pollutant mixtures ......... ....... ... .... 9-211 

9-28 Summary Table: morphological effects of ozone in experimental 

animals .... ........... ........... ...................... 9-226 

9-29 Summary Table: effects on pulmonary function of short-term 

exposures to ozone in experimental animals ...................... 9-230 

9-30 Summary Table: effects on pulmonary function of long-term 

exposures to ozone in experimental animals ...................... 9-232 

9-31 Summary Table: biochemical changes in experimental animals 

exposed to ozone ................................................ 9-237 

9-32 Summary Table: effects of ozone on host defense mechanisms in 

experimental animals ......................... ....... ... ... 9-242 

9-33 Summary Table: extrapulmonary effects of ozone in experimental 

animals ..... .. ... ..... .. .....:...... .. ... .. ............:.. . ..... 9-251 

9-34 Summary Table: interaction of ozone with other pollutants in 

experimental animals ............................................ 9-254 

viii 
LIST OF FIGURFS 

Figure Page 

9-1 Predicted tissue dose for several tracheal 0 3 concentrations 

for rabbit and guinea pig ...................................... 9-12 

9-2 Tissue dose versus ozone for rabbit and guinea pig and tissue 

dose versus airway qeneration ~ for human. Tracheal 0 3 concentration is 500 pglm (0.26 ppm) ................................ 9-13 

9-3 Intracellular compounds active in antioxidant metabolism of 

thelung ....................................................... 9-73 

9-4 Summary of morphological effects in experimental animals 

exposed to ozone ............................................... 9-225 

9-5 Summary of effects of short-term ozone exposures on pulmonary 

function in experimental animals ............................... _ 9-229 

9-6 Summary of effects of long-term ozone exposures on pulmonary 

function in experimental animals ............................... 9-231 

9-7 Summary of biochemical changes in experimental animals 

exposed to ozone ............................................... 9-236 

9-8 Summary of effects of ozone on host defense mechanisms in 

experimental animals ........................................... 9-241 

9-9 Summary of extrapuimonary effects of ozone in experimental 

animals................................. ..................... 9-250 

9-10 Summary of effects in experimental animals ~ exposed to ozone 

combined with other pollutants ................................. 9-253 

ix 
LIST OF ABBREVIATIONS 

A-V Atrioventricular 

ACh Acetylcholine. 

AChE Acetylcholinesterase 

AM Alveolar macrophage 

AMP Adenosine monophosphate 

ATP Adenosine triphosphate 

ATPS ATPS condition (ambient temperature and pressure, 

saturated with water vapor) 

BTPS BTPS conditions (body temperature, barometric 

pressure, and saturated with water vapor) 

CC Closing capacity 

Cdyn Dynamic lung compliance 

CHEM Gas-phase chemiluminescence 

CL Lung compliance 

CLst Static lung compliance 

CMP Cytidine monophosphate 

CNS Central nervous system 

CO Carbon monoxide 

COHb Carboxyhemoglobin 

COLD Chronic obstructive lung disease 

COMT Catechol-o-methyl-transferase 

CO2 Carbon dioxide 

CPK Creatine phosphokinase 

CV Closing volume 

DL Diffusing capacity of the lungs 

D LCO Carbon monoxide diffusing capacity of the lungs 

DNA Deoxyribonucleic acid 

E Elastance 

ECG, EKG Electrocardiogram 

EEG Electroencephalogram 

ERV Expi'ratory reserve volume 

FEFinax The maximal forced expiratory flow achieved 

during an FVC test 

x 
LIST OF ABBREVIATIONS (continued) 

FEF Forced expiratory flow 

FEF200-1200 Mean forced expiratory flow between 200 ml and 

1200 ml of the FVC [formerly called the maximum 

expiratory flow rate (MEFR)]. 

FEF Mean forced expiratory flow during the middle 25-75% half of the FVC [formerly called the maximum 

mid-expiratory flow rate (MMFR)]. 

FEF75% 

FEV 

FIVC 

fR 

FRC 

FVC 

G 

G-6-PD 

Gaw 

GMP 

GS-CHEM 

GSH 

GSSG 

Hb 

Hct 

HO• 

H 20 

IC 

IRV 

IVC 

K 

0 

LDH 

LD50 

LM 

LPS 

Instanteous forced expiratory flow after 75% of 

the FVC has been exhaled. 

Forced expiratory volume 

Forced inspiratory vital capacity 

Respiratory frequency. 

Functional residual capacity 

Forced vital capacity 

Conductance 

Glucose-6-phosphate dehydrogenase 

Airway conductance 

Guanosine monophosphate 

Gas-solid chemiluminescence 

Glutathione 

Glutathione disulfide 

HemoglobinHematocrit 

Hydroxy radical 

Water 

Inspiratory capacity 

Inspiratory reserve volume 

Inspiratory vital capacity 

Average mucous production rate per unit area 

Lactate deyhydrogenase 

Lethal dose (50 percent) 

Light microscopy 

Lipopolysaccharide 

xi 

0 
LIST OF ABBREVIATIONS (continued) 

MAO Monamine oxidase 

MAST KI-coulometric (Mast meter) 

max VE Maximum ventilation 

max V02 Maximal aerobic capacity 

MBC Maximum breathing capacity 

MEFR Maximum expiratory flow rate 

MEFV Maximum expiratory flow-volume curve 

MetHb Methemoglobin 

MMFR or MMEF Maximum mid-expiratory flow rate 

MNNG N-methyl-N'-nitrosoguanidine 

MPO Myeloperoxidase 

MVV Maximum voluntary ventilation 

NBKI Neutral buffered potassium iodide 

(NH4 ) 2SO4 Ammonium sulfate 

NO2 Nitrogen dioxide 

NPSH Non-protein sulfhydryls 

02 Oxygen • 

02- Oxygen radical 

03 Ozone 

P(A-a)02 Alveolar-arterial oxygen pressure difference 

PABA Para-aminobenzoic acid 

PACO2 Alveolar partial pressure of carbon dioxide 

PaCO2 Arterial partial pressure of carbon dioxide 

PAN Peroxyacetyl nitrate 

PA02 Alveolar partial pressure of oxygen 

Pa02 Arterial partial pressure of oxygen 

PEF Peak expiratory flow 

PEFV Partial expiratory flow-volume curve 

PG Prostaglandin 

pHa Arterial pH 

PHA Phytohemagglutinin 

P L Transpulmonary pressure 

PMN Po7ymorphonuclear leukocyte 

PPD Purified protein derivative 

xii 
LIST OF ABBREVIATIONS (continued) 

P st Static transpuimonary pressure 

PUFA Polyunsaturated fatty acid 

R Resistance to flow 

Raw Airway resistance 

RBCs , Red blood cells 

Rcoll Collateral resistance 

RL Tota'l pulmonary resistance 

RQ, R Respiratory quotient 

Rti 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 

Sa2 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 

VA Alveolar ventilation 

1/A/Q Ventilation/perfusion ratio 

xiii 
LIST OF ABBREVIATIONS (continued) 

VC Vital capacity 

VCO2 Carbon dioxide production 

VD Physiological dead space 

VD Dead-space ventilation 

VD anat Anatomical dead space 

VE Minute ventilation; expired volume per minute 

VI Inspired volume per minute 

VL Lung volume 

Vmax Maximum expiratory flow 

. 

V02 Oxygen uptake 

V021 02 Oxygen consumption 

125I Radioactive iodine 

5-HT 5-hydroxytryptamine 

6-P-GD 6-phosphogluconate dehydrogenase 

MEASUREMENT ABBREVIATIONS 

g gram 

hr/day hours per day 

kg kilogram 

kg-m/min kilogram-meter/min 

L/min liters/min 

ppm parts per million 

mg/kg milligrams per kilogram 

mg/m3 milligrams per cubic meter 

min minute 

ml milliliter 

mm millimeter 

pg/m3 micrograms per cubic meter 

pm micrometers 

NM micromolar 

sec second 

xiv 
AUTHORS, CONTRIBUTORS, AND REVIEWERS 

Chapter 9: ToXicological Effects-of Ozone and Other Photochemical Oxidants 

Principal Authors 

Dr. Donald E. Gardner Mr. James A. Raub 

Northrop Services, Inc. Environmental Criteria and 

Environmental Sciences Assessment Office 

P.O. Box 12313 MD-52 

Research Triangle Park, NC 27709 U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

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 

Laborat.ory of Environmental Toxicology 

and Pharmacology 

Duke University Medical Center 

P.O. Box 3813 

Durham, NC 27710 

Dr. John H. Overton, Jr. 

Health Effects Research Laboratory 

MD-82 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

Dr. Stephen C. Strom 

Department of Radiology 

Duke University Medical Center 

P.O. Box 3808 

Durham, NC 27710 

Dr. Walter S. Tyler 

Department of Anatomy 

School of Veterinary Medicine 

University of California, 

Davis, CA 95616 

xv 
Contributina Authors 

Mr. James R. Kawecki 

TRC Environmental Consultants, Inc. 

701 W. Broad Street 

Falls Church, VA 22046 

Dr. Frederick J. Miller 

Health Effects Research Laboratory 

MD-82 

U.S. Environmental Protection Agency 

Research Triangle Park, NC. 27711 

Ms. Elaine D. Smolko 

Laboratory of Environmental Toxicology 

and Pharmacology 

Duke University Medical Center 

P.O. Box 3813 

Durham, NC 27710 

Dr. Jeffrey L. Tepper 

Northrop Services, Inc. 

Environmental Sciences 

P.O. Box 12313 

Research Triangle Park, NC 27709 

xvi 
Authors also reviewed individual sections of the chapter. The following additional 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 Hospita•1 

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. 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. George L. Carlo Dr. Steven M. Horvath 

Dow Chemical, U.S.A. Institute of Environmental Stress 

1803 Building, U.S. Medical University of California 

Midland, MI 48640 Santa Barbara, CA 93106 

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. George J. Jakab 

Department of Environmental 

Health Sciences 

Johns Hopkins Schooi of Hygiene 

and - Publ-i-c—Heal th 

615 N. Woife St. 

Baltimore, MD 21205 

Dr. Robert J..Kavlock 

Health Effects Research Laboratory 

MD-67 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

xvii 
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, MD 21205 

Ms. Beverly E. Tilton 

Environmental Criteria and Assessment 

Office 

MD-52 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

Dr.• Jaroslav J. Vostal 

Executive Department 

General Motors Research Laboratories 

Warren, MI 48090 

xviii 
SCIENCE ADVISORY BOARD 

CLEAN AIR SCIENTIFIC ADVISORY COMMITTEE 

The substance of this document was reviewed by the Clean Air Scientific 

Advisory Committee of the Science Advisory Board in public sessions. 

SUBCOMMITTEE ON OZONE 

Chairman 

Dr. Morton Lippmann 

Professor 

Department of Environmental Medicine 

New York University Medical Center 

Tuxedo, New York 10987 

Members 

Dr. Mary 0. Amdur 

Senior Research Scientist 

Energy Laboratory 

Massachusetts Institute of Technology 

Cambridge, Massachusetts 02139 

Dr. Eileen G. Brennan 

Professor 

Department of Plant Pathology 

Martin Hall, Room 213, Lipman Drive 

Cook College-NJAES 

Rutgers University 

New Brunswick, New Jersey 08903 

Dr. Edward D. Crandall 

Professor of Medicine 

School of Medicine 

Cornell University 

New York, New York 10021 

Dr. James D. Crapo 

Associate Professor of Medicine 

Chief, Division of Allergy, Critical 

Care and Respiratory Medicine 

Duke University Medical Center 

Durham, North Carolina 27710 

Dr. Robert Frank 

Professor of Environmental Health 

Sciences 

Johns Hopkins School of Hygiene 

and Public Health 

615 N. Wblfe Street 

Baltimore, Maryland 21205 

Professor A. Myrick Freeman II 

Department of Economics 

Bowdoin College 

Brunswick, Maine 04011 

Dr. Ronald J. Hall 

Senior Research Scientist and Leader 

Aquatic and Terrestrial Ecosystems 

Section 

Ontario Ministry of the Environment 

Dorset Research Center 

Dorset, Ontario POAlEO 

Dr. Jay S. Jacobson 

Plant Physiologist 

Boyce Thompson Institute 

Tower Road 

Ithaca, New York 14853 

xix 
Dr. Warren B. Johnson 

Director, Atmospheric Science Center 

SRI International 

333 Ravenswood Avenue 

Menlo Park, California 94025 

Dr. Jane Q. Koenig 

Research Associate Professor 

Department of Environmental Health 

University of Washington 

Seattle, Washington 98195 

Dr. Paul Kotin 

Adjunct Professor of Pathology 

University of Colorado Medical School 

4505 S. Yosemite, #339 

Denver, Colorado. 80237 

Dr. Timothy Larson 

Associate Professor 

Environmental Engineering and 

Science Program 

Department of Civil Engineering 

Unfiversity of Washington 

Seattle, Washington 98195 

Professor M. Granger Morgan 

Head, Department of Engineering 

and Public Policy 

Carnegie-Mellon University 

Pittsburgh, Pennsylvania 15253 

Dr. D. Warner North 

Principal 

Decision Focus Inc., Los Altos 

Office Center, Suite 200 

4984 E1 Camino Real 

Los Altos, California 94022 

Dr. Robert D. Rowe 

Uice President, Environmental and 

Resource Economics 

Energy and Resources Consultants, Inc. 

207 Canyon Boulevard 

Boulder, Colorado 80302 

Dr. George Taylor 

Environmental Sciences Division 

P. 0. Box X 

Oak Ridge National Laboratory 

Oak Ridge, Tennessee 37831 

Dr. Michael Treshow 

.Professor 

Department of Biology 

University of Utah 

. Salt Lake City, Utah 84112 

Dr. Mark J. Utell 

Co-Director, Pulmonary Disease Unit 

Associate Professor of Medicine and 

Toxicology in Radiation Biology 

and Biophysacs 

University of Rochester Medical 

Center 

Rochester, New York 14642 

Dr. James H. Ware 

Associate Professor 

Harvard School of Public Health 

Department of Biostatistics 

677 Huntington Avenue 

Boston Massachusetts 02115 

Dr. Jerry Wesolowski 

Air and Industrial Hygiene Laboratory 

California Department of Health 

2151 Berkeley Way 

Berkeley, California 94704 

Dr. James L. Whittenberger 

Director, University of California 

Southern Occupational Health Center 

Professor and Chair, Department of 

Community and Environmental Medicine 

California College of Medicine 

University of California - Irvine 

19772 MacArthur Boulevard 

Irvine, California 92717 

Dr. George T. Wolff 

Senior Staff Research Scientist 

General Motors Research Labs 

Environmental Science Department 

Warren, Michigan 48090 

xx 
PROJECT TEAM FOR DEVELOPMENT 

OF 

Air Quality Criteria for Ozone and Other Photochemical Oxidants 

Ms. Beverly E. Tilton, Project Manager 

and Coordinator for Chapters 1 through 5, Volumes I and II 

Environmental Criteria and Assessment Office (MD-52) 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

Mr. Norman E. Childs 

Environmental Criteria and Assessment Office (MD-52) 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

Dr. J.N.B. Garner 

Coordinator for Chapters 7 and 8, Volume III 

Environmental Criteria and Assessment Office (MD-52) 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

Mr. Thomas B. McMullen 

Environmental Criteria and Assessment Office (MD-52) 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

Mr. James A. Raub 

Coordinator for Chapters 10 through 13, Volumes IV and V 

Environmental Criteria and Assessment Office (MD-52) 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 

Dr. David T. Tingey 

Coordinator for Chapter 7, Volume III 

Environmental Research Laboratory 

U.S. Environmental Protection Agency 

200 S.W. 35th Street 

Corvallis, OR 97330 

xxi 

9. TOXICOLOGICAL EFFECTS OF OZONE AND OTHER PHOTOCHEMICAL OXIDANTS 

9.1 INTRODUCTION 

This chapter discusses the effects of ozone on experimental animals. 

Carefully controlled studies of the effects of ozone on animals are particularly important in elucidating subtle effects not easily found in man through 

epidemiological studies and in identifying chronic toxicity not apparent from 

short-term controlled human exposures. Animal studies allow investigations 

into the effects of ozone exposure over a lifetime, uncomplicated by the 

presence of other pollutants. In the animal experiments presented here, a 

broad range of ozone concentrations has been studied but emphasis has been . 

placed on recent studies at 1960 pg/m 3 (1 ppm) of ozone or less. Higher 

concentrations 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. 

A majority of the literature describes the effects of ozone on the respiratory tract, but extrarespiratory system effects have now been noted and are 

documented in this chapter. Most of the studies utilize invasive methods that 

require sacrifice of the animals on completion of the experiment; thus, the 

studies would be impossible to perform in human subjects. Noninvasive methods 

of examining most of these endpoints are not readily available. 

Emphasis has been placed on the more recent literature published after 

the prior criteria document (U.S. Environmental Protection Agency, 1978); 

however, older literature has been reviewed .again in this chapter. As more 

information on the toxicity of ozone becomes available, a better understanding 

of earlier studies is possible and a more detailed and comprehensive picture 

of ozone toxicity is emerging. The literature used in developing this chapter 

i s. .set _out .i n a seri es of tab.l es . Not al l of,_ the . l i terature ci ted i n the 

tables appears in the detailed discussion of the text, but citations are 

provided to give the reader more details on the background from which the text 

is drawn. 

In selecting studies for consideration, a detailed review of each paper 

has"been completed. This review included an evaluation of the exposure methods; 

the analytical method used to determine the chamber ozone concentration; the 

9-1 
calibration of the ozone monitoring equipment and the analytical methods used 

(wherever possible); the species, strain, age and physical characteristics of 

the animals; the technique used for obtaining samples; and the appropriateness 

of the technique used to measure the effect. In interpreting the results, the 

number of animals used, the appropriateness and results of the statistical 

analysis, the degree to which the results conform with past studies, and the 

appropriateness of the interpretation of the results are considered. No 

additional statistical analysis beyond that reported by the author has been 

undertaken. Unless otherwise stated, all statements of effects in the text 

are statistically significant at p< 0.05. Many reports, especially in the 

older literature, do not present sufficient information to permit the assessment 

described above. However, should a particular study not meet all of these 

criteria, but provide reasonable data for consideration, a disclaimer is 

provided in the text and/or tables. 

In this chapter, a discussion of the regional respiratory dosimetry of 

ozone in common laboratory animal species is presented and compared to human 

dosimetry. Morphological alterations of the lungs of animals exposed to 0 3 

are described, followed by the effects of ozone on the pulmonary function of 

animals. The biochemical alterations observed in the ozone-exposed animals 

are then related to morphological changes and to potential mechanisms of toxicity and biochemical defense mechanisms. The influence of dietary factors, 

such as vitamins E and C, in animals is discussed with consideration of potential roles in humans. It should be stressed, however, that no evidence for 

complete protection against ozone toxicity has been found for any factor, 

dietary or.therapeutic. The effects of ozone on the defense mechanisms of the 

lung against respiratory infectious agents are discussed using the infectivity 

model system and effects on alveolar macrophages as examples of experimental 

evidence. This section is, followed by a discussion of ozone tolerance in 

animals. Last, the effects of ozone on -a - number of extrarespiratory organ 

systems are discussed to provide insight into potential effects of ozone 

inhalation in the respiratory system beyond those now well documented. 

A brief discussion of the available literature on the effects of other 

oxidants likely to occur in polluted air as a result of photochemical reactions 

or other sources of pollution•is presented. Peroxyacetyl nitrate, hydrogen 

peroxide, and automobile exhaust are the principal pollutants studied in these 

experiments. This section is short because of the general lack of information 

9-2 
in this area, but its brevity does not necessarily reflect a generai lack of 

importance. 

A summary is provided for all of the sections of the chapter to set the 

tone for a clearer understanding of the effects of ozone on animals. The 

major emphasis of this chapter is to provide evidence for the toxicity of 

ozone which can not, ethically or practically, be obtained from the study of 

human subjects. The overali health effects of ozone can be judged from three 

types of studies: animal exposures; controlled human exposures, and epidemiological studies of adventitious human exposures. No single method alone is 

adequate for an informed judgment, but together they provi.dea reasonable 

estimate of the human health effects of ozone on man. 

9.2 REGIONAL DOSIMETRY IN THE RESPIRATORY TRACT 

A major goal of environmental toxicological studies on animals is the 

eventual quantitative extrapolation of results to man. One type of information 

necessary to obtain this goal is dosimetry, which is the specification of the 

quantity of inhaled material, in this instance ozone (0 3 ), 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 i.s 

sufficiently advanced for discussion here. Until. both elements are'advanced, 

quantitative extrapolation cannot be conducted. 

At present, there are two approaches to dos.imetry, experimental and 

deterministic mathematical modeling. Animal experiments have been carried out 

to obtain direct measurements of 0 3 absorption; however, experimentally obtaining 

local lower respiratory tract (tracheobronchiai 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 0 3 are 

general to all mammals, a model that uses appropriate species andlor diseasespecific anatomicai and ventilatory parameters can be used to describe 0 3 absorption 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 additaonal 

9-3 

~ 
research, and to test our understanding of 0 3 absorption in the respiratory 

tract. 

9.2.1 Absorption in Experimental Animals 

There have been very few experiments in which measurements of the regional 

uptake of 03 or other reactive gases have been determined. Of the several 

results published, only one is concerned with the uptake of 0 3 in the lower 

respiratory tract; the others deal with nasopharyngeal uptake. 

9.2.1.1 Nasopharyngeal Absorption. Nasopharyngeal removal of 0 3 lessens 

the quantity of 03 delivered to the lung and must be accounted for when 

estimating the 03 dose responsible for observed pulmonary effects. Vaughan 

et al. (1969) exposed the isolated upper airways of beagle dogs to 0 3 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 difference 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 03 concentration. For example, 

with nose breathing and an 0 3 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 percent to 60 percent. Their data, however, indicate that the tracheal concentration increases with increased nose or mouth concentrations. Thev also demonstrated that the concentration of 03 reaching the.trachea depends heavi-ly 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 03 in the nasopharynx of acutely 

and chronically exposed dogs. Beagles chronically exposed (18 months) to 1 to 

S ppm of 03 under various daily exposure regimes had significantly higher tracheal concentrations of 0 3 than animals tested after 1 day of exposure to corresponding regimes. Moorman et al. (1973) suggested that the differences were 

9-4 
due to physiochemical alterations of the mucosal lining in the chronically exposed beagles. When dogs were exposed for 18 months to 1 ppm for 8 hr a day, 

they had significantly lower tracheal 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 3 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 03 concentrations significantly greater than 

those observed with acute exposure. 

Nasopharyngeal removal of 03 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 3 concentration in these two species was markedly similar at a given inhaled 

concentration and was iinearly 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). Caution needs to be exercised in applying the above results to 

relate ambient and tracheal concentrations of 0 3 since the effects of nasopharyngeal volume and the cyclical nature of breathing are not taken into 

account. For example, if the tidal volume was less than the nasopharyngeal 

volume and convection was the only process of axial transport, then no 0 3 

would be delivered to the tracheal opening, regardless of the percent uptake 

measured for unidirectional flow. 

9.2.1.2 Lower Respiratory Tract Absorption. Morphoiogical studies on animals 

suggest that 0 3 is absorbed along the entire respiratory tract; it penetrates 

further into the peripheral nonciliated airways as inhaled 0 3 concent'rations 

increase (Dungworth et al., 1975b). Lesions were found consistently in the 

trachea and proximal bronchi and between the junction of the conducting airways 

and the gaseous exchange area; in both regions, the severity of damage 'decreases 

distally. In addition, several studies have reported the most severe or 

prominent lesions to be in the centriacinar region (see section 9.3). 

No experiments determining 0 3 tissue dose at the generational or regional 

level have been reported; however, there is one experiment concerned with the 

uptake of 03 by the lower respiratory tract. Removal of 0 3 from inspired air 

9-5 
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 

08 concentrati ons studi ed, 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/ 

mi n. Thi s estimate of uptake appl i es to the 1 ower respi ratory tract as a 

whole; it does not describe uptake of 0 3 by individual regions or generations. 

9.2.2Ozone Dosimetry Models 

9.2.2.1Modeling Nasal Uptake . LaBelle et al. (1955) considered the absorption of gases in the nasal passages to be similar to absorption on wetted 

surfaces of distillation equipment and scrubbing towers and applied the theory 

and models of these devices to the nasal passages of rats. By associating 

biological parameters of rats with the chemical engineering device parameters 

of the model, they calculated the percent of penetration of several gases to 

the lung. They concluded that Henry's law constant is the major variable in 

determining penetration. Based on these calculations, The National Academy of 

Sciences (National Research Council, 1977) concluded that the model predicts 

99 percent penetration for 0 3 . This is much more than that measured by Yokoyama 

and Frank (1972) or by Miller et al. (1979). Several possible reasons for the 

differences were discussed (National Research Council, 1977), but the major 

factor was considered to be that the model does not account for the reactions 

of 03 in the mucus and epithelial tissue. 

Aharonson et al. (1974) developed a model for use in analyzing data from 

experiments on the uptake of vapors by the nose. The model was based on the 

assumptions of quasi-steady-state flow, mass balance, and proportionality of 

flux of a trace gas at the air-mucus interface to the gas-phase partial 

pressure of the trace gas and a"local uptake coefficient" (Aharonson et al., 

1974). The model was applied to data from their own experiments , on the removal 

of acetone and ether in dog noses. They also applied the model_ to the 0 3 

uptake data of Yokoyama and Frank (1972) and concluded that the uptake coefficient (average mass transfer coefficient) for 0 3 , as well as for the other 

gases considered, increases with increasing air flow rate. 

9.2.2.2Lower Respiratory Tract Dosimetry Models . There are three models 

for which published results are available. The model of McJilton et al. 

(1972) has been discussed and simulation results for 0 3 absorption in each 

generation of the human lower respiratory tract are available (National Research 

•. 
Council 1977; Morgan and Frank, 1977). Two models have been developed by 

Miller and co-workers.. A detailed description,of the formulation of the first 

and earliest mathematical model of Miller and co-workers is found in Miller 

(1977) and major features are given in Miller et al. (1978b) and Miller (1979). 

Results, using this model, of simulations of the lower respiratory tract 

absorpti on of. 0 3 i n humans , rabbi ts , and gui nea pi gs are i n Mi 1 l er (1977, 

1979) and Miller et al. (1978b). The formulation of the second model of 

Miiler and co-workers, as well as results of simulating lower respiratory 

tract absorption of 03 in humans, appears in Miller et al. (1985). 

Because all of the above modeis were developed to simulate the local 

absorption of 03 , they have much in common. Thi.s is especially true with 

respect to the following areas: formulation of 03 transport in the airspaces 

or lumen of the airways, use of morphometric models of the lower respiratory 

tract, and inclusion of a liquid lining that coats the tissue walls of the 

airspaces or.lumen of the airways. 

In each model the descriptions of 0 3 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 3 by absorption at 

the gas-liquid in.terface. The use of a one-dimensional approximation has been 

very common in modeling the transport of gases such as 0 2 , N2 , etc.,'in the 

lower respiratory tract (see Scherer et al., 1972; Paiva, 1973; Chang and 

Farhi, 1973; Yu, 1975; Pack et al., 1977; Bowes et al., 1982). The.approximation is appropriate for 03 as well. 

The models of Miller and co-workers took into account effective axial 

dispersion in the airways by using an effective dispersion coefficient based 

on the results of Scherer et ai. (1975). McJilton's model did,not take this 

factor into account (Morgan and Frank, 1977). However, this may not be an 

important difference since Miller et al. (1985) report that.results are little 

affected by different values of the coefficient. Also, Pack et al. (1977) and 

Engel and Macklem (1977) reported results that indicate an insensitivity of 

airway concentrations to the effective dispersion coefficient. 

Airway or morphometric zone models such as those of , Weibel (1963) and 

Kliment (1973) were used to define the lengths, radii, surface areas, crosssectional areas, and volumes of the airways and air spaces of each generation 

or zone. The breathing pattern was assumed sinusoidal; however, dimensions 

were held constant throughout the breathing cycle. The phys.ical properties of 

9+-7 
the liquid lining were assumed to be those of water. The lining thickness 

depended on generation or zone, being thicker in the upper airways than in the 

1 ower. 

The fl ux of 0 3 from the 1 umen or ai rspaces to the 1 i qui d 1 i ni ng was 

defined in each model in terms of a mass-transfer coefficient. McJilton et 

al. (1972) made the assumption that radial mass transfer was controlled by the 

absorbing niedium and estimated the transfer coefficient from empirical data on 

the physical properties (not chemical) of the medium and of 0 3 (National 

Research Council, 1977). In the first model of Miller and co-workers 

(Miller, 1977; Miller et al., 1978b) the radial dependence of the luminal 0 3 

concentration was assumed to vary quadratically with the radius. From this 

formulation, the gas-phase mass transfer was determined. For their later 

model, the gas phase mass transfer coefficient was defined in terms of a 

Sherwood number. In both models the gas phase coefficient was combined with 

the mass transfer coefficient of the absorbing medium (which depended on the 

chemical and physical properties of the absorbing medium and of 0 3 ) to-obtain 

the overall transfer coefficient. However, Miller et al. (1985) conclude, 

based on the data available for the absorbing medium, that radial mass transfer 

is controlled by the medium, making specification of the gas phase mass transfer coefficient unnecessary. 

The main differences in the models are the mechanisms of absorption and 

their formulation. In the model of McJilton et al. (1972) and in the early 

model of Miller and co-workers (Miller, 1977; Miller et al., 1978b) there is 

only one compartment, the liquid lining, which can absorb unreacted 0 3 . In 

the later model of Miller et al. (1985) there are three absorbing compartments,. 

liquid lining, tissue, and capillary or blood (in the pulmonary region where 

the air-blood tissue barrier is very thin). Further, 0 3 is known to react 

chemically with constituents of the absorbing medium(s). This aspect, included 

by Miller and co-workers, was not included in McJilton's model. The inclusion 

resulted in significant differences between the tissue dose pattern curves in 

the tracheobronchial region predicted by the models. In addition, McJilton's 

model predicts a dose curve (equivalent to a tissue dose curve because of no 

mucous reactions) in the tracheobronchial region that has its maximum at the 

trachea and decreases distally to the thirteenth or fourteenth generation (see 

Figure 7-5 in National Research Council, 1977). By contrast, the models of 

Miller and co-workers predict the tissue dose to be a minimum at the trachea 

and to increase distally to the pulmonary region. 

. ; 
The concentration of ozone in tissue and at the liquid-tissue interface 

was assumed to be zero by McJilton et al. (1972) (National Research Council, 

1977; Morgan and Frank, 1977) and in the first model of Miller and co-workers 

(Miiler, 1977; Miller et al., 1978b). The interpretation was that this boundary 

condition means that 0 3 reacts (chemically) instantaneously when it reaches 

the tissue. Miller et al. (1978b) define the tissue dose as that quantity of 

03 per unit area reacting with or absorbed by the tissue at the liquid-tissue 

interface. 

The first model of Miller and co-workers took into account the reaction 

of 03 with the unsaturated fatty acids (UFA) and amino acids in the mucousserous lining. Reactions of 03 with. other components (such as carbohydrates) 

were not included in the model because of insufficient information (Miller, 

1977; Miller et al., 1978b). The 03-UFA and 03-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 tracheal mucous flow data, 

the surface area of the tracheobronchi.al region, the concentrations of the 

specific reactants known to react with 0 3 , and the'assumption that the production rate decreased distally (Miller, 1977; Milier et al., 1978b). 

Although the- instantaneous reaction scheme is a good preliminary approach 

to treating 03 reactions in the mucous-serous lining, its use is not completely 

justifiable. Second-order rate constants of 0 3 with some of the UFA present 

in mucus indicate that although they are large (Razumovskii and Zaikov, 1972), 

they are less than the diffusion-limited rates necessary for the instantaneous 

reaction scheme. Experimental evidence (Mudd et al., 1969) suggests that the 

reactions of 0 3 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 approach to chemical reactions used in the later modei (Miller et 

al., 1985) goes a long,way in addressing the above criticisms. The reaction 

of 03 with biochemical constituents is assumed to be bimolecular; however, the 

concentration of the constituents is considered to be large enough so as not 

to be depleted by the reactions. Hence, the model uses a pseudo first order 

reaction scheme in which the pseudo first order rate constant is the product 

. . 
of the bimolecular rate constant and the concentration of the biochemical 

constituents that react with 0 3 . 

For modeling purposes, Miller et al. (1985) consider that only the reaction 

of ozone with the UFA is important, using the 0 3-oleic acid rate constant of 

Razumovskii and Zaikov (1972) for the 0 3-UFA reaction. They point out that 

although 0 3 reacts with amino acids and other constituents, rate constants are 

not known and that Bailey (1978) estimates the reaction of 0 3 with UFA to be 

—103 times faster than the 03-amino acid reaction, justifying omitting amino. 

acids from consideration. 

9.2.3 Predictions of Lower Respiratory Tract Ozone Dosimetry Modeling 

The predictions of lower respiratory tract dosimetry models are reviewed 

by illustrating the results of simulations, by comparing predictions to experimental observations, and by describing uses for dosimetry models. 

The following discussion of modeling results of lower respiratory tract 

absorption is based mainly on simulations using the first model of Miller and 

co-workers (Miller, 1977; Miller et al., 1978b). This is because the model 

includes the important effects of 0 3 reactions in the mucous-serous lining and 

because simulations of 03 absorption in laboratory animals are available. 

Simulations of 0 3 absorption in different animals can be carried out by 

modifying input parameters of the computer program that solves the mathematical 

equations. These input parameters, which characterize an animal, include the 

number and dimensions of the airways, tidal volume, length of time of one 

breath,°etc. The airway and alveolar dimensions of Weibel (1963) were used 

for the simulation of 0 3 uptake in humans. For the rabbit and guinea pig, 

Miller and co-workers used the morphometric zone models of Kliment (1973). 

The zone model is a less detailed model than the generationally based airway 

model of Weibel (1963) since more than one generation corresponds to a zone in 

an animal; they were used because they were the only complete (tracheobronchial 

and pulmonary regions) "airway" models available at the time. However, Schreider 

and Hutchens (1980) criticize the guinea pig model of Kliment (1972, 1973) as 

having a lung volume that is too low, suggesting the possibility of incomplete 

casts. Since the same method also was used by Kliment (1972, 1973) for the 

rabbit, this criticism may also apply to this model. 

To illustrate simulation results, two aspects of the simulations by 

Miller and co-workers are considered: (1) the effect of various tracheal 

9-10 
concentrations on the tissue dose pattern (tissue dose as a function of zone 

or generation) in guinea pigs and rabbits; and (2) the similarity between the 

dose patterns of guinea pigs, rabbits, and humans. 

9.2.3.1 Illustration of Dosimetry Simulations. Figure 9-1 is a set of 

plots of the tissue dose , for one breath versus zone for various tracheal 03 

concentrations for'the rabbit and the guinea pig. A11 curves have the same 

general characteristics. Independent of the inhaled concentration, the model 

predicts that the first surfactant-lined zone (first non-mucous-lined or first 

zone in the pulmonary region), zone 6, receives the maximal dose of 0 3 . 

Although the model predicts uptake of 0 3 by respiratory tissue (zones 6, 7, 

and 8) for all tracheal concentrations studied (62.5 to .4000 pg/m 3 ), the 

penetration of 03 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 9-1 for the tracheal 03 concentration of 1000 pg/m 3 , no 

03 reaches the airway tissue of the rabbit until zone 3, whereas 0 3 is predicted 

to penetrate to the tissue in guinea pig airways in all zones. However, at 

the two lowest tracheal concentrations plotted, 250 and 62.5 pg/m 3 , no penetration occurs until zones 4 and 6, respectively, for both animals. The dependence 

of penetration on tracheal 03 concentration is a result of the instantaneous 

reaction scheme used to describe chemical reactions. Penetration does occu.r 

in the simulation of uptake in humans using the newer model of Miller and 

co-workers (1985), as depicted in Figure 9-2, and can be expected to occur in 

future animal simulations. 

The similarity of the predicted dose patterns in rabbits and guinea pigs 

extends to the simulation of 0 3 uptake in humans. Figure 9-2 compares the 

tissue dose for the three species for a tracheal concentration of 500 pg/m 3 

(0.26 ppm) using results of the first model for the three species and the 

newer model resul-ts for- man. In Fi gure-- 9-2-, -the--guinea pi g and rabbi t t-i ssue 

doses are plotted in the form of a histogram to allow a comparison to human 

dosimetry data that are expressed as a function of airway generation. 

For the earlier model, the dose patterns of the three•species peak at the 

fi rst surfactant-1 i.ned zone (6) or i n generati on 17 (whi ch i s i n zone 6). 

Also, 03 penetrates to the tissue everywhere in the pulmonary region (zones > 

5 and generations > 16); however, 0 3 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. 

9-11 
r 

a 

d 

.n 

I: 

U 

~ 

ZL 

O 

w 

O 

w 

f/! 

O 

0 

w 

D 

U) U) 

z 

10•5 

10°s 

10°7 

10•4 

TRACHEAL 

~ O, CONC. 

μg/m3 PPm 

® ~ ■ 4000 2.041 

~ •1000 0.510 

.O®--■ A 1 • 250 0.128 

I ♦ 62.5 0.032 - 

%A % / 1 1 

~ 1 1 

~ ~~ • 1 1 

~ ® 

1 

. ~ ~ `` ' • 

1 ~ 1 

1 

, 1 ~ 

/ 

1 . . ~ 

10°8 

0 1 2 3 45 6 7 8 MODEL ZONE 

TRACHEA BRONCHI—ObI BRON- 1 A.D.1 A.S. MORPHOMETRIC ZONE 

CHIOLES 

Figure 9-1. Predicted tissue dose for several tracheal .0 3 

concentrations for rabbit (---) and guinea pig (- ). 

See text for details. (A.D. = alveolar duct; A.S. = alveolar 

sac). 

Source: Adapted from Miller et al. (1978b). 

9-12 
10-5 

r ~ 

ca 

a~ ~ 

M 

N 

E 

U 

~ 

Z 

n 

0 

LL 

0 

w 

~ 

n 

C 

w 

:n 

~n 

~ 

10-s 

10-7 

10-$ AIRWAY 

0 2 4 6 8 10 12 14 16 . 18 20 22 GENERATIOl11 

0 1 2 ~0-- 3 --~-- 4 —No 

1 

 00-- 5 6--~~t– 7---wi 8 ZO N E 

TRACHEOBRONCHIAL* –w-PULMONARY 

Figure 9-2. Tissue dose versus zone for rabbit ( p) and guinea pig (m); 

and tissue dose versus airway generation for human ( , earlier 

model; ---, newer model). Tracheal 0 3 concentration is 500 μg/m 3 

(0.26 ppm). See text for details. 

Source: Guinea pig, rabbit and earlier human simulations adapted from 

Miller et al. (1978b); newer model results for human adapted from 

Miller et al. (1985). 

9-13 
A11 of the earlier simulations presented by Miller and co-workers (Miller, 

1977; Miller et al., 1978b; Miller, 1979) share the following characteristics: 

(1) the maximal tissue dose occurs in the first surfactant-lined zone or 

generation; (2) 0 3 penetrates to tissue everywhere in the pulmonary region 

(zones >5), decreasing distally from the maximum; and (3) the onset of 0 3 

penetration to mucous-lined tissue, as well as dose in general, depends on 

tracheal 03 concentration, animal species, and the breathing pattern. These 

general characteristics of tissue dose pattern are independent of the two 

airway models used. 

Results using the newer model to simulate tissue dose in humans are also 

illustrated in Figure 9-2. Comparison of dose values for the two human 

simulations show_the most notable differences in the conducting airways (generations 0 to 16). The new and old results are similar in that both models predict 

relatively low doses in the upper airways, a maximum in the first pulmonary 

generation, and then a rapid decline in dose distally. There is no reason to 

assume that these features would be missing in simulation results for laboratory 

animals using the newer model. 

9.2.3.2Comparison of Simulations to Experimental Data . There are no quantitative experimental observations with which to compare the results of modeling 

the local uptake of 0 3 in the lower respiratory tract. Yokoyama and Frank 

(1972) observed 80 to 87 percent uptake of 0 3 by the isolated 1ower respiratory 

tract of dogs. With the earlier model, Miller et al. (1978b) predict a 47 

percent uptake for humans; the newer model predicts 89 percent (Mi11er et al., 

1985). Because of differences between the two species, comparing the experimental and simulated results is most likely inappropriate. 

Morphological studies in animals report damage throughout.the lower 

respiratory tract. Major damage, and in some cases the most severe damage, is 

observed to occur at the junction between the conducting airways and the gas 

exchange region, and to decrease distally (see Section 9.3.1.1). For the 

animals simulated by Miller and co-workers, the maximal tissue dose of 0 3 is 

predicted to occur at this junction, with the dose curve decreasing rapidly 

for more distal regions. Thus, in the pulmonary region, the model results are 

in qualitative agreement with experimental observations. 

Damage is also observed in the trachea and bronchi of animals (see Sections 

9.3.1.1.1.2 and 9.3.1.1.1.3). In the animals modeled, the early model of 

Miller and co-workers either predicts significantly less tissue dose in the 

9-14 

9 
upper airways compared to the dose in the first zone of the puimonary region 

or it predicts no penetration to the tissue in the upper portion of the conducting airways (see Figure 9-2). Based on simulation results using the newer 

model for humans (Miller et al., 1985), one can"infer that this model will 

predict non-zero tissue doses in the upper airways of animals, but that these 

doses also will be significantly less than predicted for the centriacinar 

airways. The observations of damaged upper and lower respiratory tract airways 

in the same animal and the predictions of significantly different tissue doses. 

in the two regions appear inconsistent. However, much of the reported damage 

in the trachea and bronchi is associated with the cilia of ciliated cells, 

which in current model formulations are not part of the tissue. The cilia 

extend into the hypophase (perciliary) portion of the mucous-serous layer, and 

the dosimetry models do not distinguish the cilia of the ciliated cells as a 

separate component of this layer. Thus, relatively low predicted tissue dose 

should not be interpreted as predicting no damage to cilia. Likewise, the 

frequent reporting of cilia being damaged following 0 3 exposure should not be 

interpreted necessarily as an indication of 0 3 tissue dose since the model 

definition of tissue does not currently include cilia. The inclusion of a 

"cilia compartment" in future dosimetry models may be helpful. There are also 

other factors that complicate our understandirig of ozone toxicity, such as the 

possibility of 0 3 reaction products being toxic or differences in cell sensitivity that may prevent explanations of observed effects based on dosimetry 

modeling alone. 

9.2.3.3 Uses of Predicted Dose. Model-predicted doses can be used to estimate 

comparable exposur.e levels that produce the same dose in different species or 

different members of the same species for use in comparing toxicological data. 

One can simulate tissue dose for several species for the same time for a range 

of tracheal 03 concentrations. The doses for a specific zone or generation, 

for each species, can be plotted versus tracheal or ambient 0 3 concentration. 

By using such plots and information on nasopharyngeal removal, the ambient 

concentration necessary to produce the same dose in different species can be 

estimated. Also, the relative quantity of 0 3 delivered to a zone or generation 

in a given species for the same time span and ambient concentration can be 

predicted from the same graph. If the same biological parameters have not 

been measured in these species at dose-equivalent exposure levels, the procedure 

can be used to scale data and to design new studies to fill gaps in the current 

data base. 

9-15 
To illustrate the above procedure, Miller (1979) calculated exposure 

levels of 03 , giving the same respiratory bronchiolar dose in rabbits, guinea 

pigs, and man. Considering the discussion on nasopharyngeal removal (Section 

9.2.1.1) and the question concerning the guinea pig and rabbit anatomical 

models (Section 9.2.3), these calculations are mainly useful for illustrative purposes. 

9.3 EFFECTS OF OZONE ON THE RESPIRATORY TRACT 

9.3.1Morphological 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 03 exposure are similar in many of the species 

studied (see Table 9-1), it appears likely that many of the postexposure 

biological processes of animals could also occur in man. 

9.3.1.1Sites 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 

and glands associated with the sense of smell. Significant morphological differences exist among the various animal species used for 0 3 exposures as well 

as between most of them and man (Schreider and Raabe, 1981; Gross et al., 1982). 

With the exception of the cervical trachea, these structures have received 

little attention with respect to 0 3 sensitivity, but 0 3 removal through "scrubbing" has been studied (Yokoyama and Frank, 1972; and Miller et al., 1979). 

9-16 
N 

N 

U 

C! 

d N 

U_ 

N 

4~ 

W 

~ 

V 

L 

N 

O 

Z 0 

N 0 

O 

U 

W 

W 

Q 

cD

U 

H 

U O 

_ 

~ 

~ 

ri 

Ql 

J 

¢ H 

~ ~ 

~ ~ ~ . ~ 

~ ~ ~' ~' ~ 

~ . . 

u 

aa ~al a 41 

V- G! 

~ i +~ - ~ L E ~ ~ 

~ titl 

. 

rL 

O L ~ ~ to 

U 

D U m CI U tN 

4•> 

C 

C a 

u +1 

a `tl 

N O 7 

m U 

G7 N C 

i c i E c 

U Sr C E 

+> T = 

N C C 

a O • 

w ~a 

O N 

C - U U 

7 

y ~ L +1 

> ¢ airn IF L •r 

m c 3 L N • O m C 

m.. r 

_ J IC •r C 

U O 

L ~ 

~ W 7 -~ U 

3 NU m a 

a vroclu 

E Lm a/ 

O L 

V- ¢ w 

x c ro E 

N ro U U N 

4-c m O 

O UL N ro ++ r 

' o •r c ui r- 

+> U M U 

m C 

ro 

roa 

¢ - 4- 

a x - 

i ly •r row N 

c 

U •.x- O 

m N 

N 

s w c 

41 00 

N L 

•O IL C ° L o N 

N C 

41 vfr• 

W N 

a .v~im °I 

N r L 

m C E N 

N O •r N m.rNN 

aNi 41~ 

r 7 

C r L m Ol m i L 

•V r N W 

w t a e oi++r n a •r n 

W Ul Lo O 

L rl 

~ ~ W t 

x ° a a'

J

i 

r L d•r 

N U a 

c r+ 

O L. t r ° 

a ro 

>a a

41i ro 

C •r .L1 

1~ mac 

m C 

E 

rooz - C U 

O C va d 

> •r L : 

ro a a 

L a a/ 

ro 7 ~ 

^C•r 

L u 

4•> L •r ol m ++ 

C r N 

r >m 

'SOro ai 

0-0 u

~ 

U Cm ~ 

C 41 m +> 

E OI

tm

~ 

v 3 r J 

Eol~ a 

G 

O 7 N o 

>J N r• m ro 

c' s ~ 7 m u O 

i CIN m V N N C7 

m U Cl L cl m rnm 

i 4-• C GJ 

U L m d 

C 7 L n 

H N U m 

T ~ ++ 

Y N C 

C L a C 

OC C O 

g v m m 

U 

L d-> nc 

a L ~ 

v++a 

N L 

m a - 

s% T s 

U L O C L 

H a N 

C 

m vN O C U 

o 

+~ 

•r •

u

r ' ~ ro~ 

+-+ dE ~ 

C L O f 

C) Gl •O W 

C >n!C6 ~ 

O L K 

U F L w 

rY •tn 

m•a n 

4J E d J 

m u.. 

N r N m 

.O•n~ ~ d•r U 

N 4- 

L L O 

C m r~ C 

O r O U 

i o m c 

LL ~

m 

 N 

L C m 

+1L O) L 

m m N O L ro 

a m 

d • L •r 

N N U r• 

Gl f C •r C Q H U 

i-+ 

TC/ C Y C 

C O 

~ m 

a- •O 

C NO C 

C C N m 

o r• ro 

L4- r- E 

a¢ O 3 '0 

C a•rm 

ro mro:a 

L O 

ro v 

E L m r N UC Q ^ O 

N 41 L N 

~ rorti u ~ 

~ N a 

N r 6 N a 

C U r n G l 

o 

 L

> 

tV 

n 

N

c 

+1 

V/ N Ul Y 

m aci T v •r

u

- 

Grol y 

~ OU G +OI 

'EJ L rO O m "O 

m a ° m- ~ 

L C L N; O 

N G 7 C 

> +J U O n +J 

O L 

L C^ L i C 

o N m O r 

E 4- C E E'1- 

~ 0 .

2 U n ~ 

N N +~ U i 

O ro m mLO SX SO. 7 f M m ar ro E¢o ~ 

r aU a N r 

m U m .0 G) 

Q L m.C+> U 

C 

N 1 YL ~ 

°- zu° ~ 

. 

L 4-Lr- m 

O m a Gl a i-1N(y 

rnro ~ a3i ro 0

o c r• 4- r- u 

aM: u m o T 

Na L L¢ N E a 

re 

E 01 m V- C • ~ 

41r-m O N•r ~ L 

O m C H N ~ E w L 

r- 11 V- L+J N F a• 

N•r L. O NL N 

m U 01 +~ a Gl 

L O N -7ro 

m c u v> v~ N 

m ar ua¢ o 

mf-- L U a ~ H f,7 

U = a ro C r J C 

O N Ol L N m v•r 

~ • a 41 N Oi E.a c 

L N•r 7 ro x r O 

• U N r• ~ L O• U 

+> C m•r • 4- L E 

L O N U U 1 .a 

O L L m a G y 

a L N 

•ai c. • s m. al o ro 

c aa 

N m a r N Ql Gl Nd L 

N ro 7 U r• 4141 L 

O m L U N m N 

rL a i• 1.• 3 ~ . L ym N 

a U'C Ol'N'N U N L>> 

Ol m m L .0 

C L O N L O C C 

O Gl O 

m01 c ~ 6 L G) 0 - ;•II 

r L N r V L y y 

N O m m N y N 

nm L ur-1 •N cL o 

7•r N n O n0 n 

L•r- Na ~ dC N y'p 

- 01 U i C a >T•r Gl U 

.0703 m ~ ~ _T J aci uroi N Ul L i- O' 

in tM N+> m . 41 O1 L 

O m r• O•r N C a V- d-1 Xmale=f w c ~ o W a U N U G. O" Ol •O 41 

N N 

a~ c o 

^ 

0 

3 

o ro c+°+ ~++ m4~ 

X 7 m i 

W a d 

~ O 

~ 1- U . a O 

1- U 

a 

m_ 

+, c 

O! a 

E O 

~ E M17 

N 

M: 

N 

u 0 _b *ro 

a•~ T\ ~,\ - a+1 C 

mL 

a L 

mL 

a L 

M U i- GO n 40 

t~/1 Y ^Y ^Y 

co 

T 

-m a ro N a L \ 

ro s ° L 

aL ~ 

I~ CO N 

^~ 

~ Z 

N 

O 

N 

M 

9-17 

c 

O •r E •al n 

a m n CL o ++ N C 

O N 

C E 

ou ~ 

ri N 

O O 

I ri M 

rl 

O 

l0 

r

cni 

1f) 

NMmOo 

O O`O O 

~ 

 01m00t0 

M1001 ~ 

N'D O oo 

lfl N f+7 

O O 

N t0 

M tm0 

N Ln OO 

O O O 

N O OJ 

01mID 

Mcnrl 
al 

U c a/ 

L 

N 

y•. 

N 

C 

0 

co 

m 

ri 

m 

4J 

C7 

C 

m E L ° 

O 

m 

N 

C! 

.~ 

c. tn 

I m 

C 

a 

~ 

C 

4J 

°u 

... 

W 

G 

0 

0 

~ 

U 

W 

LL 

W 

.J 

L 

U 

Un 

r. 

^.A ~ 

O 

S 

~ 

rl 

Q1 

W 

to 

¢ 

F 

N 

++ 

V 

w 

IF- 

•O 

N 

L 

a 0 

0 

n 

m rt 

. 00 n 

rn 

ro rt 

41 

C, 7 

U1 

N +1 

N ro 

m E 

Y y 

7 Y 

LL 

~ 

N J 

^Y U d•~ N N Gl '7 

N r N ^ 1 a 

~ ro 3•On. a 

x V 11) O M C 

N 

N E 

L n L ol n•a d 

G1 L 

C!O N 41 

3 ro ri • u 

O L y 

0 ox v 

a W iJ 

L L~ = 

a) o 

++ •a 

m +°1 O) 3 

E r m N v m e 

ar 3 3 

TN 

r p OIr 

iJ O. C L 

C X 3 Gl 

marrn 

u L 

vsm 

v=uar 

r rn 

c E L O 

~ O r L 

•N Gl 

N ~ O 

ra 

GJ•r C N 

> u m r, m L m 

L C w 

X ++ E 

GJ Gl L n 

U O) n L 

o •r a/ .E m 3 N ~ 

o ~ 

L oV O ~/1 

c ~ a ~ 41 

O U•O O 

u L LG) c 

'O X 

ai 

 O T 

vOC io X o 

3E arr 

i L Ny L 

01 +J •O V Y 

X O m •r m 

W GO L E O. 

N •O ro c E 

€ - 

rL 

 +1F' •X L 

n E C o O U+1 0•r 

n n° L4..~ C m i m 

1 O.•r 01 7rG.\ 

N +~ C•nw v 

C/ O N C ~. ~+~ C! • 8 i o•r n ~ u L Nr 

C! B E 1 u 7 N L a 

3 ~ • X N = v 3+N.+ iN N G1 O•n L N N N 

T 1 C7 g T mr 7Y 

•m0 ~ EJ t0 U O IV U ~ 

04J •O O N uL 

rn rvo ~ > 'o~~ r C Ol L iQ y 'O 

+i ai m•r ro\+.+v m 

m L O/ m Ol L 

Gl +~ a • O r r L 

N> m- NC! p r L N 

C C! Gl >•r •r 7 O. o N N>~ rnr L u u = a c ro u c u o 4..1 N ~ m\ C O O 9-

m C! ~ Of lL.i r L Z 0i 

~ rsa.+ E

+~ ctm..wtTmrtf n 

U 7•r m V L•r C I 

U a u n Q 1 C O r O W 

m m al c ° ++ •r •r 

r N•r 

~

VI •L La i m ara- a)NvuT 

O. L C a•r r •O. Gl u m 

7 Cl 7 m N C O .O 

•O N U+~ Of C 01 G1 1 

C O O C•r L}I+J O 

m nV C.r E\ L m Ol 

X Gl }) L C/ O L 

N Ol N'Q N G) L 41 •O 

im 

d ro r• r ~ 1 o N c 

I

C N N L L N m 

.c •O c "s0-. +L.+ c °m o 

''. U 1_ •r N Gl O N 4- N 

t G L N s 

O VO•1•/ a1 a O '- 

m d-~ m i m r N O • 

L4-1 Cl C r N 

6

ri C i m m•p r•r L 

f m N o N L c m m r y 

.Cr nU 3 U•rL C O•r 

41•

E O•Q L V L N E> L 

W•N ~ m C L+f m Or m F-r roa 

~ I 

r ro 

I •N 

m C 

N 

r I ro L 

Gl +- r L 

L L Gl .FI 

L O = . 

++ 

v m•r U 

U L r m 

•C

r}a •r 

i 41 07 L r i-~ 

ro Gl 

^ (1)l ~ ••~ 

4- L Ol 

.r~ •r 4- 

v N n c 

N.0 

++ 

o•r N Q 4- o 

d•r r N ~~ 

v c O O 

ar•r c c 

•N 3 m++ U ° rn ai ar m 

o E 7 u 

++ ° 

~

u 

o •0 T.0 D 

u .F•1• 

m t 

W •r n 

+1 N 

c 7 

L O 7 

07 L 4- O' 

O Ul E 

L N 

Gl L 

E+J 

• n O•r 

S E cm r 

0! v~- +~ ~ V i ~ C N 

L C. 

0) N'Fm a 

+~ 

r~ 

E 01 r O 

a ovi 

•O C! N 

va) 

V E 

ocru 

r 

 r v N 

.0 m U 

+1+1E m 

O 

4- N 

•O 

N O 1 >311 ~ 

L N rn 3 v n O t 

++ u•o ^•> 

C m ~ u i ar++•~ 

a L 

m Of•O 

tm. D)•r 

N r 

y 

•• N T L 

r E.r U ro 

C•r d 

V O r L 

-p G! 

t 

r~ G1 O r 

•N •r N L•r 

C)•r N U 

U m O)•'- 

Cr N nN 

O N Gl EC 

~ rov+1 

o+i.+ 

uGli v m 

• n 

•C 

+1L > To O ~ .a '_' ++ 

L•--• 3 m Cl L n O 4- G1 

nV- u O ~ +/ Y N O O ~ 

Gl •r-• N•O .C 3 m ~ O L aC C 

L y v'D 

= r- L T OJ 

O•r 

E > 

}I y O -- 

•1•1 L o 

N O O L 

o N N L L 

7 41 m 

r a U 

C 3 ~ E N 

N O m r 

o. c.~ o +.+ = 

x C. N u N a'r t Gl C GJ 41 

O. N V O +~ L i 

b a 70 ~ U 

E i•1 

L C E N O 

r L o 

n 7 

.1-1 C L m r 

O) O m J m 

47 N L m N 

i' -EC •F°'+ 3 

Gl u N •O • 

io 

d, U 1 L 

r o+L•+ 

r 

'cnf~U o. N 

C L• N L 

L m m U C 

O~ C••- O 

•N C O 

C O +~ 

O 7+1 r• }t 

S.. +~ m 

L y o L•r • 

v) •O J 3++ 

o• C 

I- 

 +1 •.- 

{.> m L d •n 

tn N++ u al 

C 

O I 

IDC 

I C 

L 

I C 

r0) L•r 

O 

•• U 

O 

U 

mc 

O 

• -T ^ O c Gl E Tv•r++ O M • . L o o 

7•r U 

•• N m 

o 

^ m N+~ r tf) 

•O •r m ro ri 

In N 

^ T 7 

In N 

T O N n•~ ~ o 

O m C+1 

In m\ 

'II L. 

T\ u+1 

m L L O G/ Gl 

ct m o 

ri •O 

ro o 

O- L. m O 

X 7 L 

L 

O O 

•O L Gl L N d-1 

X m m 

C 

^ O•r 

C L 

W~ n 

O•r 

N01N M Ol m V C nOl+1 ria1 M 

a 

lo 

c 

mv 

CEl L 

L i•~ 

•••1 

•• t C 

1--1 

^]C W 

N E ~ Z 

CQ 

ZZ CJ m 

d 

F 

O 

~ n N If] 00 - N LL'f O N O , O 

al m n o 0 0 0 o ri o ri ui c L 

o .+ 

N C 

C, u E N OCo NCG N O O 

C 11 

O 07 

C1 00 l0 

M 01K) . 01 G7 t0 

M 01Q1 

01 

M 

t0 O 

Ol 00 

U 7. rl rt rl O1 

9-18 
a 

v 

~ 

c 

YO 

U 

z 

O 

O 

N 

~C. ) 

LL 

W 

¢ 

~ 

0 

O 

1 

O 

E 

ri 

Ql 

w 

J 

¢ 

F- 

~ 

r 00 co a 

m m - r~ ~ - o. rn rn r OD m u cn 

s .- • ,- N m m Y r Y Y ro w v m m a v v Y Y Y a: QJ v C Y Y C . m. 41 ro v 

L OOa - ~ o O L E c L m 10 Y Y Ol •r m ro s O m m L L > O] U CO N 

N 

N O N N Y Y Y Y 7 iY •N . Vnj C tJ ' C K E lu d' 

O N i E 

+m•> E rt ~ T Y r- rl . ~ O E O O) ~ 

N Y C L C N m r C tYd E N Cl N L Y m Ol UJ r N r r 7 L 1 W T L Y C m tn L m+1 O 61 O•r • N i n Y'~ r O Gl O L V~ r N E 4- N N 3 C n ~ o n•r m a¢ i 01 3 • O U) r• m i ' 3•r O U> VI O 1n d4F 41 • ~ O. t N Y r- E O U X L N a o m VI tn N C m C N N r0- •r N > C y 1n V! o N m m U n i 1 L N O.0 L m i r u O7 L N i i s' w N•r ~ w L Ol 41 g u u G) 041 d•> 

. m v c v w- •r n ai m w E v u m n ai ro m c J 3+•+ c c i i o a 3 O ~n i• c 3 tn u c - L Y C7 N m m L n o a. U ro N C N o N ~II O~ m VI MO •~d u Y r- r .r- in c L C E tn 3 O E U7 i N E N L C N al o N E c 1A N o ro 7 4- v G. i ~ m L (tl > N •E •r C~ 1A O r v r ; E•r a •r O U O- L Y Y V GJ Y 3 T~n L o O Y u 1A 41 tn c N r- m d~ N U r r n r tp L~ o ro •1-~ ~. O 7>> N UI 4- >T N •> L•c >> •> VI Y Y r• X C r N U•r U L O m L N i U C a i~ O Y ~ N N Y .3Y u O o ro c• L Y • -O ~a'6 o Oa• u ^ N rl U •LV ro L i•r ro~n ~F nL N 3 L 4J O Gl a N GJ N • Vl •r ~ r E o ^ n4- o tn N u .L] o n o +> in u~ ; ~ na •r L 7 i N LL 3 v r• •tp Ql i Ql N VI 3 V1 L Ta m y m o m 7 c Y•r m n E r 7 L tn i Y Y n•Q O •N Y • 7 N L Y O~•r m O m N N V1 ~ m m N 7 m Y i Y O' L. VN n L. E 'p N p ~-T- r tN E Q1 L N r- i L o L 3 C m • U m C.1' n L 

^

m L y E N L Y N i i a i C y Vl a n N L Y > i m Q O l a 0) V 1 N L• r r Y L a r m L V1 . Ul u . r- 4- H ~n E G7 Y Y o ~n N U . lfl 1'7) Gl r p.'D ~n Y r ~n O N a O Y a m m N E L d C Ul OI V1 M•r U Y r N i U N C VI a G) r N • N O 41 L Y L•r m Y Y d U C C • L C U O N N• L - N 41 N L VI • a i VI c d~ tn +~ a r L O O 10 N N C d L Y e- c L Y 41 N o N N C r O 3 u Y N 3• 4- >•r a E Y L i U C d Y•r . ~n N o ~n 3 L Y .r 041 •V N W C 3- n O Of 3 C m m Y a V1 Ul E L r• G a L'O y L m M r• G7 m/n m C Gl G) N O U•~ 4- i a m O Y V N Y r• +~ Si m N{1 c n VI L O'O O N i o U s L c U.A ~O c.- C N 4! m• ro m>> Y N+> s`o >, ~ e O~n s m n o u • r- c~ a~ s r ~n c~ u> N f0 m r 3 QJ r- a N N o n u '6 r- a O)a ~ n N o o a > E a1 N• Y a1 N N m c E i a c Y N 3 Ul N ro o d 7 u a Ol s i aa a ro u 3 u•r •r r- o v o c v- v~ a > E ~. v~ . o c c X+> 

IA o VI m i- rl L 4- •r E c~ c O i•~ • w ro +~ VI r- N G1 m N a x u T a c o ro r- ro ro a a 3 o v w i c • id ro'o ~ m m s o o a ro 

,p s•r m ~ V1 s u G1 m L u "O r - m ~n Ss L L tT6 ~ U~ 1n 4 O n OJ N V G) ~ -•E tp tn V- • - C a N d V- m Gl o u a X E i m p- M Y L ro n tn a a 7 n V1 O 4- O) O O N 3 Y C N O(Jl f-. = C L C E u G) 4- >> E N N J 01 tn N m E~ n m L- 7•r c~• 'O L L 41 O O O m 7 C i O d-~ J a s Y m G r Y a U m O T U • N m C 4- 01 C i E U•r r m _ C~ O - C 1 Y n~ 2 'Q, Y C U1 tn C•r • d V' Gl i• ~ C4- Y m'rJC • L r m N W •Of•~ X C1 O1•r r L ~ Lr• 1nL V 'O ro w N Gl N O o o c N Y.- 3 E O> W J 4- Vf 7 Cl Y r- L C7 C C'•r L i U • c in •r ^ Ul • dl L C C N tn U• 41 Y C r- O O c Y a) m i~ O) c r- 3 3 a u• O a a al al L o 3 U•r .r ~N Y u Gi E U O) O 3 4-- m C Y o>TY o V1 C m E • L C • N i a N n 0) 3 U _ U n m•r a L E•r 3 - r- c -- a ~0 Y ro N n m f al u~ N 3 x~ C a a 41 a C II. N C V1 O +ii L L VI N N L N a Y Y tn L W M m 3 d U1 • Gl G7 V! L N N m m •i UJ N~n 7 O) O L 7 U L N~n m•r m O Y F m N E~- a• r m Vl •r i O 3 Y > N E c N L u) 7 r- M r-p . O,- p1 O Y N ln C~a roa a o ~~ > N o> o u a ~n Na w u Yr-r• >~ •> >a > nn•rr• 

. 01 • C L c a1 r- w x N SL i O L 7 C c•~ o•r C• c - m o N ro • }~ m N c >> x c o 0 c.., 3 m n m p Vl N N Vl 4- ~ v tJl a ro•r a Z U ro'O 1-~ K'! U a ~ VI r- r r F- N•r .N 

c cv a o o r ao c u Y 3ro in 

s- ami d v m Lri o 

y c r• ^ r>' tl L U ^>> w 3 T ^ 3 T ri ^ T ^ T U o ^~ a i o o VI 'II ~ lD ro N ro Y\ m V1 \ ro rl w m d' m VI ^ 4- o L~ 7•r u •Y \ . L Ta N Vf a Yo a Ta a 7 w ll1 0 f M Y a O dl s lO Y m\ Gl T\ dl >>\ - m\ ~ ^ o T^ • ri O m C}.~ N L 1 O 3 m i N m i t.[) a f- N ~ O m VI O Lf) nS- m O 3 I~a L L . ' at 3 a.0 - L G a 7 r L. 

W a X= i N C~ tD GO ^ l0 ^ L•r O T• O n tO rl d' N Y ri M N lS] M tO N I- rI r1 M ri 041 e!' 7 d O VL 

m 

._ .... _ }L. C.. . . -. .... _~..._._. .. . . -. .. , . . - ...-_ .... _.... .._._., ......_..., .. . -_ .... . .. ...... .. .. . .... ..... , . . <- . . ,... . . .._ ...... ......... va E O 

i~ W l~Y Y 

N ~ 

C 

E N NNO M M •M M MUrr0 MU7 O I.CI t0 O ' L'1 O O t.t) O t.[) O ' 

++ a v m n o oori o 0 0 0 0 000 i o0 C L Oi-~ N C O N V E O o 00 OO W 00 N tC ONOO tDG C Ol rl OJ l0 N CO CO N CO CO 1'. 1-- l0 W 00 cn U 1 V. u7 rn~ u~ Ln u7 . u"~ tD m--I r~i rl tD rn 

9-lS 
~ 

N r~•1 ~ i-~i O^i c~o 

1 - - ri T 

p~i 

ri 

d 

u 

Ol 

rt 

^ 

^ 

ri 

^ c m - 

L ++ 

+1 

m tl m 

4- i0 d d •f•> 41 

w 

d m 

E 

d 

t 

G 

E 

Y 

w 

N 

C ++ n E C C 41 

ro m • 

O 

d 

•V L 

m 

> 

1n 

~ 

O. m v7 m N W W 

01 

O d. 

IA •O i L N 6.f) .N 

^ pJ 

•R7 

d 

ro 

wo 3 r• 

.0 o m 

.O 

.G 

C 1A 

7 d 

d•~ o r' ' d-~ 

d VI +•~ m 

N•r IA 'O •^ 

~ 3.0 'O N 

Ol c 

Y C 

c o 

O. mZ d m m>> m m o VJ Nor- m O m 

y d'VN C CvE Q: fv3t00 C fv 

T ri m 

N 

m m 

il 

n d a v i O. C 

El0 N . O C X L 4d-+ 

••m-• d E m 

T•ul y d ~ O y.d

- 4-t0 m 

V-• L 1.1 m •N 'O 'O C m 'O N m a O L i }1 0) C m V 

c m u m 

t N•r r• 

d d o u 

'O r- 1n U 

d i 0_' a d••~ d i~ •N . 

m m 

'O >T 

r ttl dl 

~ L L L 07 d 

r ai4-++rocn 

o ep O.O 3 ~ aii a~i 

or mdo 

'o aCi cvi id- aii m 

c.a 

o 7 vi c 

u w-w+ 

O 

m-d 

N ~ 

onmmcn 

E7 tA d•r O. 

d u1 a+ r- ~ 'O >> 

L. 

L. 

d~ u> E 

L r~ d d d 

u C i-~ d 

m d-> 

V ~ ~ ~ 

C 1A d 

L o C U ~ ^ 

vi c tV0 

L L Ln 

• O 7 r-1 L s 

.c L. 0 06. • c +>' ~

C m L mN a d•r^ C 1n O d L 

L~ .r d d C 

7 

u 

O) d 'O vl 'O 

7 L d d 

d r• L C - L r• 

•r 

m C O ~ 

.- i C 

4- O p1 O 

.0 'O r- uf 

O E U Y L Y 

L C O O N 

Ql D) > d L 

d ~.7 •{-1 C Y m 

U 

O 

O+> n'O +~ 

r• >~ y N rN d'O 

3 u N Gl 

O 4- •N m d 

d O m Gl V 

C7 C O 

O d' m•i-~ L 

.O V- O C L•1-~ 

O L W•.- 

~ C C O mVr• 

d d u O o d 

VI L d•~ CI O m 

d m E•r 

N E 

~ a O m 

> 

~ n 3 U> 

N y 

E 1~ ol r 

•O V1 L 

~ N C E U 

d L Ua d L u 

E L d O.0 d-Y ~ /1 

Vf L. 

7 

C> d€ i~ 

d O L m N•r 

~ n L n tA 

f O X 

m N E 7 d 

\ r 7 C n L L 

C3. O+~ d~0 

O c r• O i VI 

i•~ O m•r o On L W i-~ 7 ro m .0 d O m+~ Q.1-~ d d d 7 U O ~ n W m wuG. vB 3 u€i s+~~aNi~ Y 

L d ~-• m 

o 

a n 

++a> 3 ~ n 

m d L. 

m.c 

_T E E i-~ 

r•auc ~ n 

O o m o d 

Ln 

• ~+!"O ~ n m O 

' 

C 'C d u! L o.G O 

d+, m d 1•> 

E X 

O d 

C d ~n d d 

d L V Cm i.~ 

E n +3 m O.'O 

• C E r O O Gi r• m n r• •O ~ 

Ci 

3 c 3 3.1C an 

O•r- L d Vl 

+~ 

U t+l 

r• 0.9- d m c 

r• vl O L. E`•-~ 

v~ r ~ m 

o W N L 

U U m T t 

C•r L ~ U 

O E•r E'O 

+1 nr• •r m 

N E U ~ 

S- O 3 O W 

/1 .0 ~ L o•r d 

41 p i0 1•~ 3 L€ 

d O 

C 

O L. m 

N 3 •O u1 3 C1 

E C • 

v•r O a 

O ~ 7 

L tn •.- N E 

O. U C.0 

v Gl m 

U 4- L N • 

N U O•r u~ 'O 

V 

Fj 

d-~ € tD d d•r VI d E}) N oo 

€ C 

c m u 

m d u 

r 

m 

N C ~'O 

.O m r• ¢ 

~ o~-1 U1 

s o'O •• . 

7 Ol i o 

'p m m Gl 

m m ~0 .~ 1n 

v 

d 

Of d vl d m L 

c 7 T L L m L 

O m 

d 

d s o 

s a m m o 

r a\ 

m L 

n N Gl 

N L> 4- ~ 

~ n tD ~ .0 Gl •~ 

o c.I.~ Y d-~ 

s r- ..- m s o 

D) m tn C L m n 

T.c 

T 

nE 

d d O)F- d 

3 o~ d c > 

m E•r E _ o d'O L 

u~n\ 

c Cl r-- o C 

nm m 

n o m u m 

X+~ c•r s 

~ c m•r m•r x 

3• a~ r• v 

L a-~ 

/~ ~ d O m m O 

O 

N y N E O r• O m 

m L CY 

T d L N 

E a 01L 

L L•--• C V 

N ul 41 ,C 

N a L d.> Gl 

O E n V i•r 

r• 7 i 7 m U C 

d O•r u1 7 O 

U 

O d 

r• m V1 r• L 

r• 'p d w M 

L L ~ 

O u d• E 

N m•r 

i~ r• E C 

T f-• m 3 E 

.G 7 ~ vi O 

= N'O d 

iy n C C 4- r 

> 

L 

L 1!! Y- C1 E E m 

ii V1 O C € 

r- c 

O 

d C 3 1 U i~ 1--1 1A d N' 

N G N n 

m•r v n 

m•r d Vl 1A 

d•~ u X Y T r• 

1A m r• 

'O O M • 3 O 

m L>>o•r o Vl 

•I-~ L U ~ -- ~ 

d 

VI 

C O 7 d ~ d 

O O. d J L c.v 

O E 

4- O 

O U 

rt C r C rE E"O TN 

m o o V m 

~ L•r- V N 

O). n Ul > 

V X i•~ L E 1 

v~ C 

m•r •O r ~ L. 

.0 

C 

u d •M1 d 

d L. r• 

d 

L C 

. m m m 

d d d•r 4•Y L 

m d r- . 

y Vl O 

d O_ L U 

O7'o +j u) 

7 d) m 3 7 0 

'o 0 •r 41 

3- c= d•r pl 

E 

V+1O) • 4- O 

C m d Vl O ~O i~ 

U n 

•r 

n L. C u1 d-~ 

>> d m Ol •r C 

4- m ~ 

C r- O'O 

m m M N 

E L .'O 

C T+~ O.-• 1 

r ~ c n 

C T ~ n C 7 

m Gl C 

ro 

1•~ • C tN d 3 Y 

L t'O d 

d d- U 

r d 7 G 

L C m 

m VI C+~ G) 

"O m.--• m¢ n 

N X t U r L r-• L'O 

i ~ r• ~ ,p O O 

d•r d.O •r ~ 

O 

L a 

L In E o t 

m r• d'O i~ +~ 

 L i• 

m d}~ l!7 

~-• r- >T G/ d Ul y 

L u G) 

• 

V1 1n UI 

m m N a ~ 4m- y N O U C.4- fdi .- N L. o O 

Y! M- 1-~ m iJ 

o c 

O.•r L N r- d 

}r 

 3 

O G 

c r- EC•r u~ 

 3 0 

U U+J E'O 

c Uf c 

0 0 o 

U ~ U > C'O m T C1 

3 3 L • p, L 

Cl • ~ 

O. N d ~ 

U m ~ n d-Y L 

+1 +~ E s i0 O c 

m N N•r ~ O"O 

I 

X d

.O •r 3 E 

d T•O d r U•r 

f...r 

01 3 

m C ^ T d 

•L 

N d d r- d> 

m 'O 

• d d i•~ 

d O O) L m 1A 

1n •r +1 n'O m 

c C m• m L m G/ 3 C•--• d n+1 nr• • N 

•U 

V a N +~ L 

V L O ul ro•r }1 

O€ O 7 Y- N 

O 

07r 

€ p7'O L L 

+~ d d m•r i*..~ d 

L . L u) V 

i•Y Vl •N O d 

i~ m O Gl 

.1-~ U•r L 1~ L 

i•--• r O r• 

Cl O! d 

m.0 L E U m 

C L .~-~ J O v 

r' +~ = n Vl Y^ 

N X•r •r O 7 O E n Vl d, a m m m C d 

C f n4-• 

d W C X 4- 

w C r- V U 

O 7••- m 41 C m U- N> m 

O L C.0 

• m ~O • 'O n 

Q€ Q d>•O N J W U F.O Z7 L•r o U tn •r d d E•r~ V E m•r- I- CJ m d-~ m V1 G. U m 07 ~ L m cL 

Y Y 

N 

~ 

^ d d O ap 'O Vl ~ 

L 

N 

m\ • L 

m ^ O 

~n 7 LO •• p>> 

O O Y\•o 

~ 

t0 

ri tE •.- 

N 

~ n O m 

. >> C 'O 

L 

>rt~f~\ 

E ~ 

\ >a d C . ^ ^<P 

~ 

N 

U j~ q m O 

T N'O C ~ N a C L 

x 7 L 

L 

o 

• L'O 3 d•> 

m ^ L m ^tn O ^o 0 

LLl •C C. ri tlf t0 I,LI1 t0 N 1~ 'O N O'O I_ M V Ol U.W 

a 

+a 

a

c

, v 

~ d 

1/~ E 

~ 

• 

m 

Z 

C 1 

Z 

> 0 0 

~ Z 

> m 

~ Z 

C 1 

Z 

> m 

~ Z 

SIO 

c 

4-,

~ Ch ln tn LN LfJ Ln uY 00 

d ro O. O O O O o O O O O O 

O N 

N C 

O d 

~ 

e~• 

O O1I 1- O~

i T O i~ T ~O1 ~

01 1~f7 u 3. r+ ~ 

9-20 

L 

S C 

•r~ 

♦./ C 

u° 

V 

~ 

N 

O 

LL. 

O 

H 

~i 

~ 

W 

J 

~ 

S ' 

fi. 

~ 

r•/ 1 O'1 

J 

~ 
Y 

Ln .c tL 

U 

.t 

C.C •L L 

O+1 O•I•~ .O O s N X 

C°O%.i 0•3'O

D E ro U OL 

r ^ O N O. 

"O 'O 'O L N 

rov rn ~ o.o 

r 7 ro O N(C m ~ IF 

E o N V r 

L C r m 4- W 

Ul p7 U•r L U) E O C 

In m al i- o o N WX ~ c s.N ai c 3 o s In 

O 7 >•r i O! N 

IN C r M In 1-0 r 

n O•r ro o r E 

..O.I X m r a y • C o 

. ~ N • r ro -p U 'p 

• E ~~ b 

M O. U x m 4- In •r 

C O. O V1 ~ - •r m N 

O 1 4- i r 0+1 N C 

lfl O O_•r •r m L GJ 0 L L L U 41 

G1 O .O •Q Q U+~ C C 

C O C V r r 

r C O m i O 

ro ra> m u n a a ~ ~ 

L al •O C N E • 

i cr•r ~ d V 

0 N E 4- m+J N In m 

> > O v m c ro ~ 

 Y VOi r N 4- O O Y S-. 4- 

u u o o0LU mm v E uf +~ m U~.~ •6 uf 

-o ~•~. s~- ul -p CN ~ OJ aii 

N ~ Vl mi~ ^ 7 ~ r. a 

O 1 ^ W ro m N•r M O 

O.N O r L•r . r uf 

x ronroaiair 

07 O 1N C O•r f-. .0 i m 

ro•r i U N•I-~ m+-> 

r'O Ul E U C E•r o uf 

C L 3 m O= O. L•r 

Q m m J E Z C N D_ 'O 

mTL •O L 

r-m 

.~ 

> m 

~ Z 

LfJ W 

O C. 

O W 

0 (1) ~ 

rt 

N 

C 

i-+ 

O 

U 

v 

z 

O 

N 

O 

LJ.. 

O 

Ln 

H 

W 

LL 

W 

CD 

Q 

H 

J 0 

x 

0. 

O 

f 

~ 

a 

~-+ 

U 

4-- 

w 

N 

~ 

> 

d 

0 

ri 

O1 

J m Q FL O O 

7 •r U 

tn 4J •O O 

O m C d-> 

X 7 m LO 

W ~ a 

B 

rti 

+~ 

c al a 

E o 

TO 41 

3 C/ 

n E ro O/ 

E 

c 

O 

E i N

4J

N C 

CL 

O +J 

N C 

O N 

U E 

C 

u s 

.~00 t 

cn 

~ 

Ir 

. r- C3

, 

r~ 

ro 

+1 ^ Cl 

m 

ro 

E +J ro v 

O + 

-lL Vl 

o ro 

>- J 

N 

m m C <Z' 

v 

IF C 

O ro L 07 

N tn N•'+~-.0 

41. 

c~ ro 3 aci ai u 

dl N r C Y L O 

•O D U d U 7 

o m•r In 

•O 

i C 

> r 

O "O 

UI m L O 

mr ~ n 

m m 

a M 

r O L•r O Gl 

O C 

G) •r 

io v- O O 

a 

'O U 

c u- 

> ~ 

U O In 7 m•O O m U 

01 Vl r U vl m GJ .0 IF +1 

O m IF > •F1 

m 

-O N 

s 

O> 

i-> d) w L. 

m•r tp C 

; r N 

O 

C'O 

O N 

L 

7'Q rC] U C 

Cl 

C u 

L r• pI E 

C 7 

Q•r m -D 

+> U 

Cl m 

Y d 

~ m 

C 

r G 

C 

w Ul ul N 

O L N m 

U i 

r U 

m t~d Y N+~ O] ^ 7 f0- ~ F> •'C• 

N U In C C VI'O U 

+01+c samia • coiai ~ io~ 

L u0i ~ U L Yr ~ L Ea 

N OC Q U d +1 C ~ 

~ C. 

~ L v t

L

0 

 0 3 + d E E L 

L m OVI m v 4- C Ol N•r C 

c 3 iawn ~ T3 b E3 %I 

a~ 

VI •r 

E O O 

L i 

SL ~ O Ol 

U) i L 

+1 N ro 

N r r 

t n C L U TJ N i U E r raci so- f w E f ~ c~ cn 3 u 

m a r 4- 

o a1 E 

U r O. 

n 

C1 c 

+1i E O. 

o E 

O. m Ul > v O_ N I 0_ 

N N in Ln 

w v o 0 0 

co 

rcn 

~ 

ro 

•h~ 

N 

i 

Q1 

•O 

v 

> 

m 

U 

N 

C 

41 •O •r tT 

m c ~ •r cmcMa 

+1 . 

L 

L D) 

N 'O U 

L .L o 

m L •r i •r i. 

- C E II- + O-r .O E T U 

Oa1E ~ pM~ Ul •O"O O+~ 

o.r O V.. ro o a> > c c.O ro 

r ~ m i r ro ro E, 

O C L V1 vl i 41 m C Cl .. 

L •1.~ 10 O C m ^ O L 

m N U fm-. O. M.0 m L r iJL 

O. iJ •r i O C U U O 

O m L O N N•r m N 

D. O ro U L U 4-

N 

 ^ 

VI 1. ^4- L ~+ 

+) N ~ 

L I O. O 'O E L N .G 

N 7 "O O) 7•L-~ • O O 

~ L p N C N•r C N LC r 

J O i m O r N r o ~ 

vl rn•^ o. ai U r rn a o 

TO. M• ~ - L N 1~ C U 4- .0 N 

C X O a-~ a O L 

O Ul r O tn O. rN 41 

y r i QI W v C C 

N L O N•r i Cl O O ti 

•> Y N L d Ul O_•r r U 

c.c c ~ nmm 3 ~ u roCO 

o r ro T 0) H ~ c ai 

r.1-> L C N 4- •O C• 

•u1 O Vf r• r[U cm O N O O 

lil C+I ro+1 7 L m L- N•r 

m U L f.7 i-1L C O Y 

r'O L O iT d O m 

a•r 4- •r - O v- m i 

L'p O • i L d-> •r ro I,a n E m u ror ro+> 

C VI C O N m i• r L 

o+ w rov•r c ~ a u u rn E m o m r= w ro•rC (6 L Gl r ~ F N 

~ N• L vl o n 3 

CL 

0.~ 'O n•~ +~ O. N Q.a 

tn F-V°' o~ oaN m T ~ .0 .n 

7X Gl s r r \ 

ro LN O. y ro O_ r.I.~ r D] 

U O T O r'p L m L r c 

C - L N O ro Gl C ~ Gl O 

M V1 N•r L O. O•r U r 

O ~ C N S L r 

W O U C.0 •N V! r• T cn •r Gr + c o ro m c 

0O. O tn ~ U"O N• 

C O^ a M L N C U m m m 

o C i->o dx moLL CP 

Y 

a 

~ 3 T 

L \ m 

o roL 

E 'O L 

l0 tf) lO 

f _ o v 

W ]C cn 

S m N 

U Z 2 

Iv 

U 

c a 

L 

N 

4-• 

v 

C 

~ 

~ C

, 

r, 

ro 

i~ 

d 

Y 

U 

~ n 

rn 

rorn 

f rl 

N 

N 

U 

N n y 

N 

T ~ ^ 

Y d r 

CL 7 

O C'O 

~ v m 

N 

X: 4~ ~ 

J 

i tn Q) 

av= ~ 

L O.o•r 

Cl •r Q 

'6 N •11 r 

N O N O 

L 4- 

~ ro a ~ 

+1L V 

h 

r •o ro 

NVI C 

L U 

Cl 

 A O 

N C m U 

3 O N = 

i L. E 

V L U 

m C N 

C N•r L 

7 N O! In 

r r L > C 

uf }> Ol O 

ai ai 3 ~ 41 ^ r N Ul v m ~ L 

4 0 In N U 

c E U r v~ 

N C L O 

w m s~ c 

4

(1) i +> O 

o~ ro •r 

i4- L ~ 

c lu O c a 

ro•a c ° E 

C N •r y 7 

L U w u- o a•~ m 

L 4- C 

N C C Gl L 

E O •r U N 

E •r C +1 r tn'- o ro 

L 0 U N 

L N E L 

 +•~ E L 301 

OE 00 mH cr m 

T 

~ ro vlm ^a 

TIC\ N\ a i 

•6 i '6 L m L 

L L O 

O O ct 

tD tD M M I- N 

~n Y ^Y 

O] 

a 

V1 N "O 

m ro s •O •O L 

~ C rl ~• 

r i ro N N 

tn O I LO O 

O r 

O 

i O y N 

QOi O~i OO

i ~ QNi 

ri M 

M 

M \ 

E 

O E 

uO~n u~ ~n 

O~ ri O O + ri 

W 

rn 

9-21 
N 

u 

c 

~ 

flf 

4- 

N 

C 

r, 

m 

m 

ri 

N 

~ 

L 

m 3 t 

tn 

9 

C 

m 

61 

L 

O 

O 

£ 

9 

'c 

ia 

C 

u 

v 

W 

N 

G 

LL. 0 

ui

1°-• 

CU 

~ 

W 

J 

c¢a .. 

co 

~ 

C 

U 

w 

41 

w 

d 

'O 

L 

a 

0 

G` 

H 

W -1 

d H 

4~ 

E 

N 

41 

0 

C 

0 

. N 

'u 

~ 

L 0 

i-~ 

N 

L 

41 

i 

a 

•~N C. 

~ 

O L 

C O) 

c n. O d 

C tdC) 

EO 

z m 

ll~ ro 

t O 

C L 

E L 

t0 N 

ro 

a~ 

c ci v M o m .c 

L 41 

7 C9 

~ € 

m 

£ 

G 

O E 

~ d wm d C L. 

04- 

N C 

O al u H c 

OO1 

U IZ 

m 

~ 

~ 

ri 

~ 

4~ 

N 

N 

C 

N 

L 

d 

41 

41 

N 

N 

CI 

• U 

N 

d 

N 

•1.1 

m 

C 

iY 

m 

C 

'II 

N 

w ro ++ •~ •€ T v¢r 

Ul OO L V U~

• 

] L o O •u LW 

A ~~ N ^G)d~ m0>•.G- 07•O C l0 M C ~ - C u 

c amw air" mm 

~ N L r L. r m O al 4- • 

t0 7 V- r y m U C.0 O'O L ul 

•O +J O °U 4- L C •d+~ ~ V1 .0 C 

a~ mo.c ~j rornroro ~ cai a 

a•r N~ u 

lL 0! i~ O C E G! N •• d-~ O) ro G/ 

1n tp t0 +- m•r .L] c o c Gl L L 

ro O E m ~ n E al 01 Gl L }J 

O) L r O E d T C 4-1 i1 G) O7 1~11 C c urw m Er ro¢ L m o•r 

°v m Lw+:+ ~ ~ v= d L £m 

~o ~ ro~ O r L ro b f~0 cOi Lm ~ c ad

i 

Oa'o•r L. 3.Vr.0 m 3'O N v1 

4- ••O O C O r 07 N L N C 7 i 

o Vl m C/ •r d ro G/ r 7 r r• m 

VN ITd ~> m N Q• L p1 NCl N C) o 

+J 'O a' ro r r u L. u OI.0 d 

U Q1 L r y • ro i+>> a! O w i- r• i Cl /~ ~ 'o m 

4~-• r~•i o ° aci m 3 aii s.m. c ~ ri°~ m 

aa rn c c ~ N 3 m m m v c wi-1 o m u) r L•r m•r O 

+~ m 0+~ . •O O ~v ~ r L 'O O) 

ro'O U7 ff7 YI Gl •r r 'O •u . UI G! C . 

3.~ vOi L ~ 

G ~ n ~ n •r T a7 G+a 07•r ~ 

C m a) r¢ cmi ~ " o a °c L 7 a=i w 

a7 N•V o'O i ++ ul a.Y FY L 4- •rC U m • N r ~ c~ U 

O U¢ L m^ F- tn L a O rl L•r . 

+~ rO H •T .•O v07 mL o L ~ N 

C L i a N i• S- O'Q C ~ N } m •• 

Cl 7 ~ L C 7 00 OJ m Vl ~ N •r f 

•O 3 N +J m YI t-i +~ 7 ^ i•~ J 

O V u O m N ~ Nr•rv 

v w d m c•r dv •r C U T al dJ 

E1 X t Cf U-• X C r m u Vl C 

v€ 41 01 •r CJ m•r .r

O 

 r• ~ L O 

C 7+j 0 C;1 U N r 'O N•r 

N r ul +J i•~ 01 Ul .• C 3 m O C/ y ~ 

N O o m d•r o O o L Em+~ c N 

S> dLa N dOlZi-~ Mri m•r L 

c~ 

c E L O L L 

a/ od. ~ c C.+O+ o ~e 

i u1 1 ^ • O T4- •r C 

O r Op L C Z L m ~ •r 

E ~

N.0 N 'II•rr g y 

N u O cf Vl • C r ~ r Vl i 

Nro a1 m N in ~ ma°~ u+~ o> ami 4- }~ r• m O CJ > 1A C ro d 

i m 4- •r 3 L G N u1 .0 d 

° c n ° B m> a€io ro 

L~ uf ++ O M'O M L E 

F •UL ^ L4L- i ICO_m tUtl ~ 

• i•~ ~I G O Q. L N 01_ £ L 

L L ~+~ N n'~' O-~ i-~ •~ L+~ O U L C O)117 C N TL C'O 

N Z i~ U€O~ r LLN Y W 

4- 

41 m 1N -O 041 - i•r 41 . r N O Vi 4- 1N C •VI r r - L r 4- N•r 

m r O) o ul .f.~ i1 L 

O E L Ol r C Gl W O 

m L ~ U u,r- IU o U N Vi C1 

~ C rC L U Vl ~ rl O r > 

E O c m 

O U•rrt'O •N 0) 71 L U 

W C N N L~ L ~ >di+' f~7 m 

ro 41 U O m= m F- 4- E 

ro L L Y- - r Cf R1 "O •r 

r.fa 4-

.

Oy C x 

y V- ~ r 'i i ro O 

~~ a i•> o u N i- N L r--- 

• U L C m 3 i L+~ N d m 

C7 4- L m E 4-•r U O N N N N G i 

O C/ E•r C r N L d•r O 

Cl U1 r i~ L O) T C 

N •r 'O in Gl O 

N C r O U4..~ 4- Vl G/ o r.% ai a

>i r-o m E 3 • ro a~ 

r E+~ L C1 U O. 'O > 

Ol L•r ~ N m+J •r O. N Gl ~ •r 

L O d N r E ro L ~ O)'O E ~ . G7 V- N O m 7= ro 

a>i • cm X~ 

c^ o ~ n 

g 

 c ur 

N 7 F- N E U ~ o Cf '0 A L 

a 

'O L 

a ~ 

L o N o mr a a 

C X 0 0 o a 

N N 4 O 

Gl r L y 0 

m 3 m L L O 

~ N O O O/ 

•r 

C 

•O r N rl > 

~ a w ~ ++ c 

U U M m o 

U i L u 

•• r~ .r O Gl 

N L 4- 4- L 

'II N ~ r N 

> T C L O 4= 

+ • 7 i ro 

v m d 

Gl O O O 

~ d w X m > 

r L r W C i 

u0a 

rOl . p a 

N rL n G) O 

Gl N N F- ~ N 

0-> i ~ ro 

Tr ~ 0) Vl .0 

~ mO •r L 41 r do o 

m x C L +~ 

c •r o 

x O L m ttl 0 o a Lct :; 

~ nc u°ir m 

m Vi .i~ 4! ' ~; +1 

'O C O N ~ O~ 

ro E • ~ ro. ~ 

A•r N C G L f Tmm w 

L GJ O Gl OJ I/1 

v da ro Tm J F

N N 

^ TN tT0 3 

O m 7 •O w ip arcmvo 0 

9-O O ~ N d'O 

u01Z•O o Oa'O -~ , d i 

O m C 1j Lf7 rl LJ tA dJ L 

41'O m O. MOuddN 

a 

rl 

•• i 

W L 

ch V00' 

N

^i 

 O 

a 

0 

~ Y N 

~ m N 

~ 2 S 

~0 

l7 € 

O \ 

m 

tPf tff E 

O O t r I 

0 

m 

Ol 

C! 

Z 

ch 00 

t1'7 N 

66 

oLn 

t[7 N 

O Ir-I ri 

~ 

lf7 

O 

0 

11'f 

O 

r-I 

9-22 
a 

d 

3 

C 

y 

c 

°u 

v 

w 

O 

N 

O 

O 

N 

H 

w 

LL 

LL 

J 

¢ U 

H 

C.7 

O 

C 

f 

ri 

01 

w 

co 

Q 

H 

Lr) 

~ ° ro 00 

^ co ri 

c rtl a o _ L y tl yy O Cl W N 

K C r~•i Y y O - m ro a c E c o G N 41 N N N L 7 0 I.L O] 

N / r 

O a•~ i-~ +1 CJ C N N yG] CTl U d! y O a i~ O a y O a 7 O C C i-~ C C 

vdi ~..Ei o ~.E.. o ~ v O ~~ m E ~ m E 

E a L N 

a•~ U ro a b O O N c E aa C/ +1 ~ 4- d L Ol f- 3 ul Cl 7 I N O C N C a~ O N ro N RS y ro L O X E C1 f0 r tE a L O~O ~ N r•r r N N y N C _ O N.0 4-- N a d L C y r r C 4- N Y- r 7 N 7 N L L•r a O a i E+~ O ro C • L L U ttl i6 L R7 ' O N O r 01 C C U d C L d N O.. 3 ft7 N U N C•r E 61 ~r O G! 4/ C 0110 n r~ 0)C C C Cr Oa N 7 ML N Nr N Ud'r i•r fQ i-r L a rtl Q1 C•r O N >T N'r 7 L v c C O+J L . r 

~ N L N C~ •C y y r ~ 7 N r L E r y L C E L •O C SO.. ~ O y 61 ^ ~ r•C ro d N r ro Cl C! . ~tl E GJ •r y L ro ro 7 a 7 O 7 N~ p7 E 16 E ~O r G1 N U C ~ 3 u ro L (tl t "O L U C 07 O N•r r Y L•r r E N•r Cl L N a F- M N 41 L. R! •r 61 0, ttl L G! N L r O•N •r N ~ L t0 ~ L N N C r a G) Gl L•r O r N L 4- C r fp L•r L0 y N ' L r 41 O L L fp r ; 7 y O N•N Of r U O) L L O> L .r-~ r t V1 10 ny C F- d OI Rl v O N X L ~ L L N C. a d O U•r y N y C 10 c O L X C7 L. 3 E L N 01 7 a L O ~-- O~d 07 N N U 4- N N C N•r O i•> N L N G) ' N C O 7 L•r U Cl N y r C O O y L y n U N O L > C +.~ ~ . G7 N L r 1S7 y N N Y N 7 ro ff1 a•O O a Gl E+J CJ r ro•r N a ~d Cl C y•r O O C . 7 N fp U y .•O 61 r r ro y C U r 1p N C a r Li tN Ol E a~ 7 O V- a > N O O] OJ •r U N N L > y (tl 7 N r r O Cl ~p 6,) y•r O• o L a> L C L L G) +.~ N N+~ O N N E~O ro. E L L 4- o c N X - +~ Gl N a c•r d U+> 4- u O 3 ro U L~ ~ r 3 N 4-• O y +1 O U • ro s o 'O d N E c N~tl y L c dl ~ N d L 

i ro Of y 77 va E L ro O L N U O ~--~ a Cl N 4- X N N ^ N r r T.0 y Ol I 3 t/1 O 10 N d E L 4- Cl C >~ U f0 f0 a OJ •r . C C ro N N u 41 r c ro N Gl N Cl O O d L 41 E C1 C O7 N N•r O L' C d r N L r C r 3 L N•r N f6 y~F t- tn U ro O a i-~ N U i fp y C E 4-- N N N ' a•r . L U ro o a in y G! y N O G N o U N O I ro C G! O•r O7 L N ro C 3 U C L G! N N~tl ~ L•r E C G1 Ta i-~ a d N r O. C7 n . a> r O Ol a~ .0 N r t6 N U O/ a d L v tp . O N•r . d-~ y C! ro C X. ~/ L E O ~ N N L L 7 a N N•r O r i. L L Cl L ro y L L. C fd •r C U 10 G! 4..~ a Gl Gl S- U(tl i-~ N Gl O a L d) a d(Q N•r ri f- u L> E CJ. N d Gl N d•r U C dr L r C 41 •r N> ~ E rtl > Ol Y r r 4- N C O.0 N L L ~0 Gl a C ro a Gl 4- , - G) ~6 T v ro 4- ~7 c • u~ r d.~..~ rt1 V- L.N ~ a O E 3 N N N i~ C L O 4- x L N - •N o Ol 7 ~ itl . ~C •r U G) y d s L O W r G C- ~ E"O C•r 3 

' . N> d i•r ro Of Cl >>> N T U 01.G M N W a t0 N•N O/ L d N 

W L C1 O Of (tl O 4- > L Gl •r N t N U r >~ fp C +> d t0 C G1 N L O N N Gl d 7 Gl C Gl Z O T C +1 Ci p, 03 > 1Y R7 .O +~ G/ G! N~ `- U L Cl a U T O T 

 'O •> V1 N r L d T i-~ -~0 'O U L L~•r L G a L C C O y F- Gl d 3 C N L i~ X a y t0 y•r fi7 7 10 .D u C T N ro f0 L O'r C IC O v 7 Uf t~ 1N r fl7 U N C! . > Cl 01+~. a ~ N ~- o r 3'O al r >~t Gl +1 RI i0 c N L r ^ a) ro Oo ro C al •r E 1, . L L 10 N - U a V- E r O N L L- n R1 +~ O O L O.0 rr C N L O C L L O V- y L y N N•r d L y y pJ -y L ~ N a E L 7 C N i.~ d • L~0 •r f0 U 3 L O L 

 .O Q1 O•r L 41 0 7 n•r ~ N N+~ E•r C IC c N O ro U L C L. 7 d 7 ro aV N L N+> N N n> O N'd U Ol 7 E~ N. c .0 N u a N N U•r O O a U +~ G! n GJ 

i O c L y U d L U a a c N E ro O L ro r fQ td n O G Cl •r ,p •p ~ U C Gl o T 4- 7 U U ro~ d-~ N O N N• r 01 4•. L 3 4- L C7 N N(0 • U • i6 a E~d ro 

 E C N L • L d Rf +> N L L N~O •r U~ ro L N N G r N U• a t C£ r ro Cl O N O L#•~ L E ~0 r O d L C+~ L•- +~ Al G) N 4- C ^ N~ IV 7 a 

V C O) O) a U N U 4• C) E O c] r tp y d tp W M 10 G) L o~ 01 N~ N p _ 3 N E d' O a Gl •r E ~ Cl +~ •r N L N N N N ro U a E T E ~d C L Q N ~p • G/ a L N r g y.N L t0 r o1 O G • n L N a~ ~0 L N £ 4- L N+~ OJ N y N L Gl r W U X i•~ 41 r 10 O d•r 'O L n . O'O ~0 i~ C~ r N tp d W O U ro ro L4F- ++ 3+5 J N ' - X u G) L 1 y C E O O O 3 0 F- c N L N o E ro U ~ a W N U U L r a C ~ U! t0 N CV f0 ro G7 d . E ~ Ol •r i-~ i-~ N O L 3 7 L N•3 a d C O fd GJ N I 1• f0 a N G a d L U X ~0 G! C >~ C y .(0 •r N y•r L a Gl L N N N T d ~ lO L •+1 O) o U 4) C E a C•r N E •c O M d L f0 10 ro•r 7 N i L L C y L E o •n N L c d o c y a ~0 7•r •r L. r o C) •r O~ O r CJ o s GJ 7 f0 o c u L . N o n o y•O a ro> N•r L y r y E u N o y ~ r ~ d s u u y u 3 

. N N C O C v L E N Q L C L r L~ > $ O Cl T ro N C E U Cl O i- 10 W•r •r L Gl 7 41 tU U O 

, . O U H w'r C c9 N O , c o W d rti 41 GU ~> O_ d E U•r ¢ ro r y oe E tq U U+•~ 3 E N E O i•> 

 ' 

N 

b a N L N T O ci T 

mv I L O O N N L 10 tp f0 L ' a O•r U Y T y N a a L a O \1- N~F~ ~ O Cl ro C \\ ~O \ i O ro C+1 L UJ a O i L L Gl L I~ ' L L X=t0 ~ t O 3 O E O L L TL M O W a d ct 41 M tD t0 rl tD I, r1 Op M N M 

a 

I o y.+ 

aGi a E o 

L~ 

u~i E Z Z > > > 

N I£ 

N M N 

C O Z O Z O 

~ d O E LO N Ql Ol N tn lD f+1 1~0 l~0 O~l%N t~0 Gl 10 d O O O•F O O•F N O ri O O O V' O O ~j Z N C v O C! U E OO ln cf O lO 03 a d• N O ct C tn N lO 01 t~ V• tn tn pp } C. ~ . 

IU 1 r0-I rl r^•f a r~-1 N Ln '-Ni rN-1 ~ ~Off rN-I 

9-23 
'O 

7 

0 

~ 

C 

C 

u 

v 

O 

O 

aL O 

tn 

~ 

W 

W 

-1 

w r ' 

~ I ~ 

~ 

'•i 

LLO

1 

01 

J 

~ 

ni o m o rn r~ ~ ~ ri 

n rl ^ rn 00 

u io - rn a c ++ W. 

9 C al c a ro ro m a € m € e m +1 d N L (! iJ C m d..) N m L E N ro > 

IU 

W rr J ~••~ 

I 

Gl 

n m m ovi a ' m oz 

L cum_ 

m •r v > 

ro uvv a m +~ L c G .Cl 9- m a O m Gl L. U•r C V- N +1 L N •O N •OL C Gl N N m r f-. c oa - m ro d m v a v- c y•r m a 

1O Gv ~= L c L. L 3~ ~ .- o C +1 v _+J 

~ m L N Cl GJ r 7 O E N N+J L •O i•~ O C L•O L m 7 N U r m . n L m C r~.O m C r 

'. O u•r m"O c N G1 7 Ol 7 G! o d u o u B N•r m u •O r C1 d •E •r 7 L•r U 

n. m r N 1- c m u u L. L N d s ~ x m L. ai m o O w T~c t r ai arv•uv ~ - 3-0 0 3 aE Lmr u 3 m.~[ O! G c O L 1' N 07 U •O n2' •1•~ C L L M N o ro L N W V- 41 N Gl r'O N o fo C 4- ~ tn m•O r i-+ ' c N 4- o L N c E a r-1 L T•r a . v v m r o E m s H € L. c m O N - s r r G1 y L u 7 d..~ 

L C a r• N C) E L.0 ~! U C m s m G! O u•r Cf r L N ^m •• . IV •r Y u u O. V Cl N N O . V-. •r ->~ L U•1-1 C•r •O 4- 4) h- 1.~ U~ L N d G! L €+~ •U N r O C OI O f-. Gl L. i~ m ri X L. N•O N L. .• C O C C 3 41 7 C r N 7 N 

u C.0 r N o 3 G7 N .O r r G1 ~ N•r n C u7 U L o 7 Cl m m .• 0- G7 N L d!tl y•N o C/ N L 

N .- N O Ol C N 7 L ~ N•r O t T E•r L L O) .1-1 N i-~ N X O n 7 a f17 •O r- E ro V) f0 - d N N u `~ d L a N . v iJ d-1 m O r C m u tn m L U 4..~ G) C f- m O OJ n 4.. o-. m TL • m 3 m.n v .o o c 3 ro m m+J ai In +1 x 

6/ O 41 U Of •N O m O C> N u 01 O N>j i O u o L u n. ~ C! L o € m~F• c c o Ol L 3 C N i-1 [y N Gl 

4-. N'II f17 L •i •L m rl T o _ ~ ~F E C1 d•~ U7 a C1 . 6f .-• N.FI m N 07 N N > N r L O L O m G! m• r 01 G) 4- N•r M•. 67 Cl .0 u a•O N •C a m N•r N 4- .0 r O L i-~ G ' 

vo E v+1cui c v o•rw . Y rtf u r y. Nr.rEmTs 'O 7 a C m G1 G) L C 4- r U r~ N Cl N•O L N N L O m 7 I C7 +1 X•r L r'O E 7 m O N y L C L Cl •O N~ r N U N U V•O ~ N E7 m m r• O N •O r~ U m N 41 G! L N r • L 1•O r L•r- L N C7 m m Gl m r m m+> m +1 ya O N L rl N i-1 Y f- •O L 67 El c O 01 u C/ O s Gl •r Y E u 4- r r N> ro a u u ~ 7 N N N rm C m G7 C c 3.a L r• L L L E r 7 y C X 07 a7 c.O m Of N lD .a u o > ai o ar •r m u• m o c o ~ ar ar p • N N r• (;) Z 4- L ~••• m U E C•r N N U U Ol LT. . C>aj•r N a•r d 11 € O N m L •U •C M O 'O I- D1r L 3 L t0 C1 € 4•1 C N O+1 C ^ r Gl O U N d-~ 0! m G/ ar C U O N O r . • N N Cl .. .. .. C m L i U T r• •r 1~ II i m N N N d G! U C L. (l L O m •1.~ •1-~ N r~ N C 7 i•~ T T T L 4- C) d..~ Gl T m T d•N G7 N~ N m m m 7 O i~F N • m m E Ol N u N O N r'Q a •O N 1 m i-1 N J r 7•O D1r 

7tr C/'~ •- L X m tD O O N a L N S.. '6 0! L. 1 • i-1 L N m a nT 'a o N. m v rorI o•r v m 

YI O•r m.0 7 C) ~ V- ri N T X d Gl 7 Gl •N Gl ty •.0 G) N n U •r-I L L O N O m C/ +.~ ~ N O) m C N U L•r O N 11 .0 m L O 1-1 ~ ~+.~ N C O C•r y E r• C+~ N L]C C i.~ r~ .O a N 1 ^i N 7 m a m r p) O O•r L u 

M U.0 v V... CI 00 U•r W X O O W O O .0 X L•r y L N L n G! m4F d v VI rl d U u W U U CI 4- U.G N d E O 

O/ N }1 N N } 7 N r t0 O N i G! ^ N 

kU C O o N 61 ITO U1 O O E ch N M a o L O `O •• ~ 7 L•O N L N a ^ 7 td N 7•r f.1 N C - C ^ 7\ T~ X N C N •O L (p C•O L C V! d•1 9 O O m C~a >>•r L•r O N L m N~ N T•r ^ C L N m C L•r ro+1 L+1 rl N O.0 '•O O.O +~ m•N L l0 m Op m +~ a L m O •O C C T a d N •O C Y m ^ OD C W'+C7 X 7 L O ~ O •• m XcY LO XO O O•r }1N t0 W O0p tD O a 1~ u N u t0 'O C/ N O N N ri a l, U 3 m 1, rl c!• 1-1 N ct rl u 

.0 

m +~ C 

E o a9 

7 IU m C1 

O }1 G I, S. 00 1, W OO 00 CM a O M O O O O O 

O +i N C O N 

C E ^'/~ ~ n t~D l~D l~D 

°u S `1'i r~-1 rm-1 .~-1 r~i 1•~•I 

9-24 
N 

L ~ C 

a io T N 

a~ v •r c 'o 

~ L i•~ IC U ~ F• N 

r O•r E O'O 

3G! N X C N C 

N.O+) i rou'O 

L 10 N O. N R1 

> ~ E rn~ _T • 

UIM4-'O L N r C 

O O G! a 1 d •r 

C d N r L N o r 

M i O• E U c•r y 

7 

C TL 

'OE

•rN ; 

~ i~ ~ r U N 

C S• N d W N U r 

G) N L v o r o 

~) L f4J 

a O. cOi • U 

F

N E r ri 

t6 +- 

O r

7 N 

N•r O 4! E = ^ c u n> a 

~ U U•Oro F a- m '~ 

d 'O 7 L L 

L++ d c °c) •r : io ° 

J, N N E 0 

•n f6 41 41 O•r r C 

y•r i r d i• X•r 

r o r > a o E 

m•rt d a L p F- v V) U m•O a.- 

N 

7 

O 

X 

W '. 

+~+ 

rl 

B 

7 

C 

~ 

O . 

v 

Z 

N 

O 

O 

U LU 

w

cm 

LLI 

¢ 

••-1 

O 

0 

C 

~ 

rl 

rn 

J 

2 

H 

u 

w 

~ 

N 

w 

ai 

a 

> 

N 

.O N

0 

io • ro i 

E ro 

N _r

c 

o 

 G) 

4- ~ 

O•rL 

E U 4- 

N L C O 

r~i0-~ L N a •r U 

G! QI UI 

a.0 v O 

L C ro+J 

U •r Vi N m 

G/ N ~ 'O 

~ ; r 10 0 0 

~ 7 U•r 

c• ~ cN 

~ c o 0 

r 7 L 

• O iJ 

G1 w i 

~ O U ~ 

•rN L 

v u rn 

so 0 T c LaC+1 

u .C7 Z7 7 

U C 4) N o .r 4J d 

L N L 

i 

7 N •r •r 

rJL C N ^E-roo 

E L 

L N 

U r d 4- tm 

c ro ro 

o E r N L •r Tr N 

 c.Liro33 0. 

~ Y 

N 

^ N 1 N 3 T 

 a 

o r. Y \ N N 

C~ N 'O X N 

G! i 

3 vLao .voi .. OUO O 10 n to v o• 

: ~ 

U 

cai 

L v 

N 

K 

N 

N 

•U 

a 

N 

~ 

~ 

~ 

m 

~ 

N 

i C/ 

a 

a 

O 

O. 

N 

7 •^ N 

f

tm

0 N fTtl 

ro o 

C

~n~~ 

v G 1- O 

co 

r— 

m 

~ 

a 

N 

C 

N 

L 

a 

Gl 

+1 

N 

~ 

^II O 

ln C - 

t ^N 6 O 

7 •^rl ^ N 

O) T O u'f T 

Lr tn NM 'O 

f0 N 3N ^ tL') O O 

K v Cf rl rl M a' 

r-. 

Lr) 

ro 

a 

~ 

C 

Y 

O 

11 

N 

i 

N 41 

3 641 

€iz 

O 6 

E N E 

~ L i 

r ro O 

r y C 

C7 r 

.

L u a c 

a ro c 

C! L N 

c 

•Vi 

ar 

co 

f•- N r J 

ro •r 

4~ L r 1n i• 

Cn O 

d O C! O 

~ 

01 Ur ~ L! 

L0 0 

nE_ 

C L 

+1 

~ wro 

V 

4- 

O O E E O 

C 

N X 

O 

L V 

L E L 

4- d 3 ^ a ai ai 

U +~ 

N N 

a 

t6 

L • 

iJ OJ 

C O • O • f N 

L 

N 

.i 

i 3 

C r N N 

cr ai . ai 

4J U 

rn 

G 

v- a 

~ rO X. 

a +1 

> o v ro c 

 O 0 41 rl r Nm 

41 E G! O~ L al 

o m ~ T ~ ° ~ f L 4- 41 J t0 N 

Cl N N ~ C7 

L a a c i 

N 'O 

O 

'O M N 

O 

X ~ M C X Gl 

41 

N W 

O 

O_ 

T rl 

L 

a a 

o c 

N i T i y 

8 ai o 

0 o um T ~ 

C t.°.) F 

cc 

 C LC C 

++ s°. o ~ , m rn ~ 

u~ •TV- ~ 'o - 

r E N L O 4- 

EL 

C 01 O O O) •C N N N 

r~

•r i C DI C1 ttl m 

N+ N 41 •r C .G 'E ~ a i c N 

r•ur aa 1- 4- 

d-~ N M 1,41C r- r 10 ° 

r N o L 10 ftl r u L N 

rV O •• w Cl ^ U 

c ro u rt r o 

L L r N ro•r 

G! i•~ •N r ~ Y- r• iC T MtP) 

~j •r 7•r i L•r •r L C M 

= 3 a v ro N uW 

 ' -

~ ~ 

- N ~ F• O 9- L C r• 10 

. Uf C 04F1 r O i ~ •r 7 

a O° N T r N •C i CNl d 

t0 ~ •N N V r• N N t6 L L 

-ovaocao u 

Orr yLrpp 00 f0 

o c -° aJ t- (L) -a) 

N M ^ C•r •• >r L 

Cl o M N n M V- r• O) 

417 Z O- +> N O O¢ u N 

RJ N r 

N 

Gl 

•C • +1 

rC L N ~ •O 'O L N 

ui o T ~ a 

3 ro o 0 o vC 

O Cl N N N O Ol 

"c ~ ro m rt•ac 

L Vai N N ri ro 0

ry N M N N 

 rL 

m L 1' 

L O O 

7.r u 

oroc+°+ 

X 7 ro f• 

w a c. 

a 

~ 

41 

c 

ai a 

E O 

N L 

L +J 7 Gl 

N E 

ro 

a E 

O 

••- E 

+J a 

ama Ci 

O iJ 

N C 

O N 

U E 

C 

O Of 

U O. 

M N LW 

~

a)

M•^>f 7 ~ ~d N' 

^7t0 V O L•O 7 ~D IIa 

N+~ N roN O 

 tn 

 M +~ d N d• ~ 

T a'O C 41 'O N o -ic roXCOV-co 

M U O ri v C/ N u ro rOa 

^ Y 

7 m 

~ Z 

~ 

O 

W 

t0 

N 

r-I 

Y 

CI 

z 

00 

Z 

41 

0 

E C 

n 

C! n 1-~ t!1 

m ~ aciva w 

r N E rl 7 

3 tI 

OD 

r O •r ^ F> 

•E a 01 i cV X o 

O 

r X C7 O O•r 

N GJ L 4- Z E 

lD O 

l0 

tp 

ri 

tD 

01 

rl 

N 

0 

~ c 

N N +> 

C ^m o 

rl 'a u 

9-25 
41 

T :3 r 'p 

io L •.- c m r• 

O r O 

N GJ > 

fp > r• 

u r• ro 

U IO 

O c rtf 

ul ro L 

aYf 4- C ooai 

L U 

a 

L c 

4- d •r 

C 

r- n ~ n 

fO•r Gl 

€ L 07 

r 

d

y 

•C L

O 

E No L i 5 u 

~ c B 

L~ L 

L E +1 

o X 

0p L Ol C = 

E i 3 d a7 

d'O V^ 

rl N ¢ 

•1•t ¢ 

7 

N 

cle 

Cf 

z 

O 

 0 999999 

rl 1J M rf rl rl N M 

0 000000 

lD O OD lD lD lD N m 

01 •1 ~ W Ql Ol Ql 01 Cn 

rl tn rl ri ri M u) 

u 

h 

u 

G7 

V~-• d 

v 

> 

L 

N 

O 

L1 +

O \ L L\\ ct v\\vv 

N TL L L 

r~-I 00 'D CO '~•I N 00 m 

11 11 II 11 II 

¢ m V CI w 

h 

b 

° m t` \ 

L L 

O L 

~L 

M 

n 

ai 

r• 

m 

+a 

a 

E N 

LL 

I ~ O 

U 

C 

~ 

w 

C 

1N 

a 

u 

tn 

n 

•O 

d 

7 

C 

~-1 

O 

u v 

W 

Z 0 

O 

U. 

c 

H 

6.1 

u. 

W 

-1 

¢6./ 

t7 

O 

O. _ 

C 

2 

e-i 

Q1 

W 

m 

aFs~ 0 

0 .

o

r U 

ul + O O 

O'C C 1 ~ 

~G( 7 L.A ~ 

W O n 

a 

~ 

C 

C7 'G 

€ O i Y 

~ E 

E I 

C 

O 

a:i a 

C L n 

N C 

CD U € 

C 

uo z 

~ 

n 

rn rl 

b 

+1 

ai

v 

L 

C 

d 

ro 

u 

ro 

v 

41 v ~ rn 

mc=•r 

C rtl C.7 d 

E M 7 O >> C 

a 

ro 

ro 

r N C O L 

d C7 +- 

L C! Y N 

•d+5 L t0 f0 (C C! +1 L. • U 

v .c m ai v ~ n ro c ro~ H(N C rL•r U O 

4- N O E ~ 'O 

• O i ^ ul •r 

O O 

i L 0

rocaMimrd ~ a°s 

C•r .r 0) 'O L. C O 

r E 

•U 

n f0 r 

ro"OLr•1~ y 

tp rdL O +1 O 

L L L. c ro E 

41 CJ •r U•r 

r• +1 

r N 

c dr• (C Gl V1 

E 

•U •r 

Of 

U.c N E N ; 4- 

O"O ~ C! 'O C! C O C 

++cmwa wo ~ nT i

m ro 

'O L C/ N Vf +.~ • 

M ~ n 

O 

C1 TR7 L E0 (C ~ n r• L 

Y L C U O Cl i•~ Of O 

' r d•r C N r• d•r v1 

E O U•r i0 d G7 

r L i0 tn a 

r• ++ •r ^. c(d fo 

r• > ~ n 

d•r Cl 

L i M M O d 

1n y.H r• r tfl C 

i•~ 07 

d• C 

G d C r-r- •M.r r E ~ (O 

. ~ N 2 U U 3-~ ~ •O~~/1 m u 

ai o 

J Z 

E E d d d C. N 

C ri N M 

o ~ 

ro ao 

M M 

y 

M N 

7 

w 

~ 

f — 

W ]C 

S O7 

U Z 

L 

7 W 

O Y L O 

O'00 M X+1 N 

ri tC0 N Q E {3 2 

0 0 t0O N 

~ roM 

~ 

ro 

c 

0 

E 

o. 

1 a v0, 

1 R1 

C 

C!07 

O. O

L. 

I ~ 

. 21 

L N 

U 'O 

d E U 

O C 41 

O U 

O 41 r 

dII 

~ N 2 0 

• 1p C J 

~ n O ro +1 n b 

o n+ 

n r d 

• E N 

a vi w 

41 C 

L C L 

N L p 

w Y r• 

3 II cU 

a ~^ 

W t() 

L ~ y 

+~ • ^ / 

a~ a•~ 

C N II 

o cn 

1-1 L Q 

Y U 

m Z •E .^ 

c u 

~ O 7 y L ~ 

u ++ 

C U L ai ai ro 

U 

N G! O 

N N 

C 0) > 

C r• 

E r rt1 ~ c m 11 

E U O 

O1 0 Q 

u II • ai 

N 2 r 

ro ~ •L 

o 

t 

d ^ U E 

G! C 7 

w d O 

ro oL ~ 

rn ua u 

~ 

II o r• +, 

L 0 C1 

w II U C L 

U O L 

2 E E U 

o cv E o ++ M 

I

i• E +" 11 ~ 

a u n. 

+1 +1 

0 o L v d F o tm 

-0 Ltl L o 

U L Ol C i 

L w O L II - 

N'O 7 1Uil aW 

E G1 a O T C 

O a ' L. L U O 

7 i 0 • ++ E 

toi v

ui . ..~ m_Em.i._..-o .. . 

1 Gl 7 +> •r 7 

41 r N 4a 

II o II r- i a 

C L 

N II Y II II u 

f C Z ~ ToE 

Z u1 

^ ~p 

o T o c rnN 

 n 

L •4 .c°w L • OO 

+1 Cf 4-) 7 d C 

E O E in L O i+> c U L 

c 

0 

 o•o E O CL 

a L 2 i > r i > N ` °rio vei 

uCi II ~ so.. oi a 

II 

ro = a > p 

a> ro ar° • w 

r ~ o a u w 

L O 4- M 

E O ~ 

O 3 = E E 

O Q O X N a 

C d L 

L 

iJ tN •{N N d C 

t0 N C] 

w Q 

O L Gl 

`O 

7 V 

afv- 

• O •Vl 

a d ~ •r n 

V- m 

• M 

r 10 C1 

C 

io vt0 r 

aci 3 aL• 

d L 

C O 41 C1 (7 

¢ T C 

In 0) 

roc 

'O L aim U•r 

U C 

O al • 

i c u 

O G/ •r 

c Y 'O 

a~ 

L 

+1 

d IN 

O d 

U rt1 

r L d 

t3) L. L o 

ro~ ~ 

W 

'O r• d 

L N L 

'O 

L V ~2 

L> U YI M 

tD t0 E 

r1 

~t •.C- mC1 

N U 10 Q 

L,rt1 

a M Cl = 10 L r d 

4- 

Eo s0•. 

n M 

 o E i c 

~ C. d~ L tp i

G. 

pJ Ip 

°' c axi daci m m 

r •

L. N 

r•i = rl u E z g E 

oa 

m C7 en W 

V ., 
The lower. or intrathoracic conducting airways include the thoracic trachea, bronchi, and bronchioles. Species variation of lower airway structure 

is large, as recorded at the NIH workshop on comparative biology of the lung 

(National Institutes of Health, 1983). The thoracic trachea and bronchi have 

epithelial and subepithelial tissues similar to those of the upper conducting 

airways. In bronchioles, the epithelium does not contain mucous (goblet) 

cells, but in their place are special.ized nonciliated bronchiolar epithelial 

cells, which in some species can appropriately be called "Clara" cells. 

Subepithelial tissues are sparse and do not contain glands. 

The ciliated cell is the cell in the upper and lower conducting airways 

in which morphological evidence of damage is most readily seen. This cell is 

primarily responsible for physical clearance or removal of inhaled foreign 

material from conducting airways of the respiratory system (see Section 9.3.4). 

The effects of ozone on this cell type, which is distributed throughout the 

length of conducting airways, are detected through various physiological tests 

and several types'of morphological examination (Kenoyer et al., 1981; Oomichi 

and Ki ta, 1974; Phal en et al ., 1980; Frager et al ., 1979; Abraham et al ., 

1980). 

The other portion of the respiratory system directly damaged by inhalation of 0 3 is the junction of the conducting airways with the gaseous exchange 

; 

area. The structure and cell makeup of this junction varies with the species. 

In man, the most distal conducting airways, the terminal bronchioles, are 

followed by several generations of transitional airways, the respiratory 

bronchioles, which have gas exchange areas as a part of their walls. In most of 

the species used for experimental exposures to 0 3 , (i.e., mouse, rat, guinea 

pig, and rabbit), the terminal bronchioles are followed by alveolar ducts 

rather than respiratory bronchioles. The only common experimentai animals 

with respiratory bronchioles are the dog and monkey, and they have fewer 

generations of_nonrespiratory br-onEhioles than does man as well as differences 

in the cells of the respiratory bronchioles (National Institutes of Health, 

1983). 

9. 3.1.1.1 Ai rwa rs~ 

9.3.1.1.1.1 Upper airways (nasal cavity, pharynx, and larynx). The 

effects of 03 on the upper extrathoracic airways have received little attention. 

The effect of upper ai rway scrubbi ng on the 1 evel of 0 3reachi ng the more 

distal conducting airways has been studied in rabbits and guinea pigs (Miller 

et al., 1979). They demonstrated removal of approximately 50 percent of 

9-27 
ambient concentrations between 196 and 3920 pg/m3 (0.1 and 2.0 ppm). Earlier, 

Yokoyama and Frank (1972) studied nasal uptake in dogs. They found uptake to 

vary with flow rate as well as with 03 concentration. At 510 to 666 pg/m3 

(0.26 to 0.34 ppm) of 03 , the uptake at low flows of 3.5 to 6.5 L/min was 71.7 

t 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 pg/m3 (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 3 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 

03 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 pg/m 3 (0.2 ppm) of 

03 continuously for 1 and 2 weeks, Ikematsu (1978) reported epithelial detachment and disarrangement and a slight cellular infiltration. The significance 

of these observations to the function of immune mechanisms in host defense is 

unknown. 

9.3.1.1.1.2Trachea. Tracheal epithelial lesions have been described 

in several species following exposure to less than 1960 pg/m 3 (1 ppm) of 0 3 . 

Boatman et al. (1974) exposed anesthetized, paralyzed cats to 510, 980, or 

1960 Ng/m3 (0.26, 0.50, or 1 ppm) of 0 3 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 desquamation of ciliated epithelium•at 1960 pg/m3 (1 ppm) of 0 3 , but none . at510 or 

980 pg/m3 (0.26 or 0.5 ppm). 

In rats exposed to 960 or 1568 pg/m 3 (0.5 or 0.8 ppm) of 0 31 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 

electron microscope (SEM) and were not obvious in rats exposed to 392 pg/m3 

(0.2 ppm) of 0 3 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 pg/m 3 (1 or 2 ppm) of 0 3 or sulfuric acid (H 2SO4 ) 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 3 . Animals exposed to both pollutants had lesions similar to those exposed to 0 3 alone. 

By using SEM and transmission electron microscopy (TEM), Castieman et al. 

(1977) described shortened and less dense cilia in tracheas from bonnet monkeys exposed to 392 or 686 pg/m3 (0.2 or 0.35 ppm), 8 hr/day for 7 days. 

Lesions occurred as random patches or longitudinal tracts. In these areas, 

the nonciliated cells appeared to be more numerous. The TEM study revealed 

that cells with long cilia had the,most severe cytoplasmic changes, which 

included dilated endoplasmic reticulum, swollen mitochondria, and condensed 

nuclei, some of which were pyknotic. In lesion areas, evidence of ciliogenesis 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 vari abl e and 1 ess severe i n the 392 pg/m 3 (0.2 ppm) 

group. More extensive and severe lesions of similar nature were seen in 

tracheas from rhesus monkeys exposed to 980 or 1568 pg/m3 (0.5 or 0.8 ppm) of 

03 in the same exposure regimen (Qungworth et ai., 1975b; Mellick et al. , 

1977). 

Wilson et al. (1984) evaivated the response of the tracheal epithelium 

from bonnet monkeys expased continuously for 3 or 7 days to 1254 pg/m 3 (0.64 

ppm) 0 3 using SEM, quantitative TEM,. and autoradiography. Extracelluiar space 

was increased and foci of stratified epithelium were reported. Changes in 

ciliated cells were generally simiiar, but more severe, than those reported by 

Castleman et al. (1977). These changes were more severe at 3 days than at 7 

days when both the volume percentage and population density had returned to 

control values. They also reported fewer and smaller granules in regular, as 

opposed to small -mucous- granule (SMG), mucous cells. These granuies also 

lacked the biphasic appearance of those seen in control monkeys. At both time 

periods SMG cells in exposed monkeys were more numerous and had greater numbers 

of granules than controls. 

After mice were exposed to 1568 pg/m 3 (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 su,rf4ce of _the tracheas was 

examined by SEM by Ibrahim et al. (1980). Short and normal-length cilia were 

9-29 
seen at 6 days, but at 10 and 20 days, a marked loss of cilia and few normal 

cilia were seen. Some of the nonciliated cells occurred as clusters and had 

wrinkled or corrugated surfaces. After the mice breathed filtered air for 

10 days, the surface morphology of the cilia returned to near normal, but the 

clusters of nonciliated cells were still present. Earlier, Penha and Werthamer (1974) observed metaplasia of the tracheal epithelium from mice exposed 

to high concentrations of 0 3 (4900 pg/m3 , 2.5 ppm) for 120 days. After the 

mice breathed clean air for 120 days, the metaplasia disappeared, and the 

epithelium had a nearly normal frequency of ciliated and nonciliated cells. 

9.3.1.1.1.3Bronchi . 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 conduction 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/m3 (0.5 or 0.8 ppm) of 0 3 8 hr/day for 7, 28, or 90 

days. Lesions at 7 days were similar to those previously described by Mellick 

et al. (1977) and Castleman et al. (1977), as summarized above. At 28 and 90 

days, lesions were not readily apparent by LM, but extensive damage to ciliated 

cells was seen using SEM. Uniform shortening and reduced density of cilia 

were seen in linear, parallel arrays. In these areas, the numbers of cells 

with a flat surface covered by microvilli increased. Wilson et al. (1984) 

reported similar changes in primary bronchi from bonnet monkeys exposed to 

1254 pg/m3 (0.64 ppm) 03 continuously for 3 or 7 days. 

Sato et al. (1976a,b) studied bronchi from vitamin E-deficient and control 

rats exposed to 588 pg/m 3 (0.3 ppm) of 03 3 hr/day for up to 16 days. Using 

LM, they did not see bronchial lesions with asymmetrical swelling and surface 

roughness, which were obvious with SEM. The observations of Sato et al. 

(1976a) support the concept that lesions in conducting airways are best seen 

with SEM and that LM tends to underestimate damage to these ciliated airways. 

In these short-term studies, lesions were more prominent in vitamin E-deficient 

rats. This is in contrast to later studies in which Sato et al. (1978, 1980) 

exposed vitamin E-deficient and supplemented rats to 588 pg/m 3 (0.3 ppm) of 0 3 

3 hr/day, 5 days/week for 7 months following which they did not see clear 

differences due to vitamin E with SEM or TEM. 

9-30 
Yokoyama et al. (1984) studied effects of 0 3 on "middle-sized bronchi" of 

rats fixed by immersion rather than infusion via the airways. They compared 

the effects of 3 hr/day exposures to 1960 pg/m 3 (1.0 ppm) 0 3 for 30 days with 

those following 980 pg/m 3 (0.5 ppm) 0 3 6 hr/day for 60 days. They reported 

increased mucus and irregular loss of cilia, especially on the projections of 

mucosal folds typical of this type of fixation. Changes were less severe in 

the 0.5 ppm group. 

9.3.1.1.1.4 Bronchioles. There are two types of bronchioles with 

similar basic structure: those without alveoli in their walls (i.e., nonalveolarized) and those with alveoli opening directly into the3r lumen (respiratory 

bronchioles). Man and the larger experimental animals (e.g., nonhuman primates 

and dogs) have both nonrespiratory and respiratory bronchioles, whereas most 

of the smaller experimental animals (e.g., mice, rats, and guinea pigs) have 

only nonrespiratory bronchioles. Because the types, functions, and lesions of 

epithelial cells are different, these two types of bronchioles will be discussed 

separately. 

Nonrespiratory bronchioles are conducting airways lined by two principal 

types of epithelial cells: the ciliated and nonciliated bronchiolar cells. 

The latter cell is frequently called the Clara celi. Although man and most 

animals have several generations.of nonrespiratory bronchioles, some nonhuman 

primates have only one.(Castleman et al., 1975). The last-generation conducting airway before the gas exchange area of the lung is the terminal bronchiole. 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 3 exposure occurs in the central portion of the 

acinus, the centriacinar region, which includes the end of the terminal bronchiole and the first- few generations of either respiratory bronchioles or alveolar 

ducts, depending on the species. The centriacinar region is the junction of 

the conducting airways with the gas exchange tissues. The 0 3 lesion involves 

both the distal portion of the airway and the immediately adjacent alveoli, 

the proximal aiveoli. In animals with respiratory bronchioles, the lesion is 

a respiratory bronchiolitis. Regardless of species differences in structure, 

the lesion occurs at the junction of the conducting airways with the gas 

exchange area. 

9-31 
Terminal bronchiolar lesions in rats due to inhalation of < 1960 pg/m3 

(< 1.0 ppm) of 03 for 2 hr to 1 week have been described by Stephens et al. 

(1974a), Evans et al. (1976a,c), Schwartz et al. (1976), and others (Table 9-1). 

These changes were recently reviewed by Evans (1984). Ciliated cells are the 

most damaged of the airway cells, and fewer of them are found in the bronchiolar 

epithelium of exposed animals. Those ciliated cells present tend to have 

cilia with focal blebs and blunt ends. Damaged ciliated cells are replaced by 

nonciliated bronchiolar (Clara) cells (Evans et al. ; 1976a; Lum et al., 1978), 

which become hyperplastic. The typical projection of the nonciliated or Clara 

cell into the lumen is reduced, and the luminal surface has increased 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 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 generation, more distal nonrespiratory bronchioles than in 

the lower generation, more proximal nonrespiratory bronchioles. 

In an earlier study, Freeman et al. (1974) exposed month-old rats continuously to 1058 or 1725 pg/m3 (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 

openings. In the 1725-pg/m 3 (0.88-ppm) 03 group, the fibrosis was pronounced 

and sometimes extended into terminal bronchioles. In the same study, Freeman 

et al. (1974)exposed month-old rats to a mixture of 1764 pg/m 3 3 (0.9 3p m) 03 

and 1690 pg/m (0.9 ppm) nitrogen dioxide, or to a mixture of 490 pg/m (0.25 

ppm) 03 and 4700 Ng/m3 (2.5 ppm) nitrogen dioxide. After 60 days of exposure 

to the 0.9/0.9 mixture, Freeman et al. (1974) reported that "both grossly and 

microscopically, the appearance of the lungs was characteristic of advanced 

experimental emphysema," referring to earlier nitrogen dioxide exposures 

(Freeman et al., 1972). Freeman et al. (1974) did not report emphysema in 

rats exposed to 0 3 alone, only in those rats'exposed to the 0.9/0.9 mixture. 

The topic of emphysema is discussed later (Section 9.3.1.4.2). 

Results differ in four studies of long-term (3- to 6-month) exposures of 

rats to < 1960 pg/m 3 (<1.0 ppm) for 6 or 8 hr/day. The differences appear to 

be due at least in part to the methods used to evaluate the bronchioles. When 

9-32 
using only LM to study effects in'rats exposed for 6 months to 980 pg/m 3 (0.5 

ppm) for 6 hr/day, Cavender et al. (1978) did.not find exposure-related.lesion.s. . 

Barr (1984) exposed rats to 1862 pg/m 3 (0.95 ppm) 0 3 8 hrlday for- 90 days" He 

examined the bronchioles with LM, TEM, and SEM. Using SEM he reported loss of . 

apical projections of nonciliated cells and loss of both density and height of 

cilia. Boorman et al. (1980) and Moore and Schwartz (1981) reported significant bronchiolar lesions following exposure to 980 or 1568 pg/m 3 (0.5 or 0.8 

ppm) of 0 3 8 hr/day for 90 or 180 days. In both studies, loss or shortening of 

cilia and flattening of the luminal projections of nonciliated bronchiolar 

cells were observed in terminal bronchioles at each time period, including the 

end of exposure at 90 or 180 days. Clusters of four to six nonciliated 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. After 50, 90, and 180 days of exposure, the nature 

of the junction between the terminal bronchiole and the alveolar ducts changed 

from the sharp demarcation seen in controls to a gradual transition with the 

appearance of a respiratory bronchiole. The presence of this change in distal 

airway morphology was confirmed by Barr (1984). Both ciliated and nonciliated 

bronchiolar cells were seen on thickened tissue ridges between alveoli. This 

change could result from either alveolarization of the terminal bronchiole or 

bronchiolization of alveolar ducts. Although this'change in the airway morphology persisted, the changed segment reduced in length after the 180-dayexposed rats had breathed filtered air for 62 postexposure days. The addition 

of 1 to 10 mg/m 3 H2So4 to these concentrations of 0 3 for the same exposure 

ti mes di d not potenti ate the 1 es i ons seen i n the 0 3-a1 one rats (Moore and 

Schwartz, 1981; Cavender et al., 1978; Juhos et al., 1978). 

Ozone-induced bronchiolar lesions in mice are similar to those seen in 

rats, but the hyperplasia of the nonciliated cells is more severe (Zitnik et 

_ .. _ .. . . _....__.. . .._ . . ......_.__, 

al., 1978); Ibrahim et al., 1980). Following high concentrations (4900 pg/m , 

2.5 ppm), Penha and Werthamer (1974) noted persistence (unchanged in frequency 

or appearance) or micronodular hyperplasia of noncilated bronchiolar cells for 

120 postexposure days following 120 days of exposure. At lower 0 3 levels (980 

or 1568 pg/m3 , 0.5 or 0.8 ppm), the hyperplasia was pronounced (Zitnik et al., 

1978; Ibrahim et al., 1980). Ibrahim et al. (1980) noted hyperplastic clusters 

of nonciliated cells 10 days after exposure but did not make.observations 

after longer postexposure periods. 

9-33 
Guinea pigs were exposed by Cavender et al. (1977, 1978) continuously to 

1 or 2 ppm of 03 for 2 or 7 days in acute studies and to 980 pg/m 3 (0.5 ppm) 6 

hr/day, 5 days/week for 6 months. Morphological effects were studied only by 

LM. The acute, higher concentration distal-airway lesions were similar to 

those seen in rats and included loss of cilia and hyperplasia of nonciliated 

cells. The authors reported that the long-term, lower concentration lesions 

were more severe in guinea pigs than those in rats exposed to a similar regimen. 

The lesions were no more severe in guinea pigs exposed to a combination of 

H2SO4 aerosol and 03 . 

9.3.1.1.2 Parenchyma. Ozone does not affect the parenchyma i n a uni form 

manner. The centriacinar region (i.e., the junction of the conducting airways 

with the gas exchange area) is the focus of damage, and no changes have been 

reported in the peripheral portions of the acinus. 

9.3.1.1.2.1 Respiratory bronchioles. Respiratory bronchioles are the 

focus of effects, because they are the junction of the conducting airways with 

the gas exchange area. However, not all animals have respiratory bronchioles. 

They are well developed in man but are absent or poorly developed in the 

common laboratory animals frequently used for 0 3 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 pg/m3 (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 03 concentration. 

The pathogenesis of the lesions due to 1568 - pg/m3 (0.8 ppm) of 0 3 forperiods 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 

9-34 
stem cells in renewal epithelium, and both contributed to the hyperplasia seen 

at the latter exposure times. The inflammatory exudate included both fibrin 

and a variety of leukocytes in the early phases. In the latter phases, the 

inflammatory cells were aimost 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 pg/m 3 (0.5 or 0.8 ppm) of 0 3 8-hr/day for 

90 days had a low-grade respiratory bronchiolitis characterized by hypertrophy 

and hyperplasia of cuboidal bronchiolar cells and intraiuminal accumulation of 

macrophages (Eustis et al., 1981). After the 90-day exposure, the percentage 

of cuboidal respiratory bronchiolar epithelial cells was 90 percent rather 

than the 60 percent found in controls. Intraluminal cells, mostly macrophages, 

reached a maximum of a thirty-seven-fold increase after 7 days of exposure. 

Their numbers decreased with continued exposure, but at 90 days of exposure, 

they were still sevenfold higher than those of controls. This study did not 

include a postexposure period. 

Fujinaka et al. (1985) exposed adult male bonnet monkeys to 1254 pg/m 3 

(0.64 ppm) 0 3 8 hr/day for one year. They reported significantly increased 

volume of respiratory bronchioles which had smaller internal diameters. 

Respiratory bronchiolar walls were thickened by epithelial hyperplasia and 

increased peribronchiolar connective tissue. Several small nodules of hyperplastic and hypertrophied cuboidal bronchiolar cells were reported near the 

openings of alveoli into the respiratory bronchiole. The authors interpret 

the morphometry of respiratory bronchioles as indicating an extension of 

bronchiolar epithelium into airways which were formerly alveolar ducts similar 

to the formation of respiratory bronchioles reported in 0 3-exposed rats by 

Boorman et al. (1980) and Moore and Schwartz (1981). 

In an earlier study, Freeman et al. (1973) exposed female beagle dogs to 

1960 pg/m3 (l ppm) of 03 8, 16, or 24 hr/day fQr 18 months. Dogs exposed to 

1960 pg/m3 (1 ppm) of 0 3 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 termtinal airways distorted by 

fibrosis and thickened by both fibrous tissue and a mononuclear cell infiltrate. 

Relatively broad bands of connective tissue were reported in distal airways 

9-35 
and proximal alveoli. Epithelial hyperplasia was seen sporadically in the 

bronchiolar-ductal zone. Other dogs in' that study exposed to 3920 or 5880 

pg/m3 (2 or 3 ppm) of 03 8 hr/day for the same period had more severe fibrosis, 

greater accumulations of intraluminal macrophages, and areas of both squamous 

and mucous metaplasia of bronchiolar epithelia. 

9.3.1.1.2.2 Alveolar ducts and alveoli. Alveoli in the centriacinar 

region, but not those at the periphery of the acinus, are damaged by ambient 

levels of 03 (Stephens et al., 1974b; Schwartz et al., 1976; Mellick et al., 

1977; Crapo et al., 1984). The lesion is characterized by the destruction of 

type 1 alveolar epithelial cells exposing the basal lamina; an accumulation of 

inflammatory cells, especially macrophages; hyperplasia of type 2 alveolar 

epithelial cells that recover the denuded basal lamina; and thickening of the 

interalveolar septa. In animals with respiratory bronchioles (e.g., dog, 

monkey) the alveoli involved at low concentrations are those opening directly 

into the respiratory bronchiolar lumen of low-generation respiratory bronchioles 

(Dungworth et al., 1975b). As the concentration is increased, the lesions 

include alveoli attached to but not, seemingly, opening into the respiratory 

bronchioles and extending distally to higher-generation respiratory bronchioles 

(Mellick et al., 1977). In monkeys exposed to 1568 pg/m 3 (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 invo1ved open into or are immediately adjacent to the alveolar 

ducts formed by the termination of the terminal bronchiole. 

In the centriacinar region of animals which lack respiratory bronchioles, 

damage to type 1 cells has been reported as early as 2 hours fo1lowing exposure 

to 980 Ng/m3 (0.5 ppm) 03 using LM (Stephens et al., 1974a). While not an 0 3 

concentration studied comprehensively or illustrated in that article, the same 

authors comment in the results section of their report that TEM evaluation of 

rats exposed to 392 pg/m3 (0.2 ppm) 0 3 for 2 hours revealed "..:considerable 

damage and loss of type 1 cells from proximal alveoli ... " Recovering of 

denuded basal lamina by type 2 cells has been reported to start as early as 4 

hr (Stephens et al., 1974b). The type 2 cell labeling index following tritiated 

thymidine reached a maximum at 2 days of continuous exposure to either 686 or 

980 pg/m3 (0.35 or 0.5 ppm) of 03 (Evans et al., 1976b). Although the labeling 

Tndex decreases as the exposure continues (Evans et al., 1976b), clusters of 

type 2 cells and cells intermediate between types 2 and 1 were reported following 

90 days of exposure to 1568 pg/m 3 (0.8 ppm) by Boorman et al. (1980). They 

9-36 
interpreted these intermediate cells as due to delay or arrest of the transformation from type 2 to type 1 epithelial cells. Type l cell damage and occasional 

sloughing were observed by Barry et al. (1983) in newborn rats exposed to 490 

pg/m3 (0.25 ppm) of 0 3 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 thickeir. Evans et al. (1985) studied 

this effect and suggested that 03 tolerance exists when the surface area of a 

cell is small enough so that antioxidant mechanisms contained in that volume 

can protect it from damage. Sherwin et al. (1983) found increased numbers of 

lactate dehydrogenase (LDH)-positive cells, presumed to be type 2 alveolar 

epithelial cells, by automated LM morphometry of lungs from mice exposed to 

588 pg/m3 (0.3 ppm) of 0 3 for 6 weeks. Moore and Schwartz (1981) reported 

nonciliated bronchiolar cells lined some alveoli opening into the transformed 

airways located between terminal bronchioles and alveolar ducts of rats exposed 

to 1568 pg/m3 (0.8 ppm) of 0 3 8 hr/day for 180 days. Sulfuric acid aerosol 

did not potentiate this lesion. 

The inflammatory cell response appears to'occur immediately following or 

concurrent with the type 1 cell damage and has been reported in monkeys as 

early as after 4 hr of 1568 pg/m 3 (0.8 ppm) of 0 3 exposure (Castleman et al., 

1980). In rats, the numbers of inflammatory cells per centriacinar alveolus 

appear to be related to 0 3 concentration, at levels between 392 and 1568 pg/m 3 

(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 

exposure 8 hr/day to 980 pg/m 3 (0.5 ppm) of 0 3 and after 62 days of filtered 

air following 180 days°of exposure. The addition of l mg/m3 H2SO4 aerosol did 

not result in larger increases. In monkeys, the intensity of the response was 

greater than i n rats exposed to the same 0 3 concentrati on (1568 pg/m 3 , 0.8 

ppm) in the same regimen (8 hr/day) for 7, 28, or 90 days (Eustis et al. , 

1981). Although in both species the numbers of inflammatory cells per aiveolus 

decreased with increasing length of exposure, the decrease was not as rapid in 

the monkey as in the rat (Eustis et al., 1981). 

Several investigators (Boorman et al., 1980; Moore and Schwartz, 1981; 

Fujinaka et al., 1985; Barr, 1984) have presented evidence of bronchiolization 

of airways which were previously alveolar ducts; i.e., bronchial epithelium 

replaces the type 1 and 2 alveolar epithelium typical of alveolar ducts. 

9-37 
This phenomenon is most easily seen and quantitated in species which normally 

do not have respiratory bronchioles or have one very short generation of them. 

In these species, airways with the characteristics of respiratory bronchioles 

are seen between the terminal bronchioles and the alveolar ducts of exposed 

but not control animals (Boorman et al., 1980; Moore and Schwartz, 1981; Barr, 

1984). In species which normally have several generations of respiratory 

bronchioles, bronchiolization is detected morphometrically by increases in the 

volume fraction or total volume of respiratory bronchioles. Using morphometric 

techniques, bronchiolization was reported in nonhuman primates exposed to 1254 

pg/m3 (0.64 ppm) 03 8 hr/day for one year (Fujinaka et al., 1985). 

The interalveolar septa of centriacinar alveoli are thickened following 

exposure to ambient concentrations of 0 3 . After 7 days of continuous exposure, the thickening was attributed to eosinophilic hyaline material and 

mononuclear cells (Schwartz et al., 1976). Loose arrangements of cells and 

extraceliular materials suggested separation by edema fluid. Castleman et al. 

(1980) also reported edema of interalveolar septa of monkeys exposed to 1568 

pg/m3 (0.8 ppm) of 0 3 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 3 (0.8 ppm) of 03 8 hr/day for 20 or 90 days. The 

increased total thickness was due'to thicker interstitium. Althouqh several 

components could contribute to this increased thickness, the subjective impression was one of a mild interstitial fibrosis. Crapo et al. (1984) made a 

more comprehensive morphometric study of centriacinar interalveolar septa from 

young adult rats exposed to 490 pg/m3 (0.25 ppm) 0 3 12 hr/day for 6 weeks. 

They reported significant increases in tissue thickness and suggested that the 

increased thickness was due to significant increases in all cell types except 

type 2 cells, and to increased interstitium. 

Moore and Schwartz (1981), after exposing rats to 980 pg/m 3. (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 pg/m3 (1 to 3 ppm) of 03 8 to 24 hr/day for 18 

months. 

In several biochemical studies (see Section 9.3.3.6), Last and colleagues 

have shown that 0 3 is collagenic. In one of these (Last et al.; 1979), the 

nm 
biochemical observations were correlated with histological observations of 

slides stained for collagen and reticulin. Elevated collagen synthesis rates 

were found at all concentrations and times studied. Mildly increased amounts 

of collagen were seen morphologically in centriacinar alveolar duct septa from 

most rats exposed to 980 pg/m 3 (0.5 ppm) of 0 3 24 hr/day for 14 or 21 days. 

More severe lesions were seen in ratg exposed to 1568 to 3920 pg/m 3 (0.8 to 

2.0 ppm) of.03 24 hr/day for 7, 14, or 21 days. Last et al. (1983, 1984a) 

have reported synergistic increases in morphometrically determined volume of 

centriacinar lesions, inflammatory cells, and "stainable" collagen in rats 

exposed to a mixture of 03 and ammonium sulfate aerosols. The 1984 report 

concerns this synergism in rats exposed to 1254 or 1882 pg/m 3 (0.64 or 0.96 ppm) 

03 and 5 mg/m3 ammonium sulfate for 3, 7, or 14 days. The results correlate 

well with biochemically determined apparent collagen synthesis rates (see 

Section 9.3.3.6). 

Two studies address the biologically important question of the morphological 

effects that ,follow multiple sequential exposures to 0 3 with several days of 

clean air interspersed between 0 3 exposures (i.e., a multiple episode exposure' 

regime). Plopper et al. (1978) exposed rats to 1568 pg/m 3 (0.8 ppm) 0 3 continuously for 3 days,.held them in the chambers breathing filtered air until 

postexposure day 6,.13, or 27, when they were again exposed to 1568 pg/m 3 

(0.8 ppm) of 0 3 continuously.for 3 days. Rats were also examined 2, 6, 9, 16, 

and 30 days after the first 0 3 exposure. Lungs from rats breathing filtered 

air for 9 days after one 3-day exposure had only minimal lesions and after 

30 days of filtered air were indistinguishable from controls. When the second 

3-day 03 exposure started 6 or 27 days after the end of the first exposure, 

the lesions appeared identical-to each other and to those seen at the end of 

the first exposure. Barr (1984) compared lesions in rats exposed to 1862 pg/m 3 

(0.95 ppm) 0 3 8 hours every day for 90 days with both control rats and with 

rats exposed to the same concentration 8 hours per day in 5-day episodes 

followed by 9 days of filtered air repeated 7 times during an 89-day period. 

The lesions were similar but less severe in the episodically exposed rats. 

9.3.1.1.3 Vasculature, blood, and lymphatics. Although edema is the apparent 

cause of death due to inhalation of high concentrations of 0 3 , there is very 

little morphological evidence of pulmonary vascular damage due to < 1960 pg/m 3 

(<_ 1.0 ppm) of 0 3 exposure. Bils (1970) reported capillary endothelial damage 

in mice less than 1 month of age exposed for 7 hr to 1960 pg/m 3 (1 ppm) of 0 3 , 

but this experiment has not been confirmed by others. Boatman et al. (1974) 

9-39 
did demonstrate endothelial damage in cats exposed via an endotracheal tube to 

510, 980, or 1960 pg/m3 (0.26, 0.5, or 1.0 ppm) of 0 3 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 pg/m 3 (0.4 ppm) of 03 7 hr/day, 5 days/week for 6 weeks. 

Stephens et al. (1974b) reported occasional areas of endothelial swelling but 

concluded "the endothelium remains intact and rarely shows signs of significant 

injury." Stephens et al. (1974a) reported that "endothelium retained a relatively normal appearance" in rats exposed to 980 or 1764 pg/m 3 (0.5 or 0.9 

ppm) of 0., for 2 to 12 hr or 980 pg/m 3 (0.5 ppm) for up to 6 months. In rats 

exposed by the usual methods to 980 or 1568 pg/m 3 (0.5 or 0.8 ppm) of 03 8 or 

24 hr/day, centriacinar interalveolar septa had a loose arrangement of cells 

and extracellular material, indicating separation by edema fluid (Schwartz et 

al., 1976). These investigators did not find morphological evidence of damage 

to endothelial cells. Evidence of intramural edema in centriacinar areas was 

found by Castleman et al. (1980) in monkeys exposed to 1568 pg/m 3 (0.8 ppm) 

for 4 to 50 hr, but they did not report morphological evidence of vascular 

endothelial damage. 

Arterial lesions have been only rarely reported. P'an et al. (1972) 

reported increased thickness of the media and intima in pulmonary arteries 

from rabbits exposed to 784 pg/m 3 (0.4 ppm) 03 6 hr/day, 5 days/week for 

10 months. These rabbits had evidence of intercurrent disease which was more 

severe in exposed animals. The LM description indicates "some degree of 

inflammatory infiltrate" in all lungs, and in one exposed rabbit the lesions 

were so severe that "visualization of the mural framework of the alveoli was 

difficult." The pulmonary artery media and intima were also significantly 

thickened in bonnet monkeys exposed to 1254 pg/m 3 (0.64 ppm) 03 8 hr/day for 

one year (Fujinaka et al., 1985). 

No references to morphological damage of lymphatic vessels were found. 

This is not surprising, because following nasal inhalation of < 1960 pg/m 3 

(<1 ppm) of 03 , blood capillary endothelial damage has not been reported, and 

edema has been reported only in centriacinar structures. In the more generalized edema that follows exposure to higher concentrations, Scheel et al. 

(1959) reported perivascular lymphatics were greatly distended. 

9-40 
9.3.1.2 Sequence in which Sites are Affected as a Function of Concentration 

and Duration of Exposure. The sequence in which anatomic sites are affected 

appears to be a function of concentration rather than of exposure duration. 

At sites that 'are involved by a specific concentration, however, the stages in 

pathogenesis of the lesion relate to the duration of exposure. Multiple 

anatomical sites in the conducting and exchange areas of the respiratory 

system have been studied at sequential,time periods in oniy a few studies. 

Stephens et al. (1974a), in a study of the short-term effects of several concentrations of 03 , reported finding by LM significant damage to centriacinar 

type 1 alveolar epithelial cells in rats exposed to 980 pg/m 3 (0.5 ppm) 0 3 for 

2 hours. Using TEM, they also reported, but did not document by figures, 

minimal damage to centriacinar type 1 cells in rats exposed to 392 pg/m 3 

(0.2 ppm) 0 3 for 2 hours. Boatman et al. (1974) found lesions in both the 

conducting airways and parenchyma of cats exposed to 510, 980,-or 1960 pg/m3 

(0.26, 0.5, or 1.0 ppm) via an endotracheal tube for times as short as 4.7 and 

6.6 hr. Thus, if there is a time sequence in effect at various sites, it is a 

short time. 

Increasing concentration not only results in more severe lesions, but 

also appears to extend the lesions to higher generations of the same type of 

respiratory structure (i.e., deeper into the lung) (Dungworth et al., 1975b). 

Several investigators who have described gradients of lesions have related 

them to assumed decreases in concentration of 0 3 as it progresses through 

increasing generations of airways and to differences in protec-tion and sensitivity of cells at various anatomic sites. For the conducting airways, Mellick 

et al. (1977) reported more severe and extensive lesions in the trachea and 

major bronchi than in small bronchi or terminal bronchioles of rhesus monkeys 

exposed to 980 or 1568 pg/m 3 (0.5 or 0.8 ppm) of 0 3 8 hr/day for 7 days. For 

the acinus, they noted the most severe damage in.proximal resp - ratory 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 bro- ichioles, 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 3 (0.2 

and 0:35 ppm) of 0 3 8 hr/day for 7 days. In the.centriacinar region, this 

9-41 
gradient is most easily demonstrated by the hyperplasia of cuboidal cells in 

respiratory bronchioles that extended further distally in monkeys exposed to 

686 rather than 392 pg/m 3 (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 exposure (Stephens et al., 1974a,b; Castleman et al., 1980). Repair of the damage, 

as indicated by DNA synthesis by repair cells, occurs as early as 18 hr (Castleman et al., 1980) or 24 hr (Evans et al., 1976b; Lum et al., 1978). Stephens 

et al. (1974a) reported little change in the extent of damage after 8 to 10 hr 

of exposure. Full morphological development of the lesion occurs at about 3 

days of continuous exposure (Castleman et al., 1980). Damage continues while 

repair is in progress, but at a lower rate. This phenomenon has been termed 

adaptation (Dungworth et al., 1975b). When the time frame is shifted from 

hours to days, severity of the lesion at 7 days differs little between exposures' 

of 8 hr/day and 24 hr/day (Schwartz et al., 1976). When the time frame is 

again shifted from days to months of daily exposures, the centriacinar lesions 

diminish in magnitude, but a significant lesion remains (Boorman et al., 1980; 

Moore and Schwartz, 1981; Eustis et al., 1981). 

9.3.1.3.Structural Elements Affected 

9.3.1.3.1Extent of injury to individual cell types . The exten-t of injury 

to an individual ce11 is related to the product of the sensitivity of that 

cell type and the dose of 0 3 delivered to the specific site occupied by that 

cell. Other factors, e.g. maturity of the individual cell, may also be involved. The dose to an individual cell is determined by the concentration of 

03 at that specific site in the respiratory system and the surface area of the 

cell exposed to that concentration of 03 . Thus, sensitivity and extent of 

morphologically detected injury are not the same. While 0 3 concentrations at 

any specific site in the respiratory system can not be determined using the 

usual analytical methods., the.. concentration can _be estimated using...modeling 

techniques (See Section 9.2). The literature does contain extensive information on the extent of morphologically detected injury to individual cells at 

specific sites in the respiratory system. That information is reviewed in the 

following paragraphs. Ciliated cells of the trachea and proximal, lower 

generation bronchi are subject to more damage than those lo`cated in distal, 

higher generation bronchi or in lower generation bronchioles proximal to the 

terminal bronchiole (Schwartz et al., 1976; Mellick et al., 1977). Ciliated 

9-42 
cells in terminal bronchioles of animals without respiratory bronchioles 

(i.e., rats) are severely damaged by even low concentrations of 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 aiveolar epithelial cells located in the 

centriacinar region are subject to damage by low concentrations of 0 3 , but 

those in the peripheral portions of the acinus appear undamaged by the same or 

higher concentrations (Stephens et al., 1974a,b; Schwartz et al., 1976; Castleman 

et al., 1980; Crapo et al., 1984). 

Although type 2 alveolar epithelial cells appear to be damaged less by 

0 3 , some type 2 cells in centriacinar locations develop mild lesions detectable 

with the TEM (Castleman et al., 1980). There is one report of larger than 

normal lameilar bodies in type 2 cells from rats fed a special basal diet with 

or without vitamin E supplementation and exposed to 588 pg/m 3 (0.3 ppm) 0 3 3 

hr/day for 11 or'16 days (Shimura et al., 1984). Type 2 cells are progenitor 

cells that recover basal lamina denuded by necrosis or sloughing of type 1 

cells and transform (differentiate) into type 1 cells when repairing the 

centriacinar 03 lesion (Evans et al., 1976b). Increased DNA synthesis by 

type 2 ceils, as evaluated by autoradiography, may be a very sensitive indicator of 0 3 damage. 

Although nonciliated cuboidal bronchiolar cells appear. less damaged 

morphologically by 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 0 3 damage (Schwartz et al., 1976). 

Following exposure to 1960 pg/m 3 (< 1 ppm) of 0 3 , their height is reduced and 

their luminal surface is more granular (Schwartz et al., 1976). The reduction 

in height appears to be due to a loss of smooth endoplasmic r•eticuluro (Schwartz 

et -a-1. ; 1976): 

Several investigators report that type 3 alveolar epithelial cells, the 

.brush cells, are not damaged by less than 1 ppm of 0 3 (Stephens et al., 1974a; 

Schwartz et al., 1976). No reports are available of damage to type 3 cells by 

higher concentrations. 

Vascular endothelial cells in capillaries of the interalveolar septa may 

be damaged much less than earlier reports indicated, because lesions are not 

described in detailed studies using TEM (Stephens et al., 1974a,b; Schwartz et 

al., 1976; Mellick et al., 1977; Crapo et al., 1984). In the earlier reports, 

9-43 
damaged endothelial cells were those located immediately deep to denuded basal 

lamina and resulted from sloughing of type 1 epithelial cells in the 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 0 3 exposure, inflammatory 

cells migrate into the centriacinar interalveolar septa (Schwartz et al., 

1976). Later, more collagen and connective tissue ground substance is found in 

the interalveolar septa (Moore and Schwartz, 1981). Boorman et al. (1980) 

reported centriacinar interalveolar septa from rats exposed to 1568 pg/m 3 

(0.8 ppm) 0 3 8 hr/day for 20 or 90 days had thicker.blood-air barriers which 

contained more interstitium. Crapo et al. (1984) made a more comprehensive 

morphometric study of centriacinar interalveolar septa from young adult rats 

exposed to 490 pg/m3 (0.25 ppm) 03 12 hr/day for 6 weeks. They reported 

significant increases in tissue thickness and suggested that the increased 

thickness was due to significant increases in all cell types except type 2 

cells, and to increased interstitium. 

Mucous-secreting cells in conducting airways appear relatively resistant 

to 03 . Boatman et al. (1974), in studies of cats exposed to < 1960 pg/m 3 (< 

1.0 ppm) of 03 via an endotracheal tube for short periods, did find limited 

desquamation of these cells, but the authors also observed that most appeared 

intact and increased in size. Castleman et al. (1977) noted roughened apical 

surfaces of mucous cells, which appeared to, be as.sociated with mucigen droplets near the cell surface, but did not find other alterations in pulmonary 

mucous cells from monkeys exposed to 392 or 686 pg/m3 (0.2 or 0.35 ppm) of 0 3 

8 hr/day for 7 days. Mellick et al. (1977) mention that mitochondria7 swe11- 

ing and residual bodies seen in ciliated cells were not seen in mucous cells 

in conducting airways of monkeys exposed to 980 or 1568 pg/m3 (0.5 or 0.8 ppm) 

of 03 8 hr/day for 7 days. Schwartz et al. (1976), who reported mucigen 

droplets being released from the apical surfaces -of mucous cells and mucous 

droplets trapped among cilia, did not find changes suggesting damage to organelles in rats exposed to 392, 980, or 1568 pg/m 3 (0.2, 0.5, or 0.8 ppm) of 0 3 

8 or 24 hr/day for 7 days. Wilson et al. (1984) reported only minor changes 

in tracheal mucous cells from bonnet monkeys continuously exposed for 3 or 7 

days to 1254 pg/m3 (0.64 ppm) 03 . Using TEM they reported more prominent 

9-44 
small -mucous- granule (SMG) cells which had more abundant cytoplasm and more 

specific granules. They speculated that SMG cells may relata to the repair 

process. In regular mucous cells they reported fewer mucous granules dispersed 

in more cytoplasm. The mucous granules appeared smaller and differed from 

controls in that they lacked the typical biphasic appearance and had only 

fiiamentous or granular secretory material. No reports of damage to conducting 

airways other than to ciliated and mucous cells were found. - 

9.3.1.3.2 Extracellular elements (structural proteins). Although physiologic 

and biochemical changes following 0 3 exposure suggest changes in the extracellular 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 eisewhere.in this document (See Sections 

9.3.2.2 and 9.3.3.6, respectively). Three studies provide morphological 

evidence of mild fibrosis (i.e., local increase of collagen) in centriacinar 

interalveolar septa following exposure to < 1960 pg/m 3 (< 1 ppm) of 0 3 (Last 

et al.; 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). One study (Fujinaka et al., 1985) reported 

increased connective tissue surrounding respiratory bronchioles from bonnet 

monkeys exposed to 1254 pg/m3 (0.64 ppm) 0 3 8 hr/day for one year. This 

increase was due to fncreased amorphous extraceliular matrix rather than 

stainable connective tissue fibers. Evidence of more collagen or changes in 

collagen location is in the report of dogs exposed to 1960 or 5880 pg/m 3 (1 or 

3 ppm) of 0 3 for 18 months (Freeman et al., 1973). 

9.3.1.3.3 Edema. Morphologically demonstrable alveolar edema, or alveolar 

flooding--an effect of higher-than-ambient levels of 0 3 (Scheel et al., 1959; 

Cavender et al., 1977)--is not reported after exposures to < 1960 pg/m 3 (:_!~ 1.0 

ppm) of 03 for short or long exposure periods (Schwartz et al., 1976; Cavender 

et al., 1978; Mellick et ai., 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 

exposure of monkeys or rats to < 1960 pg/m3 (< 1 ppm) of 0 3 for several hours 

to 1 week. Interstitial edema is not reported foliowing longer-term (i.e., 

weeks to months) exposure to < 1960 pg/m 3 (< 1 ppm) or less (Cavender et al., 

9-45 
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 9.3.3. 

9.3.1.4 Considerations of Degree of Susceptibility to Morphological Changes 

9.3.1.4.1 Compromised experimental animals. Compromised experimental animals 

(e.g., those with a special nutritional or immunological condition) in a 

disease state or of young or old age may respond to 0 3 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 populations. 

9.3.1.4.1.1 Vitamin E deficiency. Rats maintained on vitamin E-deficient 

diets tended to -develop more morphological lesions follbwing exposure to low 

levels of 03 than did rats on the usual rations (Plopper et al., 1979; Chow et 

al., 1981). Rats maintained on a basal vitamin E diet equivalent to the 

average U. S. human adult intake were exposed to 196 or 392 pg/m 3 (0.1 or 

0.2 ppm) of 03 24 hr/day for 7 days. According to LM studies, two of the six 

rats on the basal vitamin E had increased numbers of macrophages in their 

centriacinar alveoli, a typical response to higher levels of 0 3 (Schwartz et 

al., 1976). Of five rats on the usual rat chow diet exposed to 196 pg/m 3 (0.1 

ppm) 03 for the same period, LM revealed no increased centriacinar macrophages. 

LM analysis showed neither dietary group had lesions in the ciliated terminal 

bronchiolar epithelium. Rats in which lesions were observed by LM had increased 

macrophages, according to SEM analysis. Analysis by TEM showed that all rats 

exposed to 196 pg/m 3 (0.1 ppm) of 0 3 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 pg/m3 (0.1 ppm) of 0 3 

continuously for 7 days. The morphology of six rats from each diet and exposure 

group was studied using SEM. None of the filtered-air control anirnals had 

lesions. Of the rats exposed to 0 3 , five of the six on the vitamin E-deficient 

diet, four of six on the deficient diet supplemented by 11 ppm vitamin E, and 

one of the six on the deficient diet supplemented by 110 ppm vitamin E developed 

the typical 03 lesion as seen with SEM (Schwartz et al., 1976). 

Sato et al. (1976a,b, 1978, 1980) exposed vitamin E-deficient and supplemented rats to 588 pg/m3 (0.3 ppm) of 03 3 hr daily for 16 consecutive days or 

9-46 
5 days a week for 7 months. The short-term experiments (Sato et al., 1976a,b) 

were marred by the presence of chronic respiratory disease in the rats, which 

may explain the investigators' finding of large amounts of debris and numerous 

smal.l bodies "so thick that the original surface could not be seen" and their 

failure to find the typical centriacinar 0 3 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-depieted and supplemented, 0 3-exposed rats. They did find mild centriacinar 

0 3 lesions in exposed rats from both vitamin E-deficient and supplemented 

groups. 

Stephens et al. (1983) reported results of exposure of vitamin E-depleted 

and control young and old rats to 1764 pg/m 3 (0.9 ppm) of 0 3 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 

rather than on conventional LM, SEM, or TEM. They concluded that response to 

injury and repair of the lung was independent of the levei of vitamin E in - 

lung tissue. Most of these studies included concurrent biochemical evaluations 

of oxidant metaboiism and are discussed in Section 9.3.3. 

9.3.1.4.1.2 Age at start of exposure . Although most exposures use 

young adult experimental animals, there are a few reports of exposures of very 

young animals (i.e., either before weaning or very soon thereafter). 

Bartlett et al. (1974) exposed 3- to 4-week-old rats to 392 pg/m 3 (0.2 

ppm) of 03 for 30 days. Lung volumes, but not body weights, were significantly 

greater in the exposed rats. Light microscopy of paraffin sections of conventionally fixed lungs did not reveal differences between exposed and control 

rats in the lung parenchyma or terminal bronchioles, with the exception of two 

control animals which had lesions of "typical murine pneumonia." Morphometry 

was done on thick sections cut by hand with a razor blade from the dorsal and 

laterai surfaces of air-dried lungs rather than on the paraffin sections of 

conventionally fixed lungs. Morphometry using these nonrandom samples revealed 

significantly increased mean alveolar chord lengths and alveolar surface area, 

but no difference in alveolar numbers. 

Freeman et al. (1974) exposed month-old rats to 1058 or 1725 pg/m 3 (0.54 

or 0.88 ppm) of 0 3 for periods of 4 hr to 3 weeks. In addition to the centriacinar accumulations of macrophages and hyperplasia of distal airway epithelium 

9-47 
seen by others following exposures of young adult animals, they reported an 

increase in connective tissue elements and collagen-like strands that formed 

bridges across alveolar openings. Fibrosis was pronounced in the 1725 pg/m 3 

(0.88 ppm) group and sometimes extendpd into terminal bronchioles. Although 

fixed lung volumes were not determined, the 1725-pg/m3 (0.88 ppm) group required 

greater volumes of fixing fluid, evidence of larger lung volumes. In the same 

research report, Freeman et al. (1974) studied month-old rats exposed to 1764 

pg/m3 (0.9 ppm) of 0 3 and 1690 Ng/m3 (0.9 ppm) nitrogen dioxide combined. 

After 60 days of exposure, they observed the gross and microscopic appearance 

of advanced experimental emphysema of the type they earlier described following 

nitrogen dioxide exposure (Freeman et al., 1972). Although others have reported 

larger fixed lung volumes in exposed young adult rats (Moore and Schwartz, 

1981), reports of emphysema following 0 3 exposures are uncommon and are discussed in the next subsection of this document. 

Stephens et al. (1978) exposed rats ranging in age from 1 to 40 days old 

to 1666 pg/m3 (0.85 ppm) 0 3 continuously for 24, 48, or 72 hr. Rats exposed 

to 03before weani ng at 20 days of age devel oped 1 i ttl e or no evi dence of 

injury, as evaluated by light and electron microscopy. When exposure was 

initiated after weaning at 20 days of age, centriacinar lesions increased 

progressively, plateaued at 35 days of age, and continued until approximately 

1 year of age. 

Barry et al. (1983) exposed 1-day-old male rats and their mother to 490 

pg/m3 (0.25 ppm) of 0 3 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 discussed the possibility that much of the damage they observed may have occurred 

in the last 3 weeks of exposure (i.e., after weaning). Changes in lung function 

evaluated by Raub et al. (1983a) are discussed in Section 9.3.2. 

Bils (1970) studied the effects of 1176 to 2548 pg/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 3 , 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. 

N ~ . 
9.3.1.4.1.3 Effect of pneumonectomy. Two to four weeks following 

pneumonectomy of rabbits, the contralateral 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 

et al. (1983) exposed pneumonectomized and control rabbits to 784 pg/m 3 (0.4 ppm) 

of 0 3 7 hr/day, 5 days/week for 6 weeks. They examined the lungs with standard 

LM and TEM morphometric techniques, but not methods for alveolar numbers. 

Boatman and co-workers concluded that the lung growth that follows pneumonectomy 

occurred after 03 exposure and that no difference existed between males and 

females in this response. 

9.3.1.4.2 Emphysema following ozone exposure. The previous criteria document 

for 03 (ll.S. Environmental Protection Agency, 1978) cites three published 

research reports in which emphysema was observed in experimental animals 

following exposure to < 1960 pg/m 3 (< 1 ppm) of 0 3 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, 0 3 concentrations, and times) have been 

documented to confirm these observations: An additional consideration is the 

similarity of the centriacinar lesion following 03 exposure to that seen in 

young cigarette smokers (Niewoehner et al., 1974; Schwartz et al., 1976; Cosio 

et a1., 1980; Wright et al., 1983; Fujinaka et al., 1985) and the relationship 

between cigarette smoking and emphysema in humans (U.S. Department of Health, 

Education, and Welfare, 1967, 1969). Further, animals exposed to 1960 pg/m 3 

(1 ppm) of 03 reportedly have more voluminous lungs than controls (Bartlett 

et al., 1974; Moore and Schwartz, 1981). Morphometry was used to demonstrate 

enlarged subpleural alveoli in one of these reports (Bartlett et al., 1974). 

However, these authors indicate that these subpleural alveoli may not be 

representative of the whole lung and do not conclude that emphysema was present. 

Thus, a restudy of these three reports in the 1978 document appears appropriate. 

The precise definition of emphysema is critical to reevaluation of these 

reports. Several professional groups have presented definitions of emphysema 

(Fletcher et al., 1959; World Health Organization, 1961; American Thoracic 

Society, 1962). The most recent is the report of a National Heart, Lung and 

Blood Institute, Division of Lung Diseases Workshop (National Institutes of 

Health, 1985). In human lungs, "Emphysema is defined as a condition of the 

lung characterized by abnormal, permanent enlargement of airspaces distal to 

the terminal bronchiole, accompanied by destruction of their walls, and without 

obvious fibrosis" (National Institutes of Health, 1985). Destruction is 

9-49' 
further defined: "Destruction in emphysema is defined as non-uniformity in 

the pattern of respiratory airspace enlargement so that the orderly appearance 

of the acinus and its components is disturbed and may be lost." The report 

further indicates "Destruction ... may be recognized by subgross examination 

of an inflation-fixed lung slice ...." In order to stimulate additional 

research, the definition of emphysema in animal models was less restrictive. 

The document states: "An animal model of emphysema is defined as an abnormal 

state of the lungs in which there is enlargement of the airspaces distal to 

the terminal bronchiole. Airspace enlargement should be determined qualitatively in appropriate specimens and quantitatively by stereologic methods." 

Thus in animal models airspace wall destruction need not be present. However, 

where information from air pollution exposures of animals is to be extrapolated 

to hazards for humans, the definition of human emphysema must be considered and 

the presence of airspace wall destruction documented. 

Stokinger et al. (1957) reported emphysematous changes in lungs from 

guinea pigs, rats and hamsters, but not mice or dogs, exposed 6 hr/day, 5 days/ 

week for 14.5 months to a mean concentration of slightly more than 1960 pg/m 3 

(1 ppm) of 03 . With the exception of the dogs, mortality rates were high in 

both control and exposed animals, ranging in the controls from 25 to 78 percent 

and in exposed from 11 to 71 percent. The published report indicates that 

emphysema was present but does not further characterize it as to the presence 

of only enlarged air spaces (Fletcher et al., 1959) or enlarged air spaces 

accompanied by destructive changes in alveolar walls (World Health Organization, 1961; American Thoracic Society, 1962). The lungs were fixed via the 

trachea, makingathem suitable for studies of experimentally induced emphysema 

(American Thoracic Society, 1962; National Institutes of Health, 1985). 

Stokinger et al. (1957) attributed the emphysema in the guinea pigs to the, 

observed bronchial stenosis. Also in the guinea pigs were foci of "extensive 

linear fibrosis ... considered to be caused by organization of pneumonic areas." 

In exposed rats, the mild degree of emphysema "did not exceed the emphysema 

found in the unexposed control rats." In exposed hamsters, "mild to moderate" 

emphysema was present, but not in controls. Emphysema is not mentioned in the 

figure legends, but three of them mention "alveoli are overdistended ... 

alveolar spaces are dilated ... dilation of alveolar ducts and air sacs." 

Evidence of destruction of alveolar walls is not mentioned. Later, however, 

Gross et al. (1965), in an unrefereed publication abstracted from a presentation at the seventh Aspen Conference on Research in Emphysema, reviewed the 

9-50 
lesions in the hamsters from this exposure and described a"destructive process" 

that resulted in contraction of interalveolar septa not associated with enlargement of air spaces. 

Because the interpretation in this EPA Criteria Document differs from 

that in the previous document, the details need to be presented. The significance will be discussed in a paragraph at the end of this section. The earlier 

03 criteria document (U.S. Environmental Protection Agency, 1978) cites Stephens 

et al. (1976), a"long abstract" that appears not to be refereed. This brief 

article states "rats exposed continuously for long periods (3 - 5 months) to 

28,200 pg/m3 (15.0 ppm) NO 2 or 1568 pg/m3 (0.8 ppm) 0 3 develop a disease that 

closely resembles emphysema". but does not provide additional evidence other 

than citing five earlier studies by the Stanford group of investigators. Each 

of those five references was checked for studies of animals exposed to 0 3 . 

Three articles describe only NO 2-exposed animals. The fourth reference (Freeman 

et al., 1973) is to an exposure of dogs to 1960 to 5880 pg/m 3 (1 to 3 ppm) of 

0 3 8 to 24 hr dai 1y for 18 months and was ci ted earl i er i n thi s 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 weeks to 1725 pg/m 3 (0.88 ppm) 

of 0 3 , half of which died and had "grossly inflated, dry 'lungs." In this same 

study, they also exposed month-old rats to a mixture of 1690 pg/m3 (0.9 ppm) 

of NO2 and 03 continuously for 60 days, at which time the lungs were grossly 

enlarged, and "both grossly and microscopically, the appearance of the lungs 

was characteristic of advanced experimental emphysema" of the type they earlier 

reported following NO 2 alon6 at much higher concentrations (Freeman et al., 

1972). 

The third citation in the 0 3 criteria document (U.S. Environmental Protection 

...Agency, 1978)_..i.s to_ _P'-an- et al.- (1972). - These investigators exposed--rabbits -. . 

to 784 pg/m3 (0.4 ppm) of 0 3 6 hr/day, 5 days/week for 10 months. Tissues 

were fixed apparently by immersion rather than infusion via the trachea, which 

is not in accord with the American Thoracic Society's diagnostic standard for 

emphysema, making emphysema lesions much more difficult to evaivate accurately. The lesions related to emphysema are only very briefly described and 

illustrated in only one figure. The authors also report that "ali lungs 

showed some degree of inflammatory infiltrate" and "lungs of the sixth were so 

9-51 
congested that visualization of the mural framework of the alveoli was difficult." This is more reaction than reported in other species exposed to this 

comparatively low 0 3 concentration. The rabbits were not specified pathogenfree, nor was the possibility considered that some lesions could be due to 

infectious agents. Neither did these investigators consider the possibility 

of spontaneous "emphysema and associated inflammatory changes" which Strawbridge 

(1960) described in lungs from 155 rabbits of various ages and breeds. 

In the studies reviewed in this section, enlargement of air spaces distal 

to the terminal bronchiole have been described following 03 exposure of several 

species of experimental animals. In one study, the enlargement was quantitated 

using morphometry of air-dried lungs (Bartlett et al., 1974). Destruction of 

alveolar walls was only briefly described in two reports (P'an et al., 1972; 

Gross et al., 1965). In one of these studies (P'an et al., 1972) the lungs 

were apparently fixed by immersion rather than infusion, making a diagnosis of 

emphysema less reliable (American Thoracic Society, 1962). The other study 

(Gross et al., 1965) appears to be an unrefereed long abstract rather than a 

full research report article. Neither of these reports describes lesions 

which unequivocally meet the criteria for human emphysema as defined by either 

the American Thoracic Society (1962) the or the National Institutes of Health 

(1985). 

9.3.2Pulmonary Function Effects 

9.3.2.1Short-Term Exposure . Results of short-term 0 3 exposures of experimental animals are shown in Table 9-2. These studies were designed to evaluate the acute changes in lung function associated with 03 exposure in a variety 

of species (mice, rats, guinea pigs, sheep, rabbits, cats, monkeys, and dogs) 

when compared to filtered-air exposure. 

The effects of short-term local 0 3 exposure of the lung periphery have 

been examined in dogs by Gertner et al. (1983a,b). A fiber-optic bronchoscope _,.. 

with an outside diameter of 5.5 mm was wedged into a segmental airway and a 

continuous flow of 196 or 1960 pg/m3 (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 

1960 Ng/m3 (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 

collateral channels continued to increase throughout exposure to 1960 pg/m 3 

IMWA 
~ 

ocn 

u 

eci iO +1 ro -4 

i +) ia io a 4'i a ro a L a a a a. E 

4J n'7 

ca7 rmi ¢ ~ 3 'al-+ F >o 

I 

o~m arn rnrn 

Na M UJL •d0 •d0 •dC 

OG •U 'O fM +~ rtf ~ f0 LL~9 I ~ a a 1 - a 1 a_ 1 ,. tn O N Of C O a i~ G O CO ' G. O •7N ro ro •7m •ON >C C1 t,7 v Z U C5 v f,7 v W 

W U Lf O r v mc tm a A a c•~ r~ ~ c a2 ~ a • 4- •- G •O , i ro c 4- •~ r- ~ OC Ji0 00 m . F.- a M c w t v L o 3•.- ~n o.c ~» o e-i 1~ ro O Q C a•L 10 a O O a N O a+J 1 r C7f O. L t0 ~-1 a.) C r.'• v r- 2 f- C V- C•O M ~.•~ Li O ~ N C T7 O L u! O L N U Of O O a ro O L •• +~ L•r O C Ul a Rf a u1 7 G O +J 7 C C U 1n .0 C L a~ U .FJ 4- a a N ,L U a Lt O•1•~ v UI .- •'- E rt1 U A'F•• i U a w O +~ E a t0 _5.. ?> • O r^ +-~ d a t! O l0 7 V- a a L~n O f- CJ U ~C i ~+~ a ~/iG. O d L O -J uf •O '~ 7•.- L•r r- 7 S. E C O r i0 L f.,)G C7 V) . U rtf a tJ YL' U E +~ O C a ~ L 'O O ~f U a ~ U L 7 C a w+ +~+ C w o c d cd d N ro.N c0 > p c a ~n ~ 'O +~ M r U ttJ E ~ •O d X E> N Cl O. ~ Vf ~n a a a C O V tlf U a +J ^ drl • • a a a •• C • E c Cf X • ro a O) E V~ a d C c vl . N d +~ N V1 Ct L U E ~ ~ L 7 C a i~ a,••- G O t0 U x ro•~ ~ 4.- S. +) G.- ro 7 CfCJ •~ r• o e- O c L rtf r~ a C a i LJ_ a ~uf 7 r-1 ro a a a o u) c x ro 4- ~- Cf a U Tf t o L O CC1 a tn 'O > L o•e- ^ ~6 r• a c 'Q a G t1 > U U p7 V- •O > "O i o o a G a U•O 3'~* .E •• V~ •c a 4- •r G p fy ~ a c CL a C. uf a r^ N G U O C a A O N 03 Gl O ql '[7 M•~ 49 G~ 4C > E x+~ rt []. G t0 .^ L Rt t6 o f3. +J z L o i O .- to 2 L aJ a 10 a lU a A r"' •O . C'L7 u7 a a.^- a.. C C 'Cl 7 a a A•~ i •O L cf M17 O a/"~ Vl V1 i CAr- 'G a W Vl C r'Q 4- a C uf ~A Cf Vl U 1 7 4- V1 >> O 1^. a U C O / L 7 ~n t0 r~ O N r+ C N ~ r- d ~ G n1 V- C O O' ,Jly t[i US ttJ M ~ Q N•.- a7 •r p p~ ~n d C X A H L O M Gl 6C L p~ S.E a> LY i> • d O •'O T L- O N a C 1 M p L d '~•- O~ CL L r O a o •N x L~ ro U o TJ `f +~ O.- G +1 O. C.1 r- z rt3 a.- ~t•3 ro a O a'O G T 07 r•.- ro ro X •r• G TJ ro G o o t- i a L .- tQ c rtf .N~ Cl •E T! L a u) o a a a ~C1 •~- L C7 T.-• 07 .F, .-•• L C m• 'O ..- E ro L .a> Vl ~n ..- v1 i Vf t~7 a. W .F~ o G C r- a +1 T 3 i r 7 a('~ C O1 a a d.~ ro a ro •F~ G z ruvi•- aroi-w roroa~-+oo•- nc ac.-v~ro a wa-i ao O ~ a•~ O U O E C r G+~ O d•~ ~"~ U a- C r• L O L Ql U N •d C C C i r O. a~O •O •O r C d U G L.N ro 4- U d U•D •~ O O R1 O•r O O -DC L a C O O a X C J O 3~.G a C X G C 4- U L U~ U V- 2' a n• r-1 a•~ ~d 4F• +> > a•- r U'C a U"O H a H ep V'1 O i,. O 

N . U W IL a C O lL i O U 

W+ 7•r p G O L V1 +1 ) L .I1 t . O R7 O •E d S•. L L O 47 L L N X 7 d O N 

Q1 o2f ~ W~ m N rl N t0 N N 

J .0 m ¢ ro r C 

E 'II a O 

~+) trif W~.0 Y t~¢ Y V- (L) ~0 ~ U Z Z S Z Z 

E 

O +~ 

c E N rl o ct to d• 00 00 tn O d C N1~ 00 N rl NL[f O m tD O m U~ +1 °' o.-i o 0 0 0,-i o o r i o oH r-+ o 

C i 

N C O O a +J U C E (D Q '••i C• 00 C tD C Q Cf tD m 1- to 0 o Of lo m O tG 1- an -i Co l0 t0 lM ri d• OO U= r-i~ dG0 ~ a'N tn01r1 ~rINN ~ 

9=53. 

~~ 
.. 

v 

ar 

~ 

c 

N 

c 

O v 

v 

N 

W 

K 

~ 

O 

G. 

X 

W 

~ 

W 

F•- 

FI•- ' 

a 

O 

S 

h 

Z 0 

~ 

~ 

w 

C 

S 

~ 

~ 

C1.. 

Z 

C7 

W z 

N o . 

w 

C7 

N r 

U 

t1u 

W 

N 

1 

01 

~ 

in 

¢ 

H 

M 61 

cn ~ 

r•1 ~ 

n ~ 

C) N d 

c io m rn ~ d 

t +1 

ri rt 

• ul 

G1 4- d 

m ro 

Vf 

rt N O ~ I? 

ro 

+J 

O 

G1 O O 0. 

G.QO Y 

O 

Y O 

d ri Y } O L.

w 

4- 

7 

a 

ro 

4-) 4J 4J 

N d •r ~ 

•U 

+•> 

c0 

a 

ro 

a 

N 

c 

a 

y Y 

m 

2 

N 

i-T~ f0 

r L G1 

U 

C 

1 7 iJ 4 

N O L 

7 N•r•~ U 

N O 

Ol 

C 

N 

N 01 L O T 

7.0 ~ d.~ 

N O. G/ 4-1G1 

07 x u N C 

ro 

C L 

C 

Q'+a t r 'C • ~ 

01 ro 

0..+J GI • L .rN i. GI 10 

U 

Jo 

u 

• 

~ 

r d.1 c O Ol i-~ N f..) z 

N 

U 

L L 

i'O 

O O.4- u 

~ 

•H 

af 

'O E O•r c . 

~ N L i~~ 

td T'O m•r 

N 

N ai 

L 

t,l 

v- 4W. ++ ro lo aQ ~ 

N t0 M 

r- ro .0 3 

V- T9 O U 

tq L 

Gl O 41. 

61 3 N tD o 

N o L v 

C Zn c 

r m a O 

L N 

u O 

N 

ro 

~ r.- y v- 

•s" 

E•r a c. r 

~ .L v- E ~ ° +°a vi ~ N -° xai 0. 

L. 

07 

O i0 ro ~ 

4- ~~ [1 

•f0 r• 

ri (0,0 

 > 4- 

'O 

c Gf . 

N 

r0 

N 

.O 

o CJ u•r O. 

N C 

.V 'O 1 c 

e,f •• QI •r 

ro c N 

~,J 

Gf 

Q L N rl ~ V ~0 U C i••. ~ 4I 

r~ 41 

II 

U O'O p 'O Gl N(y ++ 1- qb N O J Z4.1 Uf C 

~ b L 

'p r U LLI 'O 

r a 

C O . TS 27 U ~ W L ~ N 4- (J 'O 

o v x c. a 

> G7 GI x N 

cv o q, rn 

u o C: c 

ra Ci 

G7 Tw 

o 

• n r 

C CJ O 

G/ O G1 C. 

O CM Cfl•r 

TI ~ C L 

L fp ^,~ 

U'O 

i1 • L. E t•r .0 4- x 

f- •1~ ++ O G! 

Cl V- 7 7 7 

G.' O a r• T1 

C C 

H r-1 •r 

tU ~ 

iJ (U N a 

e N o 

+) ~ ~ 

V) o -ro s n a 

7r~. ,NT N~ 

Vl }1 41 

O ro o 

7 

iJ m T7 L 

L 

L o 

L}1 

•L V- o Q 

X IZ. O w ~ ~ = 

W tJ 

eil 

U rl rl M t0 I•~ 6 a 7 

O 0. 

7 f0 Va O f. O 

U +J 

i•) 

C 

H O1 O 

]G C • al 

(D ° 

7 iJ 

L. .0 

W Y Y Na]HG Vr- 

~ Z 'O 

S L 

V V 4J 

.0 t L 

Ln N Ol 4- 

C E 00 6 m 

0 G0.. O ri N rl rl 

C O b C1 e0 

C L 

G7 • N 

CE7 1

'1'~ O 1~ O O N C L L rO u a G7 

~ t~0 N 

y 

Ot 

U r~-1 r~•1 t~~1 

tD l~O d rt1 GO! 

tn 

7. rl r~ f0 a U 

9-54 
(1.0 ppm) of 0 3 , but decreased again to control levels during continued exposure to 196 pg/m3 (0.1 ppm) of 0 3 . Based on these observations, the authors 

reported that tolerance appears to develop in the collateral airways to locaily 

delivered 0 3 at concentrations of 196 but not 1960 pg/m 3 (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 compliance in guinea pigs during 2-hr exposures to 431, 804, or 1568 pg/m 3 (0.22, 

0.41, or 0.8 ppm) of 0 3 . Accelerated respiration rates with no significant 

changes in tidal volume were measured during exposures to all 0 3 concentrati ons.. The onset and magni tude of these changes i n respi rati on 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 pg/m 3 (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 

essentially the same during exposure to either 804 or 1568 {ag/m3 (0.41 or 

0.8 ppm) of 0 3 . These investigators observed no significant change in pulmonary resistance during exposure to 0 3 . if anything, resistance tended to 

decrease throughout the exposure and recovery period. 

The lack of a significant increase in pulmonary resistance in the Amdur 

et al. (1978) study is in contrast to the 113 percent increase over pre-exposure 

values in total respiratory flow resistance measured in guinea pigs exposed to 

980 pg/m3 (0.5 ppm) of 0 3 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 pg/m 3 (0.26, 0.50, or 

1.00 ppm) of 0 3 for 2 to 6.5 hr. Pulmonary resistance had increased to at 

least 110 percent of control values in all animals after 105 min of exposure 

to 510 pg/m 3 (0.26 ppm) of 0 3 , after 63 min of exposure to 980 pg/m 3 (0.50 ppm) 

of 03 , and after 49 min of exposure to 1960 pg/m 3 (1 ppm) of 0 3 . Dynamic lung 

compliance was decreased during 0 3 exposure in 'the, Watanabe et al. (1973) 

study, as it was in the Amdur et al. (1978) study. However, changes in pulmonary compliance measured by Watanabe et al. (1973) occurred less frequently 

and were less severe (based on percentage changes from pre-exposure control 

values) than changes in pulmonary resistance. 

Like Amdur et al. (1978) and'Yokoyama (1969), Murphy et al. (1964) also 

measured breathing pattern and respiratory flow resistance in guinea pigs 

9-55 
during 2-hr 03 exposures. These investigators found concentration-related 

increases in respiration rate during exposure to 666, 1333, 2117 or 2646 pg/m 3 

(0.34, 0.68, 1.08, or 1.35 ppm) of 0 3 . Respiratory flow resistance was increased 

(to 148 and 170 percent of pre-exposure values) in guinea pigs exposed to 2117 

and 2646 pg/m3 (1.08 and 1.35 ppm) of 0 3 respectively. Pulmonary compliance 

was not measured. 

The variability in measurements of pulmonary resistance following 03 

exposure can be attributed to a number of factors including the following: 

frequency characteristics of the monitoring equipment and measurement techniques utilized, the influence of anesthetics, and the intraspecies differences 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 0 3 exposure was substantially 

different in the above three studies. Animals exposed by Amdur et al. (1978) 

showed little or no return toward pre-exposure values for any of the measured 

parameters during a 30-min recovery period following exposure. In guinea pigs 

exposed by Murphy et al. (1964) and Yokoyama (1969), respiration rates had 

returned almost to pre-exposure values by 30 min following exposure. The 

development of more persistent lung-function changes following 0 3 exposure in 

the Amdur et al. study (1978) may be attributed to the small size and associated 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 3 . 

Inoue et al. (1979) exposed rabbits to 470 to 2156 pg/m 3 (0.24 to 1.1 ppm) 

of 03 for 12 hr and performed lung function tests 6 hr, and 1, 3, and 7 days 

following exposure. These rabbits showed functional evidence of premature 

airway closure with increased trapped gas at low lung volumes 6 hr, 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 2O. These lung 

9-56 
function changes were greatest 1 day following exposure and had disappeared by 

7 days following exposure. Distribution of ventilation in the lung was less 

uniform in 0 3-exposed animals only at 6 hr following exposure. By 7 days 

following the initial 12-hr 0 3 exposure, the only significant functional 

change was an increased lung distensibility in the midrange of lung volumes 

(from 25 to 75 percent total l.ung capacity). 

Earlier studies by Yokoyama (1972), in which rabbits were exposed to 

1 ppm of 0 3 for 3 hr, showed a timing of lung function changes similar to that 

observed by Inoue et ai. (1979). For both studies, maximum changes in 0 3- 

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 H2O, whereas Inoue et al. (1979) found no difference in maximum lung volume. 

Yokoyama (1972) does not show lung pressure-volume curves at pressures less 

than atmospheric pressure, so premature airway closure and gas trapping cannot 

be evaluated in this study. One factor that may contribute to differences - between these two studies is the use of an excised lung preparation by Yokoyama 

(1972) compared with evaluation of intact lungs in anesthetized rabbits by 

Inoue et al. (1979). 

Yokoyama (1974) also evaluated lung function in rabbits following exposure 

to 1960 pg/m3 (1 ppm) of 03 , 6 hr/day, for 7 to 8 days. He found increased 

pulmonary resistance and decreased dynamic compliance in 0 3-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. 

9.3.2.2 Long-Term Exposure . Table 9-3 summarizes results of long-term 0 3 

exposures. Raub et al. (1983a) exposed neonatal.and young adult (6-week-old) 

rats to 157, 235, or 490 pg/m 3 (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 3 exposure. Peak inspiratory flow measured 

in these animals during,spontaneous respiration was significantly lower following exposure to 235 or 490 pg/m3 .(0.12 or 0.25 ppm) of 03. Lung volumes 

measured at high distending pressures were significantly higher in neonatal 

ani mal s exposed to 490 pg/m 3 (0.25 ppm) of 0 3 for 6 weeks than i n control 

animals. These results are consistent with increased lung volumes measured 

during lung inflation with either air or saline by Bartlett et al. (1974) 

9- 57 
d 

u 

c 

ar 

L ai 

va+ 

C 

N 

N O1 

a u ~ cu 

N G 

X 

W 

~ 

W 

F67 

Z 

O 

J 

O U o--1 1n 

F- N 

t~ U 

C7 

t=a. b•• d 

~ v 

~ O > 

Q L 

N 

~ a 

4• O 

x 0 

W 

.2 

N 

O 

U. 

O 

h 

~ 

W 

~ arco 

W L O U 

-IV z b01 i 

C7 d L L 

l W C ~ 

~ 

W 

J 

m .O 

H IO 

~ 

C 

N 

E •O 

C1 O 

L .0 

tp E 

a 

S 

G E 

O O. 

•r G 

d ro 

C L 

042 

N G 

O G/ 

u c im 

O 

u o~ 

s 

~ 

M 00 (

0

1 

00 •• . O1 1-1 

r

cn

-1 ~ •• e}' 

io n - a) vm 

ro d ~o +) ro +J 

+~ ~ ++~ d 

a~ m 3 ai 

4/ Ol ro ul 

a +~ 1W +) 4J m o • - ++ 

:3 L n uf L 00 _u .0 tn 

D: m r~•i - U Z1-~i } U-1 W 

H 

L }J y 

O ~ ~ 

d O O 

3 N 

Ac ~ 

O C! C C 

+~ m 3 ~ 

r0 c i O 

+~ 

Rl M 

+~ O 

rtl ri 

a 

10 - 

++ 

ro 

c o 

O m 

CVtD T C ~ Cv 

1 C 

ro 

+J /~ G. 

ro f..) RJ a 

Iq 

T N 

(l 

i +~ 

a) 

L MOD r d L Of' ~ C 

O •D.~ 

O tn C/ 

O) N• 

1 EV LL. O 

r Vl 1J C L 

• ro uN 7 'O 

Ul 

Gl 

V 

O 07 1 d-~ C V7 

> C ul - O 

C 1 . 

r- c 

C 7 C W 

O r i0 ta- 'O 

a Cl r ~ N 

~ L V O+~ 

L 

O. 

N 

u 

7 d'O • G. 

Cfr L Gl Y X 

'- 

T 

u O O C 

i> > V'C ro 

•uf u G! 'O L C 

C C Vl GI Of C/ Cl 

C 

ro 

c C. 1n M17 07 +~ Of Of O ~ C G) •~ 10 X > Gl C 

7 ~ O 01 

r G/ Cl O. G. Uf 

~ C C 

r 34~ N 

~..~ 'O 0)v 1p N 

OI c 

+J G7 •~ Of N L 

X ~ L. l.~ C L 

O 

V1 c X +~ M r L C 'O Y7 7 V! E O d ~ U - 7 0.0 E ~ T f0 Cl'O f0 O 

L' f11 •O 6! L 

a 7•'- 

L~ r 41 0-4 

 Ol C. 

+J O. L p.+1 

+1 N J O 

r C V 

C 

o 

O C 

u o 

O i C In u 4- •uf Ol ro f0 x 3 ro 

•o 

y E X 

~ FJ U Cl +17 o 

ro c i~ 10 'O b 

C•~ ~ M 

Ol L U 

Gl Vl CO 

G) m t7f 'D 

~O 'II c O Gl 

3 a C o aQ i-1 

d 

L 

u+~ 

f0 

i•.- in i OJ N E 

C t0 a 

•C.•O'O r C G. 

+~ J L 

VI +~ F- o 

~ 10 

L V1 O N O G1 

i~ f-. Vl 

+~ ftl 4- a+~ ro 

rtJ •r- O U cq 

f-• Y V1 rl N O 

ro ro ~ c C 

7 

V7 

O 

~ Lro+J 

+J c 

X C R1 O 'O O i O 10 N C7 O N ~ O O O- N G! 

01 R7 L ~ C7 tf) 

Gl 1D M C N 

M Z7 • 

Of ~ O ro 

C Cf L 

V C E C'O u 

O L•~ }~ y E i-1 

d a+) x N 7 

X 

N • 

U 

•N C 

~ Vf •r C O 

C iJ .G O C O 

G7 t0 Gl 4- •r 

C ro rl 

7 f-• I t 

r G,•~ y y J p• 'p Ul •p 

6.' — c 10 

> ro G! ro G7 M 

r V L d C G/ 

ro G d L O. Clf 

N 

V- d 

O> 

ro O 

7 u 

Q 

L+J C O L 

v m 30 r

•O 1n +j 

O1 •- 

va~ ..3 

c 00 G/ Gl Gl 

'oo .N LL 

f0 01 +1 U C 

v rocrnro 

L 

+» 

O 

v+~ 

C7 ~ C E O 

ui r uf •~ p• r N 

N M 

u1 O Yl C 

Gl •'- O tn U Gl 

Vl ~1 C 3 

N'O Vo L•'- C L 

1n r N N U 

U U 

Gl 

Gl 

V) •O 

ro 7 6f O.~ rl 

6f 'O € N 

L IC 7 CJ tn T O 

R7 G CI o 

G7 X E•- 

L d 7+1 

ro ro L'O •~ • 

Gl i O O M 

L+~ L 01 .+O 

ro d L•C N • 

N•~ 07 O E ~ N 

L T ~ € 7 r uf 

V- C! 

4- ~ 

G! 

ro G! 

N+J 

L 10 

u L N L U 1 ro U C N Of i lL U YI C! u 

N•r > V10 iJ ~ NO > ~ F M UO L 3LL. O H ~ dt (O >4-}1 Z> C7 t 

H 

- 01 

-~ 3 

C N 

N O 

LGI 

N T3 

47 

-T3 

01 OJ 

3 T3 O wv\ In ~ o ro\ a ro\ 

Y'O ul 

ro\ 

n'O N 

vi >ro' 

T'O 

a~ L T G! L ro 

o•.- }1 +7 x\ a 

y L. 10 

ai \ T ' G1 L Id \ T O L f0 co ro\ 

N~ L 

~ N -0 O N•GV 

3 L'O +1 tZ7 'O .0 

lD r-1 n 

GO 

N U lD 40 N'9 "D n t19 t0 t0 tD n C1 00 

x LU 

2 

U 

L 

7"~ W F- ry 

tn ]C 

O►r 

]L 

r•• . Y 1•- 

N 

r-i 

•• Y 

s 6ap m m 

_ ~. 

.......Y_ ~ m 

v

... ,° . Z Z° - O Z- .... __.. _.Z.. ~ _ . _... . ._.,., .O-z°--- 

O rNiN N N00 a• ~ LL7 m 

O O O O O O , O O O O 

^tCI M C1 T QN

1 tOD ~ OND OOo l00 

ri N sf 1+9 cn 1n 

rl 

n OD Ol tn 

ri 

9-58 
~ 

i•~ 

N 

E O 

~ 

O 

L 

d 

I = 

11 

} 

~ 

N 

U_ 

N 

u 

N 

C 

E 

7 

• E 

L 

U 

Cl a 

ro 

n 

Ul 

ro 

Of 

n 

F N 

W ~ 

x r U O 

~ 

L E 

}1 ~ N 

E N o O i

a a 

N O T 

~ d N 

~ o T y L 

U C 

L 

4) 

4- O 

+~ w• E 

E a 7 O d 

:3 ro w O L O 

U i-3 

r~•~ C1 O 

Y C •r }1 

II II • C h` rr • 

.E ~ Y4-.. ~ a 

s

v v ++ 

0 0 

4Jy44 o 

E E 4- 

41 C L 

c o ro 

G7 • Vf 

aEi e+'c o 

tn 

L L r 

7 a C7 

w • 

N N COl 

N a U 

V' d' 

~n ~o 

O O 

t~n ~ 

N~ -1 rl 

I-- ~ 

. ...S.Y___.__...- 

m 

z 

u~ o 

O rl 

~~ 

C N 

r tOl 7 

d iJ C O 

U O i0 L. 

ro c u ° '

+J .b.t f0 L 

Vl 

r 

•a . a 

0) u1 07 

LroT a 4 

r• -Or 

ro a 

ord •r 

L 5-N U d o m c 

4-01 GI 

• L L 7 

Cl 'OU Q 

°• vo,a so• a n. • " ~ 

~ X tn t 

m G) Trn 

i n O L 

U N 

C f00+~ rr L N N 

>, ro 

V 

O 

N 

L 

O 

n 

N 

ro E 

~ C 

4- 

 T /1 

•~o o ar ~ ro 

U i0 O 

r- rI C O 

L O 4-) f- l0 p

iJ iJ Vl L 

Cr ~ L 

O! Vl N t0 O 

U Gl G! 4 

L L L 

4- 7 CJ M 

OO N Y ~ - p 

G1O_ tn tq E 

uxro a. 

ro os A d N ~ U f0 l, 

v~ c L3 

.N.ro. 0 

v a •- •io 0 

L TrVI M CJ L 

ro ot ~ 

O1 L M d 10 

U Ld 

L Gl r O 

C w O d X rr ro w d Gi 

ro 

\ t~D 

L 

L O 4J 

tD 

•O 7 

C N 

ro O b 

M N• 0 

ar +~ 4-) 3 c 

>~ 

•• V -O +~ Vl GJ vl 

dC Et0 L L 01 O 

7 ro N CJ > d•F1 X 

Ul Vl r•i r• 7'O N 

O ~ Ol d v O_f 

X aoi c v eco c c 

N C•r G/ E O L•r 

d 3 c r 7 3 V- Of O•r T a ~ p O 

O E ~ E O C uf O O N+) 7 • O ' 

L.0 W L ro 4- — VC 11 ~•T •a o a •cc av .vo 0 

E N Z7 X .•~ •r 01 •- 6! •• N TLL i. V1 

tD ~n ro s0.•o 3 ~ c.aroi 

•.L-~ cL.7 U O L. f0 C L >s Ol 3ccd 

w

o ~ 

0 o•r xr > 

41 

r• F1 tA 'O ro> U ~ /1 

O ~ O 10 V- C E W (2) J 

LL. r• 3 O ro Vl LL L U 

Y . 

a 

~ N 

L -O Vl 

ro\ T 

01 i ro - 

r• p n 

F 

~ 

~ 

d 

~ 

C 

N 

C 

O 

U 

v 

N 

W 

C 

~ 

N 

0 

X 

W 

F 

O: 

W 

H 

C9 

Z 

O

O 

Z 0 

F~- 

U 

Z 

~ 

LL 

CD 

T 

Q 

Z 

E 

J 

~ 

2 

Z 

O 

W 

Z 

0 

N 0 

LL 

0 

N 

I--' 

U 

W 

W 

LL 

W 

M 

C1 

W 

J 

co 

cc 

-°Ia~ 

U 

C 

N I 

L 

O . 

w 

d 

K 

H 

N 

• U 

G1 d N 

U 

~ 

U 

GJ 

4- 

O 

v 

v 

~ 

N 

N 

a 

0 

C1 C O 

i O U 

3 •r O 

N ~ ~ 

O 'GO 

d 1-L 

X = d 

W 0 

~ 

a 

10 

i-~ 

C 

N 

E V ar o 

L L 

7 V-_ 

ro ~ 

m 

~ 

c E 00. 

r +; d 

ai Rs 

C L 04) 

N C 

O CI 

U 

C E 

O 

U Of 

1 

ro N N 

N O 

qC1 r•I 

IO YE tq 

O 

Y L 

T •r ~}• N 

Y m 

}rO11 Y N r-13 

~ 

~ 

N C 

Y C 

y t +J c O 

to 

9-59 - - 
following exposure of young rats (3- to 4-week-old) to 392 pg/m 3 (0.2 ppm) of 

03 continuously for 28 to 32 days. Moore and Schwartz (1981) also found an 

increased fixed lung volume (following lung perfusion at 30 cm inflation 

pressure with Karnovsky's fixative) after 180 days of continuous exposure to 

980 pg/m3 (0.5 ppm) of 03 . Yokoyama and Ichikawa (1974) found no change in 

lung static pressure-volume curves in mature rats exposed to 882 pg/m 3 

(0.45 ppm) of 03 6 hr/day, 6 days/ week for 6 to 7 weeks. 

Martin et al. (1983) studied the mechanical properties of the alveolar 

wall from rabbits exposed to 784 pg/m 3 (0.4 ppm) of 03 , 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 H 20 was also reporte.d following 0 3 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 9.3.1.4. 

Costa et al. (1983) evaluated lung function changes in rats exposed to 

392, 1568, or 3920 pg/m3 (0.2, 0.8, or 2 ppm) of 0 3 , 6 hr/day, 5 days/week for 

62 exposure days. (This report will not discuss effects in animals exposed to 

2 ppm of 03 ). These investigators found increased pulmonary resistance (not 

concentration-related) in rats exposed to 392 or 1568 pg/m3 (0.2 or 0.8 ppm) 

of 03 . Lung volumes measured at high distending pressure (VC and TLC) were 

increased following exposure to 1568 pg/m 3 (0.8 ppm) of 0 3 . 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 pg/m3 (0.2 or 0.8 ppm) of 0 3 . Changes in maximum flow at low lung volumes 

indicate peripheral,,airway dysfunction and_ . may be related to reduced parenchymal 

elasticity or narrowing of the airway lumen. 

Yokoyama et al. (1984) evaluated lung function in 7-week-old rats immediately after exposure to 1960 pg/m 3 (1.0 ppm) of 03, 3 hr/day for 14 and 30 

consecutive days and in rats of two different ages (4- ,and 10-week-old) one 

day after exposure to 980 pg/m3 (0.5 ppm) of 03 , 6 hr/day for 60 consecutive 

days. Pulmonary flow resistance (R L

) increased after 0 3 exposure; however, 

the pattern of this change was different between both types of exposure. 

Increased R L occurred over a wide range of - lung deflation pressures in the 

N .t 
former exposure while R L increased only at lower pressures after the latter 

exposure. The authors interpreted these changes as an indicatioh of increased 

central ai rway resi stance i n rats exposed to 1960 pg/m3 (1 ppm) of 0 3 for 

30 days and increased peripheral airway resistance in rats exposed to 980 pg/m 3 

(0.5 ppm) of 0 3 for 60 days. These changes were also consistent with morphological findings of greater mucous secretions in large bronchi of the animals 

exposed to 1960 pg/m3 (1 ppm) of 03 and in the peripheral airways of animals 

exposed to 980 pg/m 3 (0.5 ppm) of 0 3 . No changes in static deflation volumepressure curves of the lungs were found after either exposure nor were there 

any differences in effects that could be attributed to the age of rats at the 

start of exposure. 

Eustis et al. (1981) evaluated•lung function in bonnet monkeys ( Macaca 

radiata ) exposed to 980 or 1568 pg/m3 (0.5 or 0.8 ppm) of 0 3 , 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 3 exposure were generated. When pooling results from all exposure times and 

0 3 concentrations, quasi-static lung compliance was significantly different in 

0 3-exposed animals than in control animals. Compliance tended to decrease 

from pre-exposure values in control animals and increase in 0 3 -exposed animals. 

Wegner (1982) evaluated lung function in 32 bonnet monkeys, 16 of which 

were exposed to 1254 pg/m 3 (0.64 ppm) of 0 3 , 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 (DLCO), 

lung volumes, quasi-static pulmonary compliance (C st(L) ) and partial and 

maximum expiratory flow-volume curves by standard techniques, frequency dependence of compliance and resistance and pulmonary impedance from 2-32Hz were 

measured by a forced oscillation technique. The addition of these latter 

measurements may elucidate more clearly than ever before the site and nature 

of lung impairment caused by exposure to toxic compounds. 

Following six months of 0 3 exposure, pulmonary resistance'and frequency 

dependence of puimonary compliance were significantly increased. After 12 

months, the 03 exposure had significantly increased pulmonary resistance and 

inertance (related to the pressure required to accelerate air and lung tissue), 

9-61 
and forced expiratory maneuvers showed decreased flows at low lung volumes 

(12.5 percent VC) and decreased volume expired in 1 sec (FEV 1). Wegner (1982) 

suggested that because lung volumes and pulmonary compliance were not affected 

in 03-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-lumpedparameter 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 compliance tended to decrease in both 0 3-exposed and control animals. However, the 

decrease in compliance was significantly greater.in 03-exposed animals than in 

control animals.. No other significant differences were measured following the 

3-month recovery period, although values for 0 3-exposed animals remained 

substantially different from those for control animals, suggesting that full 

recovery was not complete. 

The forced oscillation technique has also been utilized in rats exposed 

to 1254 pg/m3 (0.64 ppm) of 0 3 for ei ther 7 or 20 days (Kotl i koff et al ., 

1984). In an attempt to further characterize 0 3-induced changes in central 

and peripheral distribution of inechanical properties of the respiratory system, 

impedance spectra of 0 3-exposed rats were compared to the spectra of normal 

rats. The effective resistance was higher at all frequencies in the 7-day 

exposed rats but no consistent differences were observed by - 20 days. The 

effective reactance, however, was significantly lower than control in both the 

7- and 20-day exposed rats. These changes in respiratory system impedance 

demonstrate evidence of inechanical alterations in the peripheral airways of 

rats for as long as 20 days of 0 3 exposure. 

9.3.2.3 Airway Reactivity. Ozone potentiates the effects of drugs that constrict airway smooth muscle in mice, guinea pigs, dogs, sheep, and humans 

(Table 9-4). Early experimental evidence for hyperreactivity to bronchoconstrictive drugs following 0 3 exposure was provided by Easton and Murphy 

(1967). Although much of their work was done with very high 0 3 concentrations 

(9800 to 11760 Ng/m3 , 5 to 6 ppm), they did show that mortality from a single 

subcutaneous injection of histamine was higher in guinea pigs exposed to 980 

or 1960 pg/m3 (0.5 or 1 ppm) of 03 for 2 hr (33 and 50 percent mortal i ty, 

respectively) compared with the mortality of air-exposed control animals. The 

animals appeared to die from massive bronchoconstriction, with the lungs 

remaining fully inflated instead of collapsing when the chest was opened. 

9-62 
0 

co 

c, 

~ 

~0 

4-3 

ar 

E 

ro 

L 

ro L 

d 

Q 

n 

a~ 

O 

L 

N 

0o I ori I o I o0 

LO 

i rl I ~ r~ I~ I~ rl 

L9 ri 

o r+ 

cn N 

o 14 N 

cn

rlM 

} 

I--• 

> 

M 

U 

¢ 

LU 

C 

¢ 

~ 

a 

Z 

O 

H 

~ 

Z 

~ LL 

ui

>- 

1' ¢ 

Z 

E 

a 

z 0 

Z 

N 

O 

O 

N 

U 

W 

LLW 

~• 

~ 

W 

m I 

0) U 

c 

a L 

a 4- 

v 

z 

H 

v 

.0 

m 

a 

y 

d 

H 

4J 

U 

w 

W 

N 

a 

G1 

L 

Ci 

.G 

to 

O 

L L. O U 

N 

4, 

 roo 

a fi5-

x=a 

W a ~ 

ro 41 G 

ma 

r o 

OJ L 

N E f0 

N 

S 

E c a o a 

c ro O i 

N dJ 

O C 

G! 

U 

C E 

°v rn 

~ z 

i 

a 

e 

a 

a 

c 

ro 

c 0 

a o 

i Cp 

O 01 

C7 rl 

~ 

O1 Of 

,c.o 0 roM 

a ~ co 

r C 

~ N 

~• v 

1 O 

n 

oaxii~ . 

OfCf -Al- C c c U l5n O 

33 C 1G +1 

,2 o•r E ro 

O O n m C 

v- v- roca 

t ~ ~ c L C U aJ O 

N C 00 L U 

C) rL c0 c ar ai a) 1n i.) 00 

E rC M C +J 

+~~dL L Ua 

NC CD c7 ~ L•r ro O ro 

V- L a .- OJ 

O G! C .- f.. 

+J R1 Ctl 

r L C +~ C +J rn 

a~imo ~ a 

•C a E n C

tm 

rain.o10 

V1 G. a.0 

o u co°1J Gl O Of U 

ro i o u- ++ ++ 3 0 

.O niry 

N (U vf 4- +) 

L 

L 

ri 

z 

W 

S 

U 

O 

~ 

rt o 

00 

O 

i-~ 

tD 00 

~~ 

r-I 

00 

cn 

ro 

~ 

a 

c 0 

a 

i 

O 

C7 

Of 

• a 

ro ai 

c 

.~ 

C:3 

ro 

3 

++ T d C 

4J LLLr ~ 

u row 

uroi aroi a 

L aG1 

N a L 

i-) 61 4••1 

CLO. d 

o c 

L •r v1 

rn lad L c 

_cL+) ro 

N O - 

rU 4- 

C E O c t0 •r Y O7 

Emvm 

O7 0) to L. i d ro — L+J aN GroJ O U L U •r a N N 

a C U ••- 

y O ~ ~ 

4os+» 

i t4j0 w N 

N e0 Hl O 

C1 Q. i C 

.~ 

ro rt ~ 

4-+ . 

..y 

L .G 

a~ •• c 

croro 

+JMr rl 

L W aG0 

d01 fOQI 

C7 rl Y rl 

01 

O 

CI 

N 

C a 

01 

E Y 

1 ro U r i.+ o 

•L (A 

a •r rna 

aL 

cJ•r d.

a+J C i O ) 

Ul C 4-a C 

f- o~ E d 3 

U d a U1 

r M ° aidi c 

~ Oo V m 

Y O ~ C i 

° a s°- i•}~ o a nw-'> X 

_T 

N O C U cq 

N E S.N o 

Ou! S. 4-

0. L Ol O E V- L T 

o 

U tOi a a C7 ~ G. O C Y •r 41 fa U ro rl o 

E H ~ aO ~ 

O•rL G! > +1 tA U }1 oaiT = os. 

Cf i r- a C O 

> Of io -5 +3 al 

c•r ~ ~c 

7 +1Gl.0 •r 

i iE •r E O °

~ a.N 

~ G! o ro 

C C •r 

C~ a O S rl L 

M 

co 

~ 

~ 

ro 

~ 

a~ 

Y 

ro 

N 

U 

N 

N 

a ai 

N 

L 

N 

ro 

Gl 

s 

u 

s 

~ 

C 

N 

~ 

O L 

a 

~ 

a 

m 

N 

U L 

+1 1~11 

~ n o 

E x 

w 

O L 

L 4J 

aro 

cs 

d N 

y 

i 10 

C > tO

M r• 

~ L 

i 

~ 

a 

c 

ro 

c 0 +.+ n 

Uf l0 

rorn W r-1 

Of . Q 

m d 

c 

7 

C.7 

O 

1 

s- + °.+ a a ~ > fl1 OC S. •r a 

Ui G C Url 

a 7 •r 4 ro — N vl N N O N i N 

r X c T T ~ 

N 

(D 

 t •.~- ro o. O O) U U ~ a 

i a i 

C 

 o ° •t0 axi m 3 C Gl O > a O) 

+t r ~a a ip •r 

~ ~ •3U Lr•°- 

••-' N(D E O .OV- 

~ ++ a U w .- 

 ro O i0 X i i0 •r y y L 

LO. aG7 E V Of et 

T E G L ~ N 

• +1 3a ro 

~ ++`o L °- ro 

O O O .- t0Cv w4-^ 

a +j lq d.) d 

G) 7 E Vl L+J Uf L \ JC R1 

i

~ 

s OU N N 

C}' e ~ 

,••1 N Ol LL Q~ 

L s 

N N 

~C W 

Z CS1 

~ 0 

++ 0 

a 

0 

ao cl co 

N C1 (D r1 

m 

y 

7 

O 

Z 

~ 

N 

N U 

OU 

o (-° c d 

ro n 7 E O 

u) c ~ c o 

~ ~o io•r c 

a000 ~ 

ML V-•r a 

O 4- O i•1 

4- TG aUi

U, 

y 

Er +~ •r o n U f. t0 

as•°~

) 

 ~ m 

ao E Ora•r 7 C 

O i Na •~ 

Lm~ro~ 

° ro c o ~ 

Ln L > 4F > 

O Of ~ O O 

O C +J M O 

' +J •r C ~ i) 

'. Ol ~ •>f 4- ~ 

9-}~ L 4- Y1 

7 r Gl O N 7 O a 

O uf d L X 

X GO- 7 r O 

o c o 3 T a •r L Q a 

61 E C. G7 G7 

N a AL (0 N ai r- U c a) a td O E a 0! > .0 O N OS O V1 U L. I 

N 

••10 a `~L•~ 

7 a1 c 'O n M w Of O t0 X•r L N O C . 

c14 ro c~ Q.` n ~' ntns o cn L. 0 +J to C4J N 7 ~ U N'O G! L! 

OMY- O OM ro+, L. -- 1 

U r1 O C.w v C! 'O Oo .G m I 

~ NQ 

F 

C 

E 

0 i M L L 1 O 

L 

r--4 N 

z H z W 

V 

N¢ W 

S V 

N 

+~ ro 

~ . 

~ o 

r v- 0 

~ a 

• a 

i 

E~ i 

~ o 

3 U~ 

a O 

r 

•~ 

C, 

C ~ 

•Id a 

~ n. °

L Cxl 

d C 

ro 3 

U 

C O 

rr W 

9-63 
n 

'O 

N 

7 

C 

4-) 

C 

U 

1-~ 

? 

M 

v 

~ 

Q ~ 

2 

O 

~ 

Z 

~ 

u.. ~ zg _ 

z 

0 

W 

Z I 

N 

0 

~ 

0 

N 

U 

W 

LL 

LL 

W 

V' 

1 

rn 

W 

J 

~ 

!••• 

co 

° r, 

~ ' ~ ~ ~ 

u 

c io id r1 

L 41 +1 

ar 

~ 

a~ ai ia r• 

ro ro 

a

4J

i 

si°. 

a

4-)i "ai 

~ ai al v 

¢ ¢ J J J 

tn 

G! 

u 

C 

0/ 

d 

CI 

IL d O1 O) Of Of 

d 

N 

L 

N 

C 

VI 

O 

C) O 

C 

O 

° 

X 

ro +J M 

-O 1 ~111 V) ~ 7 L ° ~ •3 

L. 

O O Gl (7 ro 

w L 

• 

L iQ 1 

O) ul Vl 1 a L 

O Gl O Of C C! 

O 

d rC uf S. ~ 

1 Ol O O O rt1 +.1 M C U -O L d 7 ~ O a-> >> S.. r- O O S. L L O. Y> .0 O7 c o 0 o d ~ L r- •r V- a 7 0>> C Vf N 7 N x f..) •r u L•r 

10 u ~1•~ • L 

E ~ •r 

Vl Ul 

S. 

o+1 ro 7 0 7 M Y- a O 

o L O O- V1 +J Gloij L a C L >> d U'II O d 0>> O L Gl O d T-O N Gl L d L Ma rtl S. 

ro L v x L a i•r C C i~ a) 1 a d•r x r- d ij ai ar ++ o ro x C10 Gl o ai o o i 0>>3 ~1~ rd m av.N/nE+ ~ ++uL aiz wi++ 

+~ 

~ •r aQ • •O ~- 

a O Gl 

C MN Gl O Gl f[1 7 

O O +~ L L+~ a 

OftC OJ d 

C•r w O 

C M•r y v -O 

a 

07 > i••i f7 e0 ••• r- C7 o Mr- U U d uf 01lO 41 0 -O •r Ql ~ 4- M Of 

•L. CI ro•r C •E 

O i-1 Gl 

• 'O w O) U 

f6 p/ CI N ro C ~ 

1+1 41 b+~ .0 u -O E+~ o ro ~ 

L L ~ F1 

c c E v- In a) t c 

r vl •r 

O E •r +j L C r- r -O 

tn 7•r rtf L • C U C IO Cl Gl N 6 t0 •r 

•; L 4- •r 

O ro t0 'O r• 

-O E}1 ~ r• 

r O N O • ~n ; L o O•r 

N 

~ 

r y Vl 

•O L~ 

d L d dr• 

7 d • 

Ql O C ~ N G7 O C.- .1•7 N L N O 

tT~ C i- O 

C N ro U 

L O O C O 1 dr-.r Gl U M >> N Ul a •r r• O r- U•r U O 01L ~ X v S. O d i> >> L 

R1 U 

fE O O Vl ro 

d Gl N 

L O N C fd 

+j V- L. L i--i O'- 

L U C C r 

+~ • r ro 

a N O O rV- C 4- N -O 

~ 1N 3 C X rl C E Rl 7 O7 C V) R1 ro L L • 07 -O M O N Of d d 

> 

w L r 

Uf •r 

• y y L 

f0 O> d 

d r- in C•r O Of L r• O•r v L f[7 

Gl O)+~ i 10 

L•r ro O> 

G) O L•r a N ~ 

~ V~ .0 +> > C L C t0 >> G) L. .F) ••- d .G •r C.L Of N> a L Gl 7 O) R1 -1 Gl Ol o i~ Ol V Gl Vf E X+J C C ro L u7 a t0 C rl +.~ C' L> 

w 

.n 

d•-•r c 

M al > m 

c4+v c>, 

ro a- d o lo 3v v lo o•r T o m++ ai•r r- v- a 3 La ~ -a 

o 0 0 

u•r ro o v 

3 d r• c L 

° L 7 ~ .^ vl ro o Vdf ~ ~ L o d 1~0 0 °-O r N~0 o N C •rC- ~ •r c d ul •r ro IA ~ d C7 •r ro i~ d +~ U C Gl N O E L t E o GJ •r ~ >> O IA L 

Io n c u G! f0 x i ro c v ai t ro o Y cn ro L m r• r•r u t i N M 

a 3 0 lo •r c o 1l1 L Ul a c r w++ u ro o 0 2 x 01 

L G1 N r• 

Gl >> c 

> ri 1~ rt1 •r 

L E•r ro+~ O 

C 1 i'O O fO r• 

o c 

r• a N O 

Vl c Gl L•r 

N L•r O L a

j 

 .F O ) 

•R) 

r \ 

•u7 ~ 

V O P7 O C 1V r- a -O ~ ¢ C a 

01 ro 

uf .0 L i U nJ U 

M~t ro a C • O U 

T7 ct Uf 

C 01 G! 

O In C •tl7 Cl 

•C. 

C L> (0 . 01 O.G U L O•- •d C •d Ol c!• O r ~ F L C f0 

o L ~ a d ro•r c 

t0 C.1j ¢ C>i C ul O 

i•r o a•r r- c 

10 •r O+~ 

i c 61 L 7 

L N i•~ 'O • O i~ Gl 

u O ul C d•r L N y C tC Y 1 L ro O • L+1 a C C>> u C C C•r 

~ i-/ ro ro 

IJ ~ F u 

OJ L -O •r L 

L. U C u 

•• ri .G 'O a L. 

r- RI G7 N a Gl Gl 

~ tp 7 td 

+~ N N 

r O i~ 7(O O O E 

• 'O U Id 

~ 

10,0 y 

W. 

C 10 ul 

>,.r d r 

10 I Of fO 

ro 3r La L fL .1.~ @ ro o a~ m v o 0 R1 C O O r• 

E o d a o L.1..~ •> c -O ~1-~ O v m•r .r d ro t v, 

a Id 7 

C! ro O 3 Gl tD c C L G> ~ Vl U•r N u/ L o'- •r O 7.- Vl O G. t U x r- VI 

o Gl BJ O 

.N r- 4•1 1 Y r• l.i •r v~ Id t0 u Of u 10 c L . @c 

o [}• C! 

Lct n7o 

• ~ .0 •r c•rxG.-000do ro 

cL LL G1Lro0oLo 

L c N i.- c L o ' zr+cn > a •~ ..u+~+ 

L x c a ro X 1n 

~ 3x ar•r ro u d•r .G •r M C Gl ¢ roo•r ro cD•r ioa a++ i 

I 

v=i .1.1 ~ o lo o >f lo 0 d L i 

~ G 

-O 

\N 

N V- N N N N 

~ 

N "C 

N,C W W W W 

D U 

~ 

N O NC, =J u U 

ro 

S 

O 

fJ O 

Ln 

Y 

CO ~ tl] tl'f O iD CO t^O Ir N 

01!4J 

O O e4 O O O . O 14 

c ro 

N 41 

O C O O a/ 

U 

iJ i-) 

O~ c0 tOO O lN0 

U 1 

CO 

Ol N 

rM•1 

rl ri rN•1 r~ - rl N 

9-64 
.. 

a ai 

~ 

C ~.1 

O 

U 

v 

1-}-• 

~ 

M 

F 

U 

W 

C 

6 

~ 

Q 

Z 

O 

1--~ U 

Z 

~ 

} 

Q 

F 

~ 

a 

Z 

0 

W 

Z 

O 

N 

O 

O 

FCU 

W 

lL 

W 

~ 

Ol 

W 

J 

en 

Q 

H 

~ 

~ ~ 

~ 

a, r4 

~ 

~, 

- ~ 

~ 

N E G! 

U 

• • - 

10 

ro 7 

O 

C fa L C 

41 +1 O +1 N Z C 

t° 

N 

o N ro

•s= L ~ro• ~o 

2~ ~ 

0p 

Or~i ~ ~' 

t. 

~ ~ 

G7 0~ ~~ 

O 

N 71 Of O1 E 

y o. c. n ~ 

~ v ro eo w 

a c c c ro w tm rn 

c'v 

+1 

~ c 

c° c'~. o n 

~ a 

a 

L 

Gl 

L ~ L 1 

W 

~ 

cu 

+~ U Q N ~ C. N L X L ~ GJ ta

0 a ~ . O•r - Cl d 

a ~ 1 

1 O E 

R) L+i 

C~ Gl O L Q i. 3 

N G! 

c 41 C +J 

ro N N f~ a L. 4..1 Rf i U O•r N L 

N4•~ C 

ro N O 

ro ~ E 

t0 Gl E 

G/ +1 7 C) +J ~ C1 L a Gl •r G/ 

y+~ i i 

C•Q t0 iY - 

N 3 • t O> ro 

i.1 Gl .- 

C•r C+J 

61 w CI 

w41 7 

~7 

c0 N, N 

N 

L . a 

N 

C 

O N•r U 

• r 

r• Y- > CV L L C O ~ C 

Cl N Gl 

C Gl Gl •rf 

a C 

O G) r•r 

Gl •r C O. 

s ro 

C L O O. 

r y U X 

~0 

~O 

ro a 

II 

i0. C O7 C i~ N O O. >f ro i-~ O Gl E i r 

- a•~ Gl C•r 

>> f0 

10 L. 41 

N G7 +.~ ~..~ 

10 N 07 

N 3 N i 

L 4- L 

O ro U i 

.0 Gl 

~X +~ +~ 

Y Oo 

~ 

U•r L Ol 

~ N U c 

N L C 7 

01 U O U 

ro 3- Cl 7 

3•r C>> 

\ C Cl 

~ L O N'O 

ro d• ro 

E r• 

Z • 

N 

+1 

7 C 

N O O•E 

C G U!d 

61•r 

ro N X 

L > o 

C L i w 01 

ro ~ a ro+1 N •-ta d 

U 

41 

O. Z ro 

L N +J 

> O O 

r.•• +1 4 1 

C~ Cl •r L. 

Q L N a 

et N 

.0 ri a N•r 

L+J G/ C~ L U 

C 

V-• 

4- 

i~ N N 

r L 

•N Gl 

C L a a 

N C>f 

a .O L 

C.~ GI >f N 

Q Tr ro i 

Q>t L 

d 7 

01 

U 

N 

•3 

>f L 

O ~ Gl G! 0! f1 ~ O. ~0 a C7 N ~- U N 

a a N O N 

V~i O 

 Ln 

 i C 

 id 

ro C d.0 ~ R7 C N n O O O>> C 

CJ 

> 

G! 9-+7 7 

N 7 o O 

Cl Q 7•r 

i x+J r 

t L 

c c a 

O r• L 

c•r •r ~ (n t,.) 

C•r +~ 

Gl N N•r 

•E 

i 

Gl 

ro N Of 47 

d O M11 C 

Cl 41 

>> i>> 

r Gl L 41 

• E+.~ ~ 

Gl E o'•- Q 

>•r +

1 

 Cf 

> N r• > 

e0 •r .r .r 

7 

r• 

N 

a 

L G. > ro 

U x 41 

ro i 

~ Gl a ~ 

O a1 O C 

+, rt1 m G! 

ro N > N 

L. 

L E L ~.1 

+1 CJ G U •B 

o N Gi s 7 

+~ c•r y > 

+~ a+~ .- O C L o ro CI 

a O U 

i a 

V- f0 N 

~ 10 

~O N 

N L GI 

w+1 • L 

C G1 •r C 

• 

r tA > GI 01, 

L GI 

•r •••+~ i 

t 

U 

' JG) N 7 

C.1 d-) C N 

O 

C i L p. 

07 Gl N d 

C L eIE •r C > 

_. L Ql i~ •r ro 

t G1 7 

i~ t. Gl 

4- •r 

~~0 O. L 

r L C1 +~ u> 

Cl O.•r pJ 

> L L 

~+~ L 7 C(0 •3 N 3 

N - 

ro 

c O al i ~ 

t+J Va~ r•~ ro+~ 

N. r• U 

ro Ol Gl C! 

r c C. a7 

•N •r o 3 

c U L 

•

3 U rt1 Cl +~ 

c a1 N c 

r i/0 •'- 

a .0 

a ~ n o•r 

N x ~0 

o PO. 

• 

"O ro RI 

C/ tV 

Q).5 ~~FF N 

s ro V- C 

o~0 +~ C 

at 7 o 

Q1 GI >> O 

i c 10 0+~ 

N 41 c ro 

o c 

N 

ro 

>> G! ro aa U U E a LLI O 

c ro a 

N. a GI •r 

m a c.~ 

7•r }7 

N N 

41 s•r ro 

i. Gl

~ 1L 

Ol 

G7 c c 

L•r a rtf 

r a~ ~-i N 

• N GJ 

i E ro O ~f i i0 0•r a Gl 

0! L 10 Cl N 

U+J ro+J 

C 4- i II 

U E G! •r a•r Gl Y L. 

~a U C C 

• 

r ro 4.1 L Z 

C ro Y r 

• r Y U O. 

y L G) Cl 

Y L Ci >> 

. 10 C r• 

~~ 

r C•r 

rt1 ••• L O 

~ N O D. 

r• Gl 

3f- • O7 

W 

S 

N O E 

~- .- O 

f- a Ql 3L - ~ 0 C N O O~ F- 

• 

~•r C 

roY~ L i.) N 

1' Gl O Cl Cl 

t~ V- C 

C C O 

t..) 

d'La U f ro ar-i 41 Q~+~•r tN E N S. i NN•rr- 

~ 

L i0./ N 

E O 

C 

N 

w Y+pJ ~ N 

v 

G 1-L 

 7 O. X 

L 

UR! 

W a 

oA L .0 t L L Y E 

r1 N N N N E ~ 

o a 

7; w 

ro ~ o 

C 

01 O 

f• •

O Y 

EO f +J 

aiL, 

L+J 

..W 

S 

v~i E ai Ci 

~ v S GI 

O 4J 

41 O 

O O. ON O NO 

C C. 

O O O ~+ 

CZ ri rl rl N M M- M M 

to 

tn 

N +1 C! O 

~ v y C7 

N N 

f.07 Ol ~ M G1 [h C~D 

OCCpp 

~pp 

O~Cpp 

I 3. ri N rl ~ tt1 tf7 tA tn IOZ ~ 

9—E5 
Abraham et al. (1980) evaluated airway reactivity in sheep from measurements of pulmonary resistance following inhaled carbachol aerosols. Carbachol 

causes bronchoconstriction by stimulating airway smooth muscle at receptor 

sites that are normally stimulated by release of acetylcholine from terminals 

of the vagus nerve. Pulmonary resistance during inhalation of carbachol 

aerosols was significantly higher than pre-exposure values at 24 hr postexposure 

but not immediately following a 2-hr exposure to 980 pg/m 3 (0.5 ppm) of 0 3 . 

This 03 exposure did not affect resting end-expiratory lung volume (functional 

residual capacity) or static lung compliance. In sheep exposed to 1960 pg/m 3 

(1 ppm) of 0 3 for 2 hr, baseline resistance (before carbachol aerosol inhalation) was elevated 24 hr following exposure, and airway reactivity to carbachol 

was increased immediately and 24 hr following 0 3 exposure. 

To determine if 0 3-induced airway secretions limit the penetration of an 

inhaled bronchoconstrictor, Abraham et al. (1984a) compared airway responsiveness to inhaled and intravenous carbachol before and 24 hours after exposure 

to 03 . Adult female sheep were exposed to 980 pg/m3 (0.5 ppm) of 03 , 2 hr/day 

for 2 consecutive days. Airway sensitivity was defined as the largest increase 

in specific lung resistance after carbachol challenge and airway reactivity as 

the slope of the dose-response curve. There were no significant differences 

between pre- and postexposure responses to inhalation challenge. However, 0 3 

exposure increased mean airway reactivity and sensitivity by 34 and 31 percent, 

respectively, using intravenous challenge. Since carbachol causes direct 

stimulation of airway smooth muscle, the authors suggested that 03 may have 

decreased penetration of the inhaled carbachol to the airway smooth muscle as 

the result-of increased airway secretion. This hypothesis is supported by a 

previous study showing that changes in airway responsiveness to inhaled histamine following exposure to 0 3 may have been related to changes in airway 

permeability to histamine (Abraham et al., 1984b). 

A study i n awake gui nea pi gs' compari ng changes = i n ai - rway-reacti vi ty wi th . . .-_ ~ 

intravenous and inhaled bronchoconstrictors after exposure to a high concentration of 03 has suggested that mechanisms other than increased airway permeability may be involved. Roum and Murlas (1984) measured changes in specific 

airway resistance with intravenous and/or inhaled acetylcholine or methacholine 

from 4 hr to 2 days after exposure to 5880 pg/m 3(3.0 ppm) of ' 0 3for 2 hr. 

Airway hyperreactivity by either route was similar within 14 hr of exposure. 

Two days after exposure airway reactivity to bronchoconstrictor inhalation 

• M. 
returned to baseline levels while responses *to intravenous acetylcholine 

persisted. The consistent early changes in airway reactivity after 03 exposure 

with either intravenous or inhaled bronchoconstrictors indicated that this 

response may be independent of the route of delivery. How-ever, it is also 

possible that there may be more than one mechanism responsible for 0 3-induced 

airway hyperreactivity depending on the concentration of 0 3 reaching the 

airway tissue and on interspecies differences in celiular responsiveness to 

03 . 

Gordon and Amdur (1980) evaluated airway reactivity to subcutaneously 

injected histamine in awake guinea pigs foliowing a 1-hr exposure to 196 to 

1568 pg/m3 (0.1 to 0.8 ppm)•of 0 3 . Airway reactivity to histamine was maximai 

2 to 6 hr following 0 3 exposure and returned to control levels by 24 hr following exposure. The histamine-induced increase in pulmonary resistance was 

greater in guinea pigs exposed to 1568 }ag/m 3 (0.8 ppm) of 0 3 than in air-exposed 

control animals. Pulmonary compliance decreased more following histamine 

injection in all 0 3-exposed groups than in air-exposed controls, but there 

were no differences in the histamine-induced decreases in pulmonary compliance 

between any of the 0 3 concentr,ations (from 196 to 1568 pg/m3 ; 0.1 to 0.8 ppm). 

Gordon et al. (1981) studied the effect of 0 3 on tissue cholinesterases 

to see if they were responsible for the bronchial reactivity observed following 

challenges with bronchoconstrictor analogs of acetylcholine. Guinea pigs were 

exposed to clean air or 196 or 1568 pg/m 3 (0.1 or 0.8 ppm) of 0 3 for 1 hr. 

After 2 hr, brain, lung, and diaphragm samples were analyzed for cholinesterase activity. Brain cholinesterase activity was not affected, but lung 

cholinesterase underwent a 17 percent decrease in activity at 196 pg/m 3 

(0.1 ppm) and a 16 percent decrease at 1568 pg/m 3 (0.8 ppm). Ozone at 

1568 pg/m3 (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'irreversibie cholinesterase inhibitor. Airway 

_ ___.... _.__. _.._.._ . . 

resistance­tended to increase following histamine challenge in the p4rathiontreated guinea pigs, but the difference was not statistically significant 

because of large variations in response. The authors suggested that cholinesterase inhibition by 0 3 may contribute to the 0 3 -induced bronchial reactivity, 

as already reported. Presumably, the. decreased cholinesterase activity could 

result in higher acetylcholine concentrations in the bronchial muscle. A 

cholinergic-related stimulus, such as occurs with 0 3 exposure, should then 

9-67 
increase the contraction of the bronchus. The persistence of this activity is 

not known. 

Gordon et al. (1984) extended these studies to determine the site of the 

airway hyperresponsiveness to histamine-induced airway constriction after 0 3 

exposure. Anesthetized guinea pigs were evaluated for response to subcutaneous 

histamine 2 hr after exposure to 1960 or 2352 pg/m3 (1.0 or 1.2 ppm) of 0 3 for 

1 hr. Respiratory resistance increased and dynamic compliance decreased in 

03-exposed animals, as previously reported (Gordon and Amdur, 1980). However, 

static compliance changes after histamine were similar in the air- and 0 3 - 

exposed animals, suggesting that the site of hyperresponsiveness was in the 

conducting airways rather than the parenchyma. In addition, enhanced airway 

responsiveness to histamine was not blocked by atropine or vagotomy, indicating 

a minimal level of vagal involvement. The significantly greater increase in 

respiratory resistance caused by efferent electrical stimulation of the vagus 

in 03-exposed animals suggested that other mechanisms, such as changes in the 

airway smooth muscle, may be responsible for the hyperexcitability following 

03 exposure. However, in avitro studies on isolated parenchymal strips removed 

from the lungs .of air- and 0 3-exposed animals failed to show any differences 

in the contractile responses to histamine or carbachol. 

Lee et al. (1977) evaluated airway reactivity in 0 3-exposed dogs from 

changes in pulmonary resistance induced by histamine aerosol inhalation. Dogswere exposed to 1372 to 2352 pg/m3 (0.7 to 1.2 ppm) of 03 for 2 hr. Airway 

reactivity to inhaled histamine aerosols was significantly greater 24 hr but 

not 1 hr after 03 exposure. Bronchodilatation induced by inhalation of isoproteronol aerosols prevented any change in resistance following histamine exposure. This experiment showed that the increased resistance normally observed 

following histamine exposure was caused by constriction of airway smooth 

muscle and not by edema or increased mucous production, which would not be 

prevented by isoproterenol bronchodilatation. Administration of atropine 

which blocks bronchoconstrictor acti-vity coming from the - vagus nerve) or 

vagal cooling (which blocks both sensory receptor activity traveling from the 

lung to the brain and bronchoconstrictor activity going from the brain to the 

lung) decreased the response to histamine both before and following 0 3 exposure 

and abolished the hyperreactive airway response. These experiments showed 

°that the increased sensitivity to histamine following 0 3 exposure was caused 

by heightened activity of vagal bronchoconstrictor reflexes. 

• .: 
Although the work of Lee et al. (1977)' provides evidence that stimulation 

of vagal reflexes by histamine is in part responsible for the increased airway 

reactivity found in dogs following 0 3 exposure, Kaplan et al. (1981) found 

that iocal responses to histamine in the lung periphery may not be mediated by 

a significant vagal component. When monodispersed histamine aerosols were 

delivered to separate sublobar bronchi in dogs through a 5.5 mm diameter 

fiber-optic bronchoscope, collateral airflow resistance increased both before 

and after bilateral cervical vagotomy. In follow-up studies that used similar 

techniques, Gertner et al. (1983a,b,c; 1984) described the role of vagai 

reflexes in the response of the lung periphery to locally administered histamine 

and 0 3 . Collateral resistance increased during separate 30-min exposures to 

either 196 pg/m3 (0.1 ppm) of 0 3 or 1.5 x10-6 mg/m3 of histamine. Nowever, 

although parasympathetic blockade (atropine or bilateral cervical vagotomy) 

prevented the responses to 0 3 , it did not prevent the responses to histamine 

(Gertner et al., 1983b). To determine if 0 3-induced increases in collateral 

resistance in the lung periphery were dependent on vagal reflexes, aerosolized 

neostigmine was administered locally to maintain parasympathetic tone. Responses to 196 pg/m 3 (0.1 ppm) of 03 in the lung periphery were enhanced only 

if the vagi were intact and were limited to the challenged region (Gertner 

et al., 1984). When larger areas were exposed, vagally mediated responses 

occurred in both lungs. In addition, the characteristics of responses to high 

concentrations of 03 differ markedly from responses to the lower concentrations. 

A 30-min exposure to 196 pg/m3 (0.1 ppm) of 0 3 did not affect the airway 

responsiveness to histamine, but when the 0 3 exposure was increased to 1960 

pg/m3 (1.0 ppm) for 10 min, histamine produced greater increases in collateral 

resistance.that were not abolished by parasympathetic blockade (Gertner et al., 

1983c). Exposure to 1960 pg/m 3 (1.0 ppm) of 0 3 for 30 min produced an increase 

in collateral resistance that was mediated by the parasympathetic system in 

the early phase of the response and related in part to histamine release in 

the late phase of the response (Gertner et al., 1983a). Results from this 

series of studies by Kaplan et al. (1981) and Gertner et al. (1983a,b,c; 1984) 

are difficult to interpret because of the small numbers of animals in each 

test group and large variations in response. In addition, because peripheral 

resistance contributes only a small part to total pulmonary resistance, the 

findings of these authors'do not necessarily contradict the work of Lee et al. 

(1977). Rather, all the studies taken together suggest that the periphery of 

Oa-69 
the lung may respond differently from the larger conducting airways during 

exposure to 0 3 and that factors in addition to vagal bronchoconstrictor reflexes 

can produce an increased airway reactivity to histamine. 

Holtzman et al. (1983a) reported the time course of 0 3-induced airway 

hyperreactivity in dogs exposed to 1960 and 4312 pg/m 3 (1.0 and 2.2 ppm) of 0 3 

for 2 hrs. Airway responsiveness to acetylcholine in 7 dogs increased markedly 

1 hr, and to a lesser extent, 24 hr after exposure to 4312 pg/m 3 (2.2 ppm) of 

03 , returning to control levels by 1 week after exposure. Ozone-induced increases in airway responsiveness to histamine were similar following exposure 

to 1960 pg/m3 (1.0 ppm) of 03 , but data were reported for only 2 dogs. The 

authors suggested that the time course of the 0 3 effect may be linked to acute 

airway inflammation. In a coincident publication, Holtzman et al. (1983b) 

found a strong association between airway hyperreactivity and tracheal inflammation in dogs 1 hr following a 2-hr exposure to 4116 pg/m 3 (2.1 ppm) of 03 . 

Airway reactivity was assessed from the increase in pulmonary resistance 

following inhalation of acetylcholine aerosols, and airway reactivity was 

increased in 6 of 10 03-exposed dogs. The number of neutrophils present in a 

tracheal 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 3-induced increases in airway responsiveness with inflammation in more distal airways (Fabbri et al., 1984). The 

number of neutrophils as well as ciliated epithelial cells i,n fluid recovered 

from bronchoalveolar lavage was increased in 5 dogs that were hyperreactive to 

acetylcholine following a 2-hr exposure to 5880 pg/m 3 (3.0 ppm) of 0 3 without 

significant changes in the numbers of macrophages, lymphocytes, or eosinophils. 

In dogs depleted of neutrophils by treatment with hydroxyurea, 0 3 exposure to 

5880 pg/m3 (3.0 ppm) of 0 3 for 2 hr caused the desquamation of epithelial 

cells but airway responsiveness to inhaled acetylcholine was prevented (0'Byrne 

et al., 1984a). This observation suggests that 0 3-induced hyperresponsiveness 

may depend on the mobilization of neutrophils into the airways. 

The authors have expanded their work to speculate that the neutrophils 

produce mediators that are responsible for the increased responsiveness of 

airways (0'Byrne et al., 1984b). In dogs pretreated with indomethacin, a 

prostaglandin synthetase inhibitor, exposure to 5880 pg/m3 (3.0 ppm) of 0 3 for 

2 hr had no effect on airway responsiveness to inhaled acetylcholine but there 

was a significant increase in the'number of neutrophils in the airway epithelium. While these results suggest that oxidation products of arachidonic 

9-70 
acid, possibly prostaglandins or thromboxane (0'Byrne et al., 1984c), may be 

released by the neutrophils, they are not conclusive. Therefore, the identity 

of the specific inflammatory cells or of the responsible mediators is still 

uncertain. 

The results of tracheal lavage in sheep exposed to 980 μg/m 3 (0.5 ppm) of 

0 3 for 2 hr suggest that the migration of mast cells into the airways may also 

_ have important implications for reactive airways and allergic airway disease 

(Sielczak et al., 1983). Nasotracheal-tube exposure to 03 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 3-induced 

bronchial reactivity reported previously in sheep- (Abraham et al., 1980). 

Additional evidence in guinea pigs suggests that 03-induced bronchial 

hyperreactivity may be due to airway mucosal injury and mast cell infiltration 

(Murlas and Roum, 1985). Specific airway resistance was measured as a function 

of increasing intravenous acetylcholine doses for varying periods of from 2 hr 

up to 4 days after exposure to 5880 pg/m 3 (3.0 ppm) of 0 3 for 2 hr. The largest 

airway response to-acetylcholine challenge occurred 2 hr after 0 3 exposure with 

complete remission by the fourth day. Neutrophil infiltration occurred later and 

lasted longer despite the remission in airway hyperreactivity suggesting that the 

influx of neutrophils was a result of the initial damage and not a direct cause of 

increased airway responsivensss. 

Increased drug-induced bronchoconstriction is not the only indicator of 

airway hyperreactivity following 0 3 exposure. Animal experiments were designed 

to investigate the mechanisms responsible for the abnormal, rapid, shallow 

breathing found in human subjects exercising during expe.rimental 0 3 exposure 

compared with subjects exercising in clean air (Chapter 10). Lee et al. 

(1979, 1980) showed that abnormal, rapid, shallow breathing in conscious dogs 

immediately following 2-hr exposures to 1100 to 1666 pg/m 3 (0.56 to 0.85 ppm) 

of 0 3 was a hyperreactive airway response. This abnormal breathing pattern 

was elicited by mild exercise, histamine aerosol inhalation, or breathing air 

with reduced oxygen (02 ) or elevated carbon dioxide (CO 2 ) concentrations. The 

rapid, shallow breathing observed in dogs following 0 3 exposure was not affected by drug-induced bronchodilatation (inhaled isoproteronol aerosols) or by 

blocking vagally induced bronchoconstriction with atropine. In all cases, 

rapid, shallow breathing was abolished by vagal cooling, which blocked the 

transmission of sensory nerves located in the airways. These investigators 

(Lee et al., 1979, 1980) suggest that the rapid, shallow breathing observed 

9-71 
following 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 

bronchoconstriction). 

Studies of lung morphology foilowing 0 3 exposure showed damage to the 

respiratory epithelium (Section 9.3.1). Damage to ILhe epithelium overlying 

sensory receptors may be responsible for the increased receptor reactivity to 

mechanical stimulation (increased ventilation with exercise, low 0 2 , or high 

c02 ) or chemical (histamine) stimulation (Nadel, 1977; Boushey et al., 1980). 

The rapid, shallow breathing observed in guinea pigs during 03 exposures (Amdur 

et al. 1978; Yokoyama, 1969; Murphy et al., 1964) may also be related to increased sensory neural activity coming from the lungs, not to an indirect effect 

of changes in airway diameter or lung distensibility as previously speculated. 

In their study of allergic lung sensitization, Osebold et al. (1980) 

showed additional functional evidence for epithelial disruption caused by 0 3 

exposure. These investigators studied the anaphylactic response of mice to 

intravenous ovalbumin injection following repeated inhalations of aerosolized 

ovalbumin. Mice were continuously exposed to 980 or 1568 pg/m 3 (0.5 or 0.8 ppm) 

of 03 for four periods of 3 to 5 days each, separated by 3 to 8 days of ambient 

air exposure. During periods of 03 exposure, mice were removed from the exposure 

chambers for short periods, and they inhaled ovalbumin aerosols for 30 min. 

Mice exposed to 03 and ovalbumin aerosols developed more severe anaphylactic 

reactions and had a higher incidence of fatal anaphylaxis than air-exposed 

rnice receiving the same exposure to aerosolized ovalbumin. In mice exposed to 

aerosolized ovalbumin, 34 percent of the 0 3-exposed mice (1568 pg/m 3 ; 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 03 exposures and exposures to sulfuric 

acid aerosols. Of the mice exposed to 980 pg/m 3 (0.5 ppm) of 03 plus 1 mg/m3 

of sulfuric acid aerosols, 55 percent died of anaphylactic shock following 

intravenous ovalbumin injection, compared with 20-percent mortality in mice 

exposed to 0 3 alone and zero mortality in mice exposed to sulfuric acid aerosols 

alone. The authors propose that these data may indicate that pollutants can 

increase not only the total number of clinical asthma attacks, but also the 

number of allergically sensitized individuals in the population. Matsumura 

9-72 
(1970) observed a similar increase in the allergic response bf sensitized 

guinea pigs to inhaled antigen following 30 min of exposure to 3920 Ng/m3 

(2.0 ppm) of 0 3 . 

9.3.3Biochemically Detected Effects 

9.3.3.1 Introduction . This section will include some studies involving concentrations above 1960 pg/m 3 (1 ppm) of 0 3 , because the direction of the 

effect is opposite that for lower 0 3 concentrations (decrease vs. increase in 

many parameters). An extensive body of data on this topic has been reviewed 

by Menzel (1983), Mustafa et ai. (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 inetabolic activity. 

This results in some degree of -artificial separation, particularly because 

many patterns of response to 0 3 are similar across the classes of inetabolism. 

Lung permeability is discussed in this section, because it is typically detected 

biochemically. The final subsection presents hypotheses about the molecular 

mechanism(s) of action of 0 3 , relying on the data presented earlier by metabolic 

class. 

9.3.3.2 Antioxidant Metabolism . Antioxidant metabolism of the lungs is 

influenced by 0 3 exposure. As shown in the schematic diagram below.(Figure 

9-3), this system consists of a number of enzymes. As shall be discussed, 0 3 

has been shown to produce several reactive oxidant species in vitrofrom com- 

-o---G-6-P___Y_NADP + "O-v-► GSH—y--[O] 

— G-6-PD G SH I GSj H 

HMP shunt reductase peroxidase 

6-PG --O~ NADPH —)%--GSSG ~ ROH 

+ 

H + 

Figure 9-3. Intracellular,compounds active in antioxidant metabolism of 

the lung. (G-6-P = glucose-6-phosphate; 6-PG = 6-phosphogluconate; 

_ 

._ .r ~.._.._......_ .._ . - G-6-PD = glucose-6-phosphate dehydrogenase; HMP shunt `'hexose - _ 

monophosphate shunt; NADP+ = nicotinamide adenine dinucleotide 

phosphate; NADPH = reduced NADP; GSH = glutath:one; GSSG = 

glutathione disulfide; [O] = oxidizing moiety [i.e., hyai•ogen peroxide, 

free radical, lipid peroxide]; GSH peroxidase = glutathione perioxidase; 

GSH reductase = glutathione reductase; and ROH = reduced form of 

[Ol)• 

Source: U.S. Environmental Protection Agency (1978). 

9-73 
pounds found in the lung, as well as in other organs. It is reasonably certain 

that 03 can produce such reactive species in the lung after in 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 3 . 

Typically, following exposures to levels of 0 3 below 1960 pg/m3 (1 ppm)., 

the activities of most enzymes in this system are increased (Table 9-5). 

Whether this increase is due to direct mechanisms (e.g., de novo synthesis 

resulting in greater enzyme activity), indirect mechanisms (e.g., an increased 

number of type 2 cells that naturally have a higher enzymi,c activity than type 

1 cells), or a combination of both has not been proven. The increase in type 

2 cells is most likely to be the mechanism, because with similar exposure 

regimens, the effects (increased enzyme activities and increased numbers of 

type 2 cells) increase, reaching a maximum at 3 to 4 days of exposure and a 

steady state on day 7 of exposure. Whatever the mechanism, the increase 

occurs at low 0 3 levels in several species under varying exposure regimens 

(Table 9-5). The net result is that the antioxidant metabolism of the lung 

is increased. Whether this is a protective or a toxic response is often 

debated. To resolve this debate scientifically will require more knowledge of 

the mechanisms involved. However, even attributing it to be a protective response implies a physiological need for protection (e.g., an initial toxic 

response occurred which required protection). A more detailed discussion of 

these effects follows. 

Acute exposures to high concentrations of 0 3 generally decrease antioxidant 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 3 levels (3920 and 7840 pg/m 3 , 2 and 4 ppm) and 

short-term (8.,or 24 hr/day, 7 days) exposures to lower 0 3 levels (392, 980, 

1568 pg/m3 , 0. 2, 0. 5, 0.8 ppm) on rats: For - nonprotei"n sul fhydryl ' 1 evel s 

(principally glutathione, GSH), decreases in the level of GSH were progressive 

with time of exposure (2 to 6 hr) to 7840 pg/m 3 (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 reductase and glucose-6-phosphate dehydrogenase (G-6-PD). After the first day of a 

7-day continuous exposure to 1568 pg/m3 (0.8 ppm) of 03 , no significant change 

was seen in the nonprotein sulfhydryl or GSH content or in the activities of 

G-6-PD, GSH reductase, or disulfide reductase. However, the levels/'activities 

9-74 
U_ 

Vl 

N 

U 

IF 

4- 

d 

a G1 

i 

N 

a O 

r-I 

co 

a, 

~ 

v 

v 

- 

I.() J 

M1 N 

1•

cn

-1 c +~ 

v m ro v 

c +) (O ro L 

a 

L 

a w vlo 10 

a) 

a 

4- O N N I

, 

O 1~ 

C U $ E r~-1 

N 

O a~ 4•1 +J 

N C C C 

C. 

N 

-a 

m ~c ~ 

wr-I 

Io 

+~ •• N C! 

7 GJ 

Z J 

b 

!Y 

aa ~ C 1 

ro ~cw . 

I C1 

x U' +•/ 0. 

N r 1 

C7 II •> tD 

C 1 4- ro E c7 

OGII 

d m -4 

> i1 m 

ro 

r II•r 

al 

a 

° ~ 3 x N o ai 

N N d Gl C 

i C7 N G-10 

C C x U 

rN OfC7 O 

N O 

U 

C 

N ~ •iO •• 

a 

0 

 aci ~ 

 w 

+J(1) II G! Q 

c c. > 

07s u u 

 1''i 

U N r- +j 

a05  E d > 

'0 O C 1 0 00 E 

w N ° L G°~. 

~ ~ U C O 

+J (V •r rl 

> • V- ri 

> uC•r Gl+1 t 

+/ +~ d +~ , U O r •r 

grtt c 

•

II3 

c ro 

r i~ •i 

G) ro +J E II 

to 

roOl~ a ° L 

L C N U 

U O a O 

~ N O N 

ai c a E 

++mEmv— 

O aCi ITr1 ° 

v rna 

~ 

4-1

II E 7 7 

6) O N .1.1 O 

i.l L 1 10 E 

C W ro 

E C +) O II 

N ^•r•r G! 

> }G1t N 

a 4- E E Groi 

7 O O. 7 t. 

N d N U 

w o rl O ~ rl U 

+~ 41 O - 

C. t N 1J 

•> 7 ~ ~ C1 

E N 3 ^ o 

0 0 

t0 U E0u0i CI tocc.loloC G/ C 01 G1 7 LOf L L r 

+~ TN U U 

3 X C C C 

oo•r.-.•r 

I DI 

U Y 1 

~~ c a~ 

IIGl E A 

5- +1 O 

SlD C ~ N 

tnv O C LLJ 

- +j A N +~ 

u° i •3cc.•> 

XN ~ N ~ o>awv 

d ~ 7 N ~ 

nS > OU7 

S tn W 4- 

N d L 4- 

G.7 Z 4•1 +-) C 

4-4- > 3 3 

O O — N N 

N II O 7 N C 

G! C Y y 

ro~ -•

O

r 

~ Cf E vl GN7 

~ O. 

+~~c r~ U 'i air 

..4- co 

' .t0 U' 4- U 

+f y •r 

o •o ~ rn c 

0ui ICOt E ~•~ 

L 61 • 3 ~ G.+J 

U N N L C 

H+07 OC ~ E 

II C C! cp r 

i O 7 C U 

N O O 

O •r N 

W L O. 

II' 

7 >> 

O f0 

7II 

I C 

~ 

~ C i II O O 

U V3 T O f0 

C II 

^ 

I C i 

U 9- 

O N 

7 a C 

n 

~ 

C i 

U VW 

Z 

O 

N 

O 

>- 

m 

LLJ 

z 

O 

~ d 

S 

~ 

~ 

Z 

O 

U 

z 

~ 

r 

X 

O 

Cf 

Z 

Q 

f 

N 

J 

0 

H LLJ 

2 

G 

X O_ 

M-• Z Q 

C.7 Z 

J 

w 

x 1--~ Z 

f-1 

W 

C:J 

Q 

U 

tn 

Q~ 

w 

J 

m 

Q 

- 

C1 

r rC-/U 

1. 

z 

Y 

c x rtr a v ro a> 

O G1 7 N i • E> N 07 N 

U10 G1 

CD 

 C G/ L r0• 7 nG/ N 

i II U V- G! •r .0 O RJ L L 

y•r C O U+~ U a1 L U C Of 

L X O C•r O O Of C1 G7 47 •r N 

Ofo U N o>iJ c.Ca_ ro 

L Gl i ~ U ~••~ U+•1 L~ .G ~ N C.1 

O °• 3:~O~air- w I~co~Tv 

~ tN -r C Gl ft1 C G- i ~~ 'O L 

d ov c'~i~ T

c ° aXi -'v•~ vc.~~ 

L N L 10 N U+•) C N N O 

~ °I '

J

II Q mcoc •° mmmE ~ 

O i~ i•~ G) N C. N d-~ .~ Gl O CM 

.bJ•rQ N N O+J G.C.O L i 

> (6 O! G) O) L 1J E N U L N 

.0 ~~•E N ro Q ~ O b 

7 U C U+J iJ N T i C+J +3 •O 

N fE 1 C U 7 G! O 10 +> >> ' O 1•r 7 r L 4-1 U • C U C 

CII t0 II.- c•r c m N C1 1 O! '. 

X Gl 1 •• d RI O 10 •r N C>> 7 G1 N-. 

O Id ~ N i C i•~ ~ i ~~C a d 10 +3•1 

N O a 7 x•r °• T o 4- •r N c Gl 

7 i C O tn E+•1 V- O ro O.° •r ~ 

O U Id 7 f.7 N 7•r E M 7 U 

7 C C N N> N N y N U II 

c•r -• II ro C•r +J al L a/ = ai 

y}l C C7 O i-~ r +j L Id 1A 7 

R7 S. U U 7 ro II i•~ /0 

- C N rt1 O U ro N L C II'F Ol 

O C+~ U N C N N 10 V- C O+j 

U O U v N•r O d i a O 10 fO 

CJ ~-1~ C V IIII b i u0i~ L~i•T~ C 

r/o a o•r a> a~ +~ la ro> d rl •r 

+~ L L•r X 4~ N U r 07 fO 27 > L 

44 41 o ro(0 7•r L•O c T•rv 

i CS rt1 L

r 

 NII E U tG+1 •r 

O N N f- G! G! L d•r C+J •10 a U L 

lL U (.7 i-) (1 L U L tn 1-•~ (0 E v(0 3 

L 

O 

i O O 

7 jl

4- 

O M 

4~ 

}f ^ L\. 1~0 iJ ~ N C i L a C L T 

C..) V°- CO t~ U 4°- •CS 

.0 

44 

G1 

BII a/ o 

7 4J Y N Y 

b E Z H ~ Z }+ 

N Z 

C E rl 

4, ~! G O 

G! ftl C L O +- 

N C t0 C) 

 =

(n 

E -1 

I °U 1 

r•1 N I rl N I N Iff 00 

66 O C O O C 

r~l f+

M

'Y I r~ir~~7 ( C~nO~i~ 

C~ 

O 

00 

to LO 

ro 

9-75 
i N 

1 7 

tJ ' O 

7 1 7 

II C C 

i U +1 

C C 

U O O 

C7 U 

4-1U 

E m ro a 

i C r Cm 

L •r 4- 

U

• 

+ ) 

O Ol C C 

ii N D7 a 

U Gm/ N 41 

x U o E ~

' ¢ •r 

(U 

• Z 0! • C 

N C •r 

C

m 

 L r G! 

•rL L 

X N +J 

o°.. ° vm-c 

~ ~ c 3 

ca r o a n 

4- U C L 7 

° 7 O N i 

N N Q! Q! O7 

O a C II Gl 

i~ C C d i 

r m O L 7 

> r m N 

N +J 4- O 

n 

U m i •r X 

¢ iJ +3 II Gl 

ip =1 

\ N 

i N 

L C N 

d °u ro N ^ II 

O i > 

O •r 

W 4- .1J 

i 

ro i 

CaJ N 

•r m 

G1 

~ i.1 N 

m u •r 

GJ t~ 

C 4- i 

r 4• N 

• N X 

o w 

d O Nz ro m a i 7 

tJ L O 

C Cl i 

r c rn 

• 

 N 

c . 

m 0

E N 

> +~ U 

G1 C i 

r m a 

a~ caxi 

L •G10C 

m C L 41 

? • O 1~ ~~ 

O7 m•. 4 C--• 

G1 C nII uaioai 

7u u IIC N C ai o ro 

z uo.c 

i 

O 

4~- 

ro 

II N 

L b 

L II 

M -~r 

N¢Y 

S Z m

N1fIC10 

O O O 

N O N 

(h 01 N ri 

M 

Y 

m 0 

Z Z 

~ 

N tf) O N O O N N 

O O ri O O rl 14 W 

N O O N O O 

i N N 

Ol 00 tD 

mrnrn 

rl 

Gl 00 tD 

mmrn 

ri 

ul 1 , 

mo 

N 

~ 

o--• 

Y 

M Z 

tff 

N M tn CO N tn OO 

O O O O O O O 

N t0 O OD N O GO 

Q1 03CO tD Ol ab 40 fnlO011n M (n117 

i-7 rl 

n • a 

E!f 

~ 

C 

~ 

C 

O 

u v 

w 

Z 

O 

N 

O 

>• 

m 

Z 

0 

1~ 

~ 

N 

x O 

U 

x w 

CD 

x 0 

c 

S h 

m

4 

~ 

~ 

O 

.. 

X 

O 

~ I 

~'•` I 

`¢ 

t~ 

z 

~ 

1 

tz~ 

Z 

M 

N W 

t7 

I U 

vi 

0 

w 

~ 

v c d roo u rc rom aa w. •••i i ro 

y 7 y 

~ 

N C! 

y Y 

C 

U O 

Gl 'E 

G 

N 

l! 

~ 

~ 

U 

W 

d 

v ai 

O 

.O 

O 

4- 

Ln 

O 

i 

Gl •O N II . 4°- 

~ m N 

m 00 Gl 

•> •• N tn 41 

+~ w u M 

 00 

 o 

C1 m COO n 

m~ •r d 

u +7 4.1 i 

c tOtQ a 

m L U Cal 

•r Y Y O 

O7 U 4- C C 

i^ ai •c E ~ s°• 

O O ~i~ N 3 

~

i N Gl 7 

C c N m 

ro u c ai ++ 

i OoL m 

•F ~ i-- 0! 

C >> N N a+ u m ai ai r• m 

U 1 'O .0 .0 m •O 

Oa •X~~+~ •X 

C7 C1 O ~ ^ u O 

II Z Cl m d 

N •• m Cl € i f~O 

tt1 C] Ci Q. CI c 

L. ~ •t7 7 n Gl •t7 

u lD u u tn 3 u 

M(; N 00 S N 

II ' 

C 

d ro r• 

i O 

7 C U 

N O C 

G~ O 

u~ s%. a 

3 

'O 

a 

m N c 

~ t ~ Y 

N G1 

m E N 

~ 

C E 

•r n 4J 

m ro Ci o4-3 

N C 

ou E c 

O O7 

U =L 

00 

r, 

cn 

b 

^ +3 m 

io d +J 

~ 4J ° 

° ro ~ 

O .3C 1- Y 

U N r I LL 

G1 

N 

K CC Z 

N 

C 1 O 1 C i • 

U O ro i N 

i•> > U Ol N m O. 

m s a v(a ~ ~ o 

•F-) i C II r E iC O •r r Of 

C1S•r XON u ~ n ++ o a ai 

C C J m L+Ji 

C,1 w.N nrr N N 

i vOi CCl S f0• •~ G. 

G) m u t/1 1+~ X 

L II C C,7 C•r 07 

07•r O O > 

r XU 4- •r .r aJ c o O c 

ii muar 

3 a 3 N ~ m ~ ++ o •r c m •r 

G) E E 

otn r u a

N

i 

iJ U' (3)/ •> C 01 

.0 •r O C dJ 

i O~ U O U ~ 

1=A N¢ m¢N i 

o Gl O i O 

X +~ • vOi G» ~ 

61 •r C m N II 

N ~

Cl- 

 LO• +f er- (1) 

7 i.1t0 U u m•r 

0 ~t~ ~ v c+%+ 

C G! •r p 

C N m N S N N 

O ro 7 N m+J 

U G7 •• O C7 Gl rLCl 7 L. 7 

~. i U N C II U N 

O C m•r C C d 

 ti •r +a +~ m •r,i 

i 

O i 

O 

N TN VO f0 

3II ro 

a 

C II N 

C i L C 

00 

U 4- 00i- 

~ 

~ 

~ 

ro 

+~ m 

v N 

Y 

I LL 

G1 

N 

~ 

E 

C 

O 

4J4-I- E+°1 r• 

C y i.0 ~ . 

Q1 •r 4- 

N 7> 

. N Gl 

u i~ ro 00 O N 

C •r 

u ••>- VOi 

O. 

~ N O 

MU•O d 1~l1 ~ 

~ ac 

L C O 

+~ •r S. • 

N 

O N +1 

• 3 ai o. o. X~ 

N 

~ 

G. 

~ C1Nri 

O/ II r 

i O 

U t) U II ~ I~- G/ 

d C II C V- N 

i r• c m ro m 

', •O ro u7 + 3 E i 

II c aio cai ac 

v°7i°mo co r 

b U ~ C I0I 

' cii ~ v s°. 

U cP 

x 3 ~ C II i N 

L7 ° O 

++sxn. ca u 

4-) O O O •G/ 07C7 C n ^ F 

o x c o 

N C 

m m u N O. QI .0 E 

I N 

2 II LO a 

i u G. N 

U O a 

~ roc~ v oz° w -mo 

N 

i 

O ~ 

4- 'O 

roM 

II 

L L 

G. 

~ 7 [~• 

i 

O 

w 

fa0 

O N 

L ta0 

L II 

O n 

M TO 
m 

~ 

~ 

u O 

c ro 

s

4

' 

G! 

u /o 

7 1n 

y- J ^ 

n' 

cn 

O e4 

N 

Cl u d Y 

iJ 

ro 

7 Gl 0 C C 

r N F 

+J 

O 

U 

v 

N 

Z O 

n°i c a 

~ 

•o >, 

Vl U •r iJ }.1 

r ro > c o m a+ o 

L+~ +1 U C 

Z 

O 

U T U 

C U /0 

r 

H 

~ rs 

E 

v n 

Gl 

rn 

~ 

N 

N Z ro CO t 

4) N • U 

U •— V~7 C4-1 L C U 

Z 

ro•r q 7 

S+•/ N 

Ql f0 

C L 

W . u N u O O 7 Vc> O. O_ L 

X O v ZN Ont0 • vi CX! l0 E Y N 

O 

2~ U O 

ro 

G ~ L 

Z 

v 

4- 

r 

GJ tA O 7 •O 

w N E 

O +•~ 

q O N 

x 

GJ N II d 

a). ^ c s. >, 

rtl N U O E 

•E 

o 

/n 

rr 

II 

N 

S. a R3 ro 

u G• U'a 2 

v- C L 

O O 4- 

J 

O 

> 

L 

C i 1P) 

r lO -•FJ 

tN 

U• 

4•r 

NC (1) 

m 

H 

Cl 

.O 

/ o4~ ro 

O +1 U C 2 

N t0 •r y 

r M 

w O N K- Q ro N ro.0 •r 

Z 

~ a°fi 

N Ol d C•O ~ 

z iz 4 

C,> G II U N 

Q 

a 

r N•r y 

r +~ t0 u N 

y 

N N 

O C ~ O C 

4- L IE O 

H M•r i•~ U ro ro G) O L GJ •r 

X 

~ 

O> u 3 G! 

N 

d E 

N 

u N E 

r• 4J•O U U N 

C-~ N 

N i•/ O R1 

2 Q N i O ~ J •E + GC7 w 

Q 

C- Z T 

J -p •ro0 

C \ 

w aro.- iN N 

Z O•raJ N "O Oo 

H n+

, 

 O I

, 

\ Ir 

tN w L CI. O S. L i 4~ 

C.7 •O CO 4- 00 V°~ - N 

Q 

x 

U 

d 

~ 

1 

Ol 

ro 

44 

J E G 

m N O 

1— C 4J vf Y 

N a 

rO E 

q m 

E Z 

v 

Z 

~ 

c E N tn a9 00 o c. 

r n 000 O N ~r 

4••1 

to 

CL 

O +J 

N C N O N W O C. 

O u E 

cn

c0M 0

1 t~0 ~ON

1~ 

C e-~ ri [+•) i~ 

O D7 

u 3. 

0 

. ° _ ^ 

m ^ ro m 

Iv 0 +1 +J 

ro m ro ro 

a- v10w- 

+~ [7 L +~ N 1••~ d' 

7007G1 O 701 1 

rl f rl 

N 

C 

y 

111 

•r N 

•• M11 A•~ 

iJ 7 G! L ro d 

C f 7 N L ~ 

f m o ~ 

II 

N 

v, 

}1 N 

C.. ro 

C 1•~ O U+j i u 

o x x d u r u U 

+J 

•Q 

(1) 

N •• N tnc N>f 

0-CI N C7 6! a+/ 

V- CI c G/ 

r V- •r E 

n ~ x z C7 ro C7 N•r •c V- O 

7 C! ~' J •• G. ••Or S •.0 r O O N L 

N E S/X N N}t •V/ U 

c 

O 

a II 

r• C 

N t0 O O_ c o u 

H z •r ~ (0 

r T O O i+/ L 4..) 

U 0.f0 ^ U ro C N 

TU 

 N 

n 

7 N 

W C V- i. L L 

O O. Gl O+) O O "O •O) L 

O N Q! N 

C r y 

1E N lL 

N lD O ~ N N 

01 L ~ F L7 

U d r C 

C O d•~ •r O) i L d ~ 7 C 7 ro 

r C•r i-/ 

.> •r 

rC7 L ~ - f0 • Gl 

i•/ U +~ L O! > 

•O T N 

O N y 

G1 'O •r > ^ O d^ N L C N 

 N 

. /0 

I N 

c d•/ •r 

ro U+~ 

ro u 

> 01 d> >>• 

• N`~ Cl N r 

~ ro U 4JN C c 

a V•r ld 

d u+~ •r 

i L v ro u+) u O•r C 7 O X 

C i0•/ 1 y \ 7 G/ \ 

a 

 N• 1•~ ° O n ° 

• 

r C/ N N II N N Cl O N i C QI 

N c0 ro 4! -Ri a i i 

G! L C GJ U! G. a 

O. 0+1 

N c rti 

> Gl N> 1 G! O N C 

1•~ 

/o 

q 

ao 

Cl 2 O ro G/ t0 i i rLnoor• / oo Z O II•r 

zcu 

0. Ol •O CD i•r CD E - G1 'H IO U 

•O •O X II q Uf 

Gl ^ T O Gl •C G! Z ro 

O+ 7 

N 

' / 

C 

/ Gi t0 

w L d 

N y.0 N c i a L 

/0 N C1 G/ ti1 f0 G! d 

E c+7 rN 

n O+~ w 

o u L 

i•~ = U 

N<0 'O +> C! 3 C~ 

L II ftl L r N 0. 

O O 

i•> +~ 

+~ r G1 u•r C1 c u G1 N , C 0p 7 N 

ro 

L. 

•> • a c X+J •r e sn U N ro 

r O Rl U•r tp •r Df 5- 

d'O N 

~ 61 •r 

C }1 N N 

E C d-~ 

d d.~ 7 f0 1 

N N ~..~ 

II~i•~ R1 •r 

61 O 0.r• 0.•r N.. j( ^.0 lq C U Gl > 

U 

C 

d 7 0! 

O> 

G1 U N O U/ 

U 2 O'O ~ i r• 2 

ro C! i•r 

4- U+> 

O 

u 

C i O/ 

'••~ OI r 

r cn N C ro ~ d ro O N 

f(> •r f0 d' n L C C7 

M 4- C U 

O G1 •r ro 

N N N N N 

7 

C G II 

+1 

C L. 

i' 

L 

44 

C i 

L 

L L 

U ~ t0 U4°- OOw 

N 1n Op M d C• 

000 0 o c 

NO CO i-I 

M 00

1 ~ 

cli 

NCp N 

OaD 

rl 4 0 OO 

9-77 
~ i~ 

i~ 

~ I ro 

I ~ ro 

0 

I U 

4J 

z 

N N 

• 

ro

N 

 ~ ui i~ GJ i 

U t = N 

v c o m Cl >f O. -0 i N C X 

S C•.- N N C 

N •r- •IJ N O 

U ~ V C. O+J 

C! L X N ^ > 

ro° ° ~ ~ 4! 

'O r Ol •O d 

- ro44 

X L ro Ol l0 L 

O O1 C v rtf 

L •r O 

Gl G

C

)U i~ C ~ arn

~ ia € E 

N 2 N U1 O) N 

O 1 R) 7 L•rC- 4- ~ Z •~ r 

° V- 'D > E d 

Ol ° O r V1 r ~ N.0 O = d.; r U f.. CE N ai o+~ c ai •> C>1••~- O •`n L 

U r E U = ~ 

`0c o +°J s°1. 3 

'O C. = N 

~ to + ~.+ (1) o. 

ro ro ro ax 

L O LL N+J 

U V'OU I N 

C C C N d O 

.~ ro mo ~ o 

L 

W 

N 0 _ c ~ 

C O 

U M 

m 

4-) v 

a% ro 

I 4-) ~ ro' 

L 

rn a C E _ _ ~ G r N C +3 O O 

(! (I •r- .0 

.G 4- L € +J GL .. 

iJ O +) C 4-) 7 

^ C O •~ ¢ N 

° N L o N ~, G . u 

N ro C) •r Gl rtf 'O +) i V N 

a) '•- ro >, o 

L x L d .0 

U O O. L Ol C 

C N ro O • - - C O 

r L C 0 Gl 

• •C InU O N 

coric

1 

raa 

O••- 3 ro i 

+J O. O N EC 

ro E L r C•r 

.1J N G G1 

Gl O ~ U ~ C U U O1 i C O 

C 10ro •~ 

O c~l'O r L .4.>

L. 

U O C O U 0

L r ro> O +J v ro d

drz c L •.- -> ro C/ 

O L% N C • rO c ro m o O +J 0! •r U 

d L = O. L 

+J O >07~ +~T C N 

+J r X V- L O 

Q E O O U r 

L O N 

4- ro N O _ 

C N 

C ~ L 

U ~ O 

c. c. ro fv c ro 

on L 01 

U rl 

I z ro

+~ C 

i Q L 

2 N fj 

f 7 C +J ~ ~ 

ai~ 3 c°i ~ 

ro~ t~0 0x1 L 

a 

ul 

p ro v U 

O 

L 

O i rl N ~ N C 

'. C n ro L

W.4~

+i

.+ E 

vvvac a)(M 

N 10 4.1 'O 7 U O= 

. U' ro •F ~ •r C- 1- N r 

Y.0 +~ O! L 4- 

 ° 1D G1(M O O 4- 0 

N O O. r U O N 

~ lO 41 N L G1 UI E 

~ ro V+

3 4) 

.O) IV •> Cf C 4- L> .F; C 

~ n. L o 3 a~ •~; ai 

IU ~ ro C(n O +~ r 

t•7 Ol •r ro r U yJ 

c L c -°r 

c r- .•- 

ro T 

a)-x++ 

O/ N M •O O N 

ao 

G! (J 

fN0 i%~ T~ aii o 1NQ ~ 

0/ U ~ 7 O. ~'~I N O) 

hC. 

a) 

 f~0 +~ C. ~7 ~Q C m 

= o 

041 = N 

c ~ ro 

N N N = a 

7 •ro0 

c n 

C L ~ G i O O O U 4-M C - °7 °4 

Y ]~C 

m m 

z z 

n co ao 

O O O 

O 00 W 

I~ ut0~ 

ri 

t0 

~r-1 

H 

O~ 

O N 

00 O t0 N 

LO 

r-I M 

_ Lr, 

~ c, iro i (V 

4-3 ~ Gn .a) 9 C1 i to 

i0 

c a a.~ c 

m m to 

w ` o 

J

o c"n

o 0 n 

~ CGr~

I U U r0•1 

(A 

~ 

V N al U 

m 

Y 

c c. 

4-3 o ro 

41 v) ¢ a z z 

c 

°u 

11 

ui 

Z ~ 

N ro 

O 

} •F~ 

L w 

m C N+3 

L 

G~. G•• C - 

O +1 O tD C1 A 

{- .G Cf 4- C7 Ol Cf ~ 

n N ° > 3 > 

S ln 'O N Gl Gl Nl 4- 

Vl 

= v i ar NN G1 O ro .c ~ 

U ~O L G! -° ° N O 

z M Cr o V GCl ~ W fJ_ 

Ln 

2 

Ln 

 L>> C1 

X v b O ~ 4- 

O U 9 

C. N7 

V- ••Ca'a0 

G C7 i~ O 

N i0 d•~ U 

f.7 C N•r 

~ 4- ro S. N C 4- 2 ~.C E

°1 a-T+ N ai ro O ~ v- ^ S 

y~ d 

J > 

•>n 

r• X 

~ aT 

•> 

a tro 

O 

6] 

i 

d 

•N G7

Cl 

r- tD Y 

d~ 1 C 

4.1 dl O 

- N C r 

~ n ro o 0 m

0 

>~ - ro++ 

S ~ O 1, C C c U= Gy N r 

~ v) ~ .- ro• ~ - ro ~ Nrow v 

~ 07 N O1 N U d L L N•ri 

fN0 N 1N0N 40- (NtlN C ~ N 

x 

O 

Oi 

i Vl 

_ G! C7 V- S. 

Gl 

C7 C7 .- a r 

L • • d 

~ U T U•O C1 •O Cl L> 

~ 

W V°-• 

_- 

61 ro — 

~ c u 

N N>~ 

o~7 

U1 Uf N Vl 

o N o ro 

tgL 1•=9 OX_ ir•~ O 

W

M 

 p. 

C t~ 

C 

\ T 

L'ro0 

C!f7 C Ih 

OC N L C L. C L 

~ C7 •O U w GO t~ U 4°-• U 4°- 

z U~ 

M 

J tn 

~ 

w io 

m m 93. s+ 

c 

J E •p 

1•-• 

41 O 

= •_ bC Y 

N d 

ro 

CI m O] . 

~ 

c ~ 0 0. 

tn 

co 

r O 

N f~p 

O O O O 

O 41 

N C 

O eU 

C P 

N 

GO W 

O 

c0 

Ol 

N c0 

I , t0 

O 01 

~ rl 

U =• 

1 
a 

U c 

c 

C! 

W 

GI 

C 

a 

v 

~ 

~ 

iJ 

C 

O 

U 

v 

N II 

N 

~u 

m d N 

U 

N 

41 

U d 

~ 

ar 

a 

d 

L 

v 

L 0 

W 

LLJ 

H 

Z 

N 

C7 Z 

Q 

S 

V 

ui 

c, 

w .-j 

m 

6 

H 

a C 

y m r• 

L o 

~ C U 

O r i4 

x ro z w L a 

•a 

a 

ro 

4J 

c ai 

E a 

N O 

L L 

~ +J 

NCJ 

-to .r= .. 

GI 

2: 

= E o d •r d 

~ d ro 

c s o +j 

N C 

O N 

U E 

C 

O Of 

U =. 

N U) 

.+ 

~ E 

N 

=o 

d a 

X O L 

ro 

n 

~ 

N 

4- 

O 

•~ 

.> 

4J 

U 

ro . 

a a N rt 

G1 

U 

c 

H 

U) T 

7 'OD 

C 

n 

C L. 

U w 

Z 

ro ro 

4J 4J a m 

m ro 

ua JLn u 

C1 rl n rl 

~ 4- 

z 

tu 

~o 

4-) 

N 

w ro I 4J ~ I N 1~ ' 

7 ~ 

$ '•i 

ro

~ 

z 

~ r 

1 

a 

a 

roN N O~ 

W rl 

~ 

O a 

p1 r• 

iJ O O co 4

~ T 

b 

+J ui 

•a 

ro N N /b 

W r0l 

d 

Ln 

7 0 

~ 

O 00 

O N i N e'7 O 

~ ao 0 0 o w 

tD N N co t0 

r`-i o c°.m ' m ~r~i 

9-79 

L 

O L 

L O 

t . 

N O Ld 

V w ~ Cl 

L O 

O E 4- C9 4- n N 

n MN 

O N O O 

v- c v- +J o •r 

o ro 

E G) 

E a f]. 01 da nc 

n N R1 ro d L 00 L .yU 

o v Q o 

4-) - G) c 

ro 

L S 

aL L H 

G1 L () 

Uc0 ~ 4- 

G/ L O 

w V 4-

0 

 r 

c O E > 3 w n Gl 

r E O.,- 

dnva a) 

S'1 L i 

tnL U 0-Ly 

Z O OOa 

L 

L L 

L 

N N 

V V 

M.. 

00 tl) 

o ri v 

ao o ° 

tD -d- s!• 

t19 Q1 00 r-1 N 1 , 

aa c rn 

ro ro 

1 U N •~ ~ 

1 

l0 O 

C7 > 

61 

1 Z • i ~ N 

co cuvi a 0 r 

~ CI 

ro d 

c 

tn 

SrCl ~ tn y C N•r 

N 

c '~ ro r• 

4J 

r 

ro•r cud 

y> 

s •r 

r 3 p 

a T 

N 

U+~ a 01 L N 

H UN N Lv N U r r 

u01i N 

a ro 

ai c 

N E 

x 

v7 

0/ iJ N > 0,2 

C 7 N ~~ E C 

(q a > u r N 

.0 Gl i ~ 

U 

S O C 

N+~ •r 

Gl rVl d 

c T ~o > 

o uv cD c u s%

2 

N E L ~ C Cf0 CUi G) 

Cl O 61 . roL •Oa 

N L ar r U L 

—uro aio m 

o C C/ N Z o a 

=vi a — ° m~ +~Ii 

O) U E 1-7 N > O 

c o ua v 

7 C c GJ 

~ ro• 

O - c L+~ C

I ro ~,E 

i 

L 

~ °0 c 

V O E 

~ 

~ ~ M 

. N . - 

1 •O 

O a Gl 

4 r p N 

O a O. M1S Rf 1 N 

O N S L l0 L 

mroasc

I c 

a ~ v ic +- a •r N 

NN ••m ro c 

ro nr ro 

ro s 

a+-a v 

r fp I • N N 

X 'O d ro +3 

a 

o +J 1 a) •o ro 

U C7 N•ri 

u v°/ - v- oa 

o 

1 

ro G! 4-. O a 

O a a) N.i0.~ 0 1 L o ro T ro>, N 

ri L z L ro c ro 

U •O •r- a ' 'G 

O a U 1 C 

T 61 0rl 01 7 ro 

t^ C b C NC V1 ^ 

Nrn° t0 ai ro ro ~ 

/0 O O ~r N 1 i. 

a~ 01 N cu 

N ^a. ~ 

X T C}.1 L C r 

O L•r R1 u•r O 

y 

aiaaa•ra T +~ 

+1 Cl C7 61 f0 L 

c~ ~7 NN aii ro •O E ro a a+J vao 

•U i f.. L_IC L rl L 

U u 61 U O1 

7 •r ~ ~ L a c 

N U • Of'Q •r S 

i• 

N 

n 

~ 

~ 

~ 

0 

co 

t0 

~ 

vi a 

1 C 7 G1 

nroa ar• 

N U L f0 Gl 

L O 2 d G N 

C7 1 O 

+•~ L t0 •- 

v..m•rcn+> C1 N L 0 

•U N ro 4- 

•r ~ C! L o Of 4- L. o c 

N E u N •r a v 3 (L) c 

a 'O •r C 

. ro r- Cl L • > G! 

W tOnN i•~ 

+)

a . 

cL O3 roai 

- +J L O! Vf 

•E •'- ~ Of C 

4J ~ 3 b rc ' > /~ L Gl • 

N u N w 

ro ro iJ 07 Gl O d Gl C> N C i L 

+Jra •r C C 

N T•r •r 

C N X +~ 

O~i O•r N L 

U L > > +> 

a a •r •r .•- 

I

ro~ du N 3 

3aci vzi roL c°Z 

+1 E C7 N Of ~ 

O OC 10 L ~ 

'. C1 d•r }~ D. t0 I 

N N 

~ T O t0 

C 4,1 Ln 

C L I 00 

u V- 

~ 

~ 

C~ 

O 

n 

tD 

`n ri
N 

ui t~ 

Z 

Y V 

ui 1 

cn 

LU J m 

1.¢.• 

U_ 

v 

4-+ U

w 

d 

.Q 

N 

N 

a 

O 

v c 

7 c U 

N O O 

O •r •Fa 

W L O. 

7 

~ 

c. a °i 

0.d tif c 

rl C t ~ o 

L t0 U 

O.0 C! 

tL 

S 

U O tn, 

 tl 

tn O Z 

IF- CN II 

O • N C 

E Cp7 1 ro 1]. 1x 

t1 0- 

O l0 Vl 

t0 ^ 

cp ° rtf n. x m 1 O tn 

W t0 L ro 

c c1,7 a s0.. 

E 'O S C M 

a~ N C7 O 

>

rtf O L o/ L ro al 

+1 U 4- 

C C tli N f0 

f0 •r N G }) f0 

N 4.1 O o rC U N L /L 

OG! 7 f0 Oi > 

UGl U + •F ~ ~ ro L v'°I ro 

3 •S Cl'atn 

+J tn C7 L .0 O 

ar x aJ c 

C C 

p•r•r(D

tn 

 t7

O

p r

7 

N N 

3 

O 10 

7 'O 

C 

•r tCl 

C L 

U Vv 

m 

N N 

a~ ro 

N O 

i~ C1 i 

U G/ 

> +~ C tA 

1-~ •> •r C 

U •r G1 Gl 

f0 4•1 N OI 

v ¢ c °s 

~ Y T t0 • L 

U d v G1 

U T b '0 ^ N > G7 

c = +.+ 

~ U 0. E 

acimcv 

> E r0 c 

L 'O Gl ro 

0. C tn d 

ioc°~ o °o 

+~ O U N 

ra 

L 1 7 

0. to 4- O 

W p L i 3 

IL t1 U 

C 1 N N 

r tp O 4- 

E 1 L v- 

~ C O Gl 7 4- 4- O > O O Z 

N N 

~ 

7 Q 

C 

•r t~ 

C L 

U 40-• 

0 

3 LO vOi 

°1 v , ° ro aroi 

+Joalv U v- ol c 

7 C •r o 

a

-ro 

i p7 U C 

ro ro 

S O 

N F C 

C.7 O N 

4- N .. b 

O >>p O ro n. N 'O 1 Lf1 

N plD I 

+' r-1 cD ro 

>als 

L O d 

U 4- LLtd 

10 4) N 

O aL U 

ul t]. 4J "O 

to 14- 

~ 1O c`°i ui 

v~ c°1 ro ro I N N ~ O '0 

'O IT7 N t!7 

L•O G! N M ro .r N >ro, 

al0i-o c 

+J L s tt1 

GJ U O 

O 0.Crltn 

C N 

L 

L 

01 

.G 

ro 

C 

E~ V 

i L 1-1 f F--• 

U g 

E 

c E 00 CO 

o IS 

~ a o 0 

d ro 

o +L

i 

o aci ~ 

E 

~ 

c pr•1 rW-1 O 

U 

cm 

7. 

rn o 

0 0 

~ 

~ 

to 

~ 

G/ io o°_ ' . 

ro 4J ~ ro 

c v c d 

IT9 M 

ru 

tL ~ M O N V- 

(1) 

N qO 

Wr0-1 

O 1- N 00 

Url HrMi 

'O 

O O 

4'1 U7 rl N 

c a E ~ z

Lo v 

•r N 

a~ 

c 0 

v 

~ 

ro 

~ 

ar 

c 

ro~ 

C t~ 

! J rl 

'O 

O O 

In 

1 N 

m •~ 41 x4- 

co ro 6 0 

rv w Gl j~ 

.0 10 p

O_ L CJ }f 'O 

1 U L 

lp •r ~ ~ 

1 L N tL MN 

0 3 O OI . 

L X ro7 C 

Gl GJ N V- O O 

cA 44 o a •r 

roro Gl N ro ~ 

o -3 ro c+'o c N O O 

>, ro . ~, c°i u 

ro'o N l3'f L y 

OGl rl i L 

tt1N O 7 

O M ro O U N 

N G) Y U O 

3 i-O/ U O O X 

O 0. 9 ri ~ N 

G! 7 i-1 1 N a alocn ~ 

L •r C 1 O 

+J d C7 ~ 

U +~ ~ dJ C 

d 4- O 

M 

 C •r a- ror•a•r N 

'F N Ov C 

N LU O 

Gl N N IA U 

z~ f~0 M Gro! L 

'O L L Gl 

N O U O 

d O C C 

L N t17 L.f) •r 7 

L R1 N 

O~l U N t~0 E NO 

~ 

v 

E 

O 

4J 

a 

> 

~ 

II N 

> ro 

p ~ 

D) 

G7 • O 

U L 

C "O 

U L 

N G/ 

N 9 

C 

N 

~ m 

U 

e ro 

a r 

L U II 

v x p 

J 0 N ~ 

N N ro ro a c 

II cl 

f L 

LU 

U L 

N 

ci a 

'II d 

~ ro 

O L 

a 

E o vOi 

~ Lro 

N ~O 07 

+J dLO.. 

0. O T 

O 7 N 

s rn ~ 

~ II ro 

t U 

l0 7 

ro 

L (7 m 

i•> O 7 L 

C7 N 0. 

C CN 

II o L 

H d }1 CL 

X 'O N ~D 

m iJ 

o r 

~ O)p 

~ v i C7 

G! e O 1 

G/ a 7 Ol 1 

+1•r r U 1 

NL N 7l0 

E U N O 

N t6 G! ..• 

N'O O L N 

z4~ 

c II L 

v O 'O C ] O C O1 tn >> 

i II 40) c7 ~ 

4~ G 4- • j 

N 

o • - O I! 

U 

7•r N LS 

O L L +1tn 

j~ d 7 }O

i . 

H E N 7 N ]C O C IO~ 

¢N II Y =t 

T~ ~-• Z CN.7 ~ 

3 

N 

O 'O 'O C 

O O Gl •r 

L .0 N CI 

iJ dJ 7 +~ 

N dl O 

E E N L 

C G 

+J C o 1 

aci ° +> o 

L L > II 

~ a a 

N L S 

ro a N v mae E ci ¢ z t0 a U 

L 

L 

l0 

O1 

nl 

all 
of these constituents increased by day 2 and remained elevated for the remainder 

of the exposure period. Comparable results were reported with similar (but 

not identical) exposure regimens by DeLucia et al. (1972, 1975b) using rats 

and Fukase et al. (1975) using mice. 

Investigators (Table 9-5) have found that for lower levels of 0 3 , increases in antioxidant metabolism are linearly related to 0 3 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) 

for 7 days to 392, 980, or 1568 pg/m 3 (0.2, 0.5, or 0.8 ppm) of 0 3 and found a 

concentration-related linear increase in activities of GSH peroxidase, GSH 

reductase, and G-6-PD., Significant increases occurred for all measurements, 

except G-6-PD at continuous exposure to 392 pg/m 3 (0.2 ppm). Although the 

difference between continuous and intermittent exposure was not examined statistically, no major differences appeared to exist. Schwartz et ai. (1976) 

made similar observations for G-6-PD activity when using identical exposure 

regimens and found concurrent morphological changes (Section 9.3.1). Mustafa 

and Lee (1976), also by using identical exposure regimens, found similar effects 

for G-6-PD activity. DeLucia et al. (1975a) found simiiar changes and increased 

nonprotein sulfhydryls at all three concentrations of 0 3 . A similar study was 

performed in mice by using a ionger exposure period of 30 days (Fukase et al., 

1975). The increase in GSH level was related to concentration and time of 

exposure. Fukase et al. (1978) also observed a linear concentration-related 

increase in GSH levels of mouse lungs exposed 3 hr/day for 4 days to 392, 980, 

or 1960 pg/m (0.2, 0.5, or 1.0 ppm) of 0 3 3 . Exercise enhanced the effect. At 

the lower 0 3 level, the increase in GSH was significant only in the exercising 

mice. 

The influence of time of exposure was examined directly by Ohow arid 

-Tappel (1973). Rats - werE exposed continuously to 1470 pg/m 3 (0.75 ppm) for 1, 

3, 10, or 30 days, at which times measurements of GSH reductase, GSH peroxidase, G-6-PD, pyruvate kinase, and 6-phosphogluconate dehydrogenase activities 

were made. No statistical tests or indications of data variability were 

presented. A few of the enzyme activities (GSH peroxidase and 6-phosphogluconate 

dehydrogenase) may have decreased at day 1 of exposure. All enzyme activities 

except GSH peroxidase increased by day 3 and reached a pe-ak at 10 days and 

then began to return toward control values. GSH peroxidase activity continued 

01 ; 
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 

pg/m3 (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 pg/m 3 (0.8 ppm) 

of 03 , allowed to remain unexposed for 6, 13, or 27 days, and then re-exposed 

for 3 days to the same 0 3 level (Chow et al., 1976b). Immediately after 

the first 3 days of exposure•, the activities of GSH peroxidase, GSH reductase, 

and G-6-PD were increased, as was the nonprotein sulfhydryl content. By 2 

days after this exposure ceased, recovery had begun; control values were 

completely reached by 9 days postexposure. Following a 30-day recovery period, 

no changes were observed. When re-exposure commenced on day 6 of recovery (at 

which time incomplete recovery was observed), the metabolic activities returned 

to levels equivalent to those of the original exposures. Similar findings 

were made when re-exposure commenced on days 13 and 27 days of the recovery 

period. 

The influence of vitamin E, an antioxidant, on 0 3 toxicity has been 

extensively studied, because it typically reduces the toxicity of 0 3 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 

continuous exposure to 1960 pg/m 3 (1 ppm) of 0 3 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 

(lung edema) on continuous exposure to 1568 pg/m 3 (0.8 ppm) of 0 3 or higher 

for 7 days (Fletcher and Tappel, 1973). Vitamin E protection against 0 3 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 

Ng/m3 (0.8 ppm) of 03 continuously for 7 days also had changes in 6-phosphogluconate dehydrogenase activity. Rats were exposed to 1372 to 31,360 pg/m 3 

(0.7 to 16 ppm) of 0 3 while being fed diets containing ascorbic acid, dlmethionine, 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 a-tocopherol (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 peroxidizability of the lung by feeding either low or 

high polyunsaturated fats (PUFA). Mice were continuously exposed to 1960 

pg/m3 (1 ppm) of 0 3 . The mortality (LT50 of 29 to 32 days) was the same, 

regardless of the large differences in peroxidizability of the lungs of animals 

fed high- or low-PUFA diets. High supplemental levels (105 mg/kg) of vitamin E 

acetate were protective and delayed the LT50 to 0 3 by an average of 15 days. 

Although these experiments demonstrate clearly the protective effect of vitamin 

E agai nst 0 3 toxi ci ty, they do not support the hypothesi s that changes i n 

fatty acid composition of the lung will increase 0 3 toxicity. The results 

could be interpreted to indicate that the scavenging of radica1s by vitamin E 

is more important than the relative rate of oxidation of PUFA. These findings 

led to biochemical studies that used graded leve1s of dietary vitamin E. 

Plopper et al. (1979) corre1ated biochemical and morphological (Section 

9.3.1) effects in rats maintained on a synthetic diet with 11 mg kg/vitamin E 

(equivalent to the average U.S. adult intake) or commercial rat chow having 66 

mg/kg vitamin E. The 11 mg/kg vitamin E group was exposed continuously for 7 

days to 196 or 392 pg/m3 (0.1 or 0.2 ppm) of 0 3 , 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 3 (0.2 ppm) of 0 3 

was roughly equivalent to the magnitude of the responses of the low vitamin E 

group exposed to 196 pg/m3 (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 pg/m 3 (0.1 

ppm) of 03 continuously for 7 days. In the 0-ppm vitamin E group, 0 3 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 9.3.1), only 1 of 6 rats of the 110-ppm 

vitamin E group had lesions, whereas more rats of the two other groups had 

lesions. These lesions became more severe as the vitamin E level decreased. 

They occurred at the bronchio-alveolar junction and were characterized by 

disarrangement of the bronchiolar epithelium and an increase in the number of 

alveolar macrophages. 

Chow and Tappel (1972) exposed rats continuously to 1372 pg/m 3 (0.7 ppm) 

of 03 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 concentration. 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 increase in GSH peroxidase activity increases iipid peroxide catabolism. 

Chow and Tappel (1973) observed the typical protection of vitamin E(0 

and 45 mg/kg diet a-tocopherol) from the effect of 0 3 (1568 pg/m3 , 0.8 ppm; 7 

days, continuous) on increasing G-6-PD activity in rat lungs. Similar findings 

occurred for 6-phosphogluconate dehydrogenase and malic enzyme activities. 

The activities of two glycolytic regulating enzymes, phosphofructokinase and 

pyruvate kinase, were increased by 0 3 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 containing 55 ppm vitamin E with either 0 ppm or 1 ppm of Se and exposed to 1568 ± 98 

pg/m3 (0.8 ppm) of 03 continuously for 5 days. In these mice, Se deficiency 

• ;, 
caused a sevenfold decline in Se level and a threefold decline in GSH peroxidase activity in the lung. Other enzyme activities (e.g., GSH reductase, 

G-6-PD, 6-phosphogluconate dehydrogenase) were not affected by dietary Se. 

After 03 exposure, the GSH peroxidase activity in the Se-deficient group 

remained unstimulated and was associated with a lack of stimulation of GSH 

reductase, G-6-PD, and 6-phosphogluconate detiydrogenase activities. In contrast, the 0 3 -exposed Se-supplemented group exhibited increases in 6-phosphogluconate dehydrogenase and G-6-PD activities. Dietary deficiency or supplementation of Se,vis-a-visalteration of GSH peroxidase activity, did not appear to 

influence the effects of 0 3 exposure as assessed by other parameters. Although 

the ani mal s recei ved the same 1 evel of di etary vi tami n E, after ai r or 0 3 

exposure, the Se-deficient group showed a two-fold increase in lung vitamin E 

levels relative to the Se-supplemented group, suggesting a complementary 

relationship between Se and vitamin E in the lung. This sparing action between 

Se (i.e., GSH peroxidase activity) and vitamin E might explain similar effects 

of 0 3 exposure in Se-deficient and supplemented mice. 

Several investigators have studied the responsiveness of different species 

to the effect of 0 3 on antioxidant metabolism. DeLucia et al. (1975a) exposed 

both Rhesus monkeys and rats for 7 days (8 hr/day) to 1568 pg/m 3 (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. 

(1975). Animals were exposed to 980 pg/m 3 (0.5 ppm) of 0 3 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 relatively 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 

apparentiy 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 

after exposures for 8 hr/day for 7 days to levels as low as 392 pg/m 3 

(0.2 ppm). However, no data for G-6-PD were presented, and the description of 

these results was incomplete. 

Mice (Swiss Webster) and 3 strains of rats (Sprague- Dawl ey, Wistar, and 

Long Evans) were-compared after a 5-day continuous exposure to 882 pg/m 3 (0.45 

ppm) of 0 3 (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 those , in exposed rats. 

At present, it is not possible to determine whether these apparent species 

differences in responsiveness were due to differences in the total deposited 

dose of 0 3 , an innate difference in species se,nsitivity, or differences in 

experimental design (e.g., small sample sizes, insufficient concentrationresponse studies). 

Age-dependent responsiveness to 0 3-induced changes in GSH systems has 

been observed. Tyson et al. (1982) exposed rats (5 to 180 days old) to 1764 

pg/m3 (0.9 ppm) of 0 3 continuously for 96 hr, except for suckling neonates (5 

to 20 days old) which received an intermittent exposure (4 hr of exposure, 1.5 

hr no exposure, 4 hr exposure). Given that others (Mustafa and Lee, 1976; 

Chow et al., 1974; Schwartz et al., 1976) have observed no differences between 

continuous and intermittent exposures for these enzymatic activities, this 

difference in regimen can be considered inconsequential. A11 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 sensitivity. Weanlings (25 and 35 days old) and nursing dams (57 and 87 days old) 

had hi gher 1 ung to body wei ght rati os. General ly, 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 3 exposure, the trend was a decrease 

in activities at and before 18 days of age, followed by increases thereafter. 

For G-6-PD, this trend was most pronounced; at 5 and 10 days of age, no significant 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 pg/m 3 (0.9 ppm) 

of 03 (Lunan et al., 1977). Ages of rats reported are presumably ages at initiation 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. 

Elsayed et al. (1982a) exposed rats of various ages to 1568 pg/m 3 (0.8 

ppm) of 0 3 continuously for 72 hr. Ages given are those at initiation of 

exposure. Ozone increased lung weights, total lung protein, and total lung 

DNA in an age-dependent fashion, with the older (90-day-old) rats being more 

affected than 24-day-old animals. For isocitrate dehydrogenase,activity, no 

effect was seen in the 24-day-old rats., but an increase was observed in the 

90-day-old animals. For G-6-PD and 6-phosphogluconate dehydrogenase, increases 

were observed in the 24- and 90-day-old rats, with a greater magnitude of.the 

effect occurring in the 90-day-old group. Younger rats (7 to 18 days old) 

were also examined. The exposure caused >60 percent mortality to the 7- and 

12-day-old rats. Glucose-6-phosphate dehydrogenase activity decreased in both 

the 7-and-12-day old groups, with the younger rats being more affected. The 

18-day-old rats had an increase in this activity. These trends were similar 

am 
to those observed by Tyson et al. (1982), although the exact age for significant 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 3 . The 

decreased activities observed in the neonates are reminiscent of the decreased 

activities that occur at higher 0 3 levels in adults (DeLucia et al., 1972, 

1975a; Fukase et al., 1975). The increased activities in the later stages of 

weanlings or in young, growing adults is consistent with the effects observed 

in other studies of adult rats (Table 9-5). 

Mustafa et al. (1984) are the only researchers to report the effects of 

combined exposures to 03 and nitrogen dioxide on the GSH peroxidase system. 

Mice were exposed 8 hr/day for 7 days to either 9024 pg/m3 (4.8 ppm) of nitrogen dioxide, or 882 pg/m3 (0.45 ppm) of 03 , 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-transferase were unchanged. Tissue 0 2 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 2-), a toxic oxidant species thought to be formed from 

03 exposure, and is thus involved in antioxi.dant metabolism. Rats exposed 

continuously for 7 days to 1568 pg/m 3 (0.8 ppm) of 03 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 

for 3 days to 1568 pg/m 3 (0.8 ppm) and then various days of combinations of 

2940 pg/m3 (1.5 ppm) and 5880 pg/m3 (3 ppm) of 03 , SOD activity also increased. 

Bhatnager et al. (1983) studied the time course of the increase in SOD activity 

after continuous exposure of rats to 882 pg/m 3 (0.45 ppm) of 0 3 . On day 2 of 

exposure, there was no effect. On days 3 and 5, activity had increased; by 

day 7 of exposure, values were not different from control. 

9.3.3.3.Oxidative and Energy Metabolism . Mitochondrial enzyme activities 

are typically studied to evaluate effects on 0 2 consumption, which is a fundamental parameter of cellular metabolism. Mitochondria are cellular organelles 

. .. 
that are the major sites of 0 2 utilization and energy production. Many of the 

enzymes in mitochondria have functional sulfhydryl groups, which are known to 

be affected by 0 3 , and mitochondrial membranes have unsaturated fatty acids 

that are also susceptibie to 0 3 . The patterns of 03 effects on 0 2 consumption, 

as will be discussed, are quite similar to effects on antioxidant metabolism 

(Section 9.3.3.2). 

Mustafa et al. (1973) showed in rats that acute (8-hr) exposure to a high 

concentration of 0 3 (3920 pg/m3 , 2 ppm) decreases 0 2 consumption using the 

substrates succinate,"a-oxoglutarate, and glycerol-l-phosphate. Similar 

findings were made by DeLucia et al. (1975a). Decreases in mitochondr.ial 

total sulfhydryl levels were also observed (Mustafa et al., 1973). Equivaient 

changes occurred in whole-lung homogenate and the mitochondrial fraction. No 

change in malonaldehyde levels was found. When rats were exposed to high 0 3 

levels (5880 pg/m3 , 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 

20-day continuous exposure to a lower 0 3 concentration (1568 pg/m 3 , 0.8 ppm) 

caused an increase in 0 2 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 ce11s, which contain 

more mitochondria than type 1 cells. Thus, .the increase in 0 2 consumption 

appears to r-eflect changes in cell.populations. 

Schwartz et al. (1976) exposed rats for 7 days continuously or intermittently (8 hr/day) to 392, 980, or 1568 pg/m 3 (0.2, 0.5, or 0.8 ppm) of 03 . 

Succinate oxidase activity increased linearly with 0 3 concentration. No major 

differences were apparent between continuous and intermittent exposures. No 

statistical analyses were reported. Concentration-dependent morphological 

effects were also observed (Section 9.3.1). When using rats and an identical 

exposure regimen, Mustafa and Lee (1976) found similar responses for succinate 

oxidase and succinate-cytochrome .c reductase activity. These increases were 

statistically significant. Mustafa et al. (1973), when using 7 days of continuous exposure, also showed that 0 2 consumption of rats increased with increasing 0 3 level (392, 980, 1568 pg/m 3 , 0.2, 0.5, 0.8 ppm). 

Although concentrati-on appears to be a stronger determinant of the effect, 

time of exposure also plays a role (Mustafa and Lee, 1976). Rats were exposed 

. ; . 
to 1568 pg/m3 (0.8 ppm) of 03 continuously for 30 days, and 0 2 consumption was 

measured as the activities of succinate oxidase, 2-oxoglutarate oxidase, and 

glycerol-l-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 

30 days of exposure. Mitochondrial succinate-cytochrome c reductase exhibited 

a similar pattern under similar 0 3 levels for 7 days of exposure. Equivalent 

results occurred in rats during a 7-day continuous exposure to 1568 pg/m 3 

(0.8 ppm) of 03 (DeLucia et al., 1975a). 

In rats, recovery from an ozone - induced (1568 pg/m3 , 0.8 ppm; 3 days, 

continuous) increase in succinate oxidase activity occurred by 6 days postexposure (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 03 on 02 consumption. 

After 7 days of continuous exposure to 196 or 392 pg/m (0.1 or 0.2 ppm) of 

0

31 the lung homogenates of rats maintained.on diets with either 11 or 66 ppm 

of vitamin E were examined for changes in 0 2 consumption (succinate oxidase 

activity) (Mustafa, 1975; Mustafa and Lee, 1976). In the 11-ppm vitamin E 

group, increases in 0 2 consumption occurred at both 0 3 levels. In the 66-ppm 

vitamin E group, only 392 pg/m 3 (0.2 ppm) of 0 3 caused an increase. Mitochondria 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 03-exposed rats of 

the 11-ppm vitamin E group did increase (15-20 percent). 

Similar to earlier discussions for antioxidant metabolism (Section 9.3.3.2), 

responsiveness to effects of 0 3 on 02 consumption is age-related (Table 9-5). 

Elsayed et al. (1982a) exposed rats of various ages to 1568 pg/m 3 (0.8 ppm) of 

03 continuously for 72 hr. The 0 3-induced increase in the activities of 

succinate oxidase and cytochrome c oxidase increased with age (from 24 to 90 

days of age), with no significant change at 24 days of age. When younger rats 

were examined, succinate oxidase activity decreased in both the 7- and 12-dayold animals, with the younger ones more affected. The 18-day-old rats had an 

increase in this activity. 

. .E 
Species and strain differences were observed after a 5-day: continuous 

exposure to 882 pg/m3 (0.45 ppm) of 03 (Mustafa et al. , 1982). Mice and 

Sprague Dawley rats (but not other strains of rats) had an increase in activity of succinate oxidase. Cytochrome c oxidase was increased in mice, LongEvans rats, and Sprague Dawley rats, but not Wistar rats; tho increase in the 

mice was greater than that in the rats. 

Rats have also been compared to two strains of monkeys after a 7-day (8 

hr/day) exposure to various concentrations.of 0 3 (Mustafa and Lee, 1976). 

Rats were exposed to 392, 980, or 1568 pg/m 3 (0.2, 0.5, or 0.8 ppm) of 0 3 and 

exhibited increases in succinate oxidase activity. Rhesus monkeys exposed to 

either 980 or 1568 pg/m3 (0.5 or 0.8 ppm) of 0 3 had an increase in this enzyme 

activity only at the higher exposure concentration. When Bonnet monkeys were 

exposed to 392, 686, or 980 pg/m3 (0. 2, 0. 35, or 0.5 ppm) of 0 3 , succi nate 

oxidase activity increased at the two higher 0 3 levels. The number of animals 

used was not specified, which makes interpretation difficult. At the 392-pg/m 3 

(0.2 ppm) level, the increase in rats was to 118 percent of controls (significant); in Bonnet monkeys, it was to 113 percent of controls (not significant). 

At the 980-pg/m3 (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 2 consumption is typically attributed to concurrent 

morphological changes (Section 9.3.1) in the lungs, principally the loss of 

type 1 cells and the increase of type 2 cells and the infiltration of alveolar 

macrophages. Several investigators have made such correlated observations in 

rats (Plopper et al., 1979; Chow, et 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/m3 (0.8 ppm) of 0 3 (DeLucia et al., 1975a). The increase in the homogenate was about double that of the isolated mitochondria (on a per mg of 

protein basis). As mentioned previously, this indicates that an increase in 

4 • 
the number of mitochondria, rather than an increased activity within a given 

mitochondrion, in exposed lungs is the probable dominant cause. 

9.3.3.4Monooxygenases . Multiple microsomal enzymes function in the metaboiism 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 3 exposure (Table 9-6). 

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 tiiAO 

activity is located principally in the mitochondria, it also is found in 

microsomes. Activity levels of MAO in rats were determined after exposure to 

3920 pg/m3 (2 ppm) for 8 hr or 1568 pg/m 3 (0.8 ppm) continuously for 7 days. 

Substrates used included n-amylamine, benzylamine, tyramine, and 3-hydroxytyramine; three tissue preparations were used (whole lung homogenate, mitochondria, and microsomes). The acute high-level exposure reduced MAO activity in 

ali tissue preparations. The longer exposure to 1568 pg/m 3 (0.8 ppm) increased 

MAO activity in all tissue preparations. This pattern is similar to that 

found for mitochondrial enzymes and antioxidant metabolism (Sections 9.3.3.2 

and 9.3.3.3). 

The cytochrome P-450-dependent enzymes have been studied because of their 

function in drug and carcinogen metabolism. Palmer et al. (1971, 1972) found 

that hamsters exposed to 1,470 pg/m 3 (0.75 ppm) of 03 for 3 hr had lower 

benzo(a)pyrene hydroxylase activity in the lung. Goldstein et al. (1975) 

showed that rabbits exposed to 1,960 pg/m 3 (1 ppm) of 03 for 90 min had 

decreased levels of lung cytochrome P-450. Maximal decreases occurred 3.6 days 

following exposure. Recovery to control values occurred.somewhere between 8 

days and 45 days. Cytochrome P-450-mediated activity of benzphetamine Ndemethylase in the lung was lowered by a 24-hr exposure to 1,960 pg/m 3 (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 b 5-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 3 . 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 

. .; 
N I W 

N ~ 

¢ z ' w 

} 

o ! 

O ' 

Z ' 

0 Z 

t0 

~ On 

LLJ 

J 

m 

H 

r1 N 

n n 

N 

r

cn-1 r

cn-1 

^ ^ ^ 77 

C7 N -J 4i ^ N J s io io 'O ro In i0 itl m ro 

ro m 'O 

G7 ro.0 rM

'~ G~l +~ +~ a~i a~i n ~ai ro 

c 

Ww 

ro vui ^ ro 

.4- 

°J 

L. 

0

1 

L. 

ro 

4- 

ro r~i ro cs 

VL. ro• r- 10 N GJ 10 •U •U i0 ro 

GJ 

4- 

+ ) tD N+4 1~ 

Vl I~ N Iz 

E E +a 1 , 

Vl (, 

7 N 

J n 

7 tA 

J^ 

+) tO 

Ul n 

N 

6.' 

7 01 + ~ 

Z rl Gl E ri 1 

f0 

6. 

7 01 

= rl 

N 01 

G rl 

O1 01 

C rl 

7 01 

Z rl 

L 

u ai' c+o ~ ~ ~ ro ro o ro 

a o: x z O: 

N 

L 

N Of 

i 7 

c ~ 

1 7 

~ 

1 O 

~ t 

U Gl t0 

c e61 ~ 6 

al 

C.- 

al 

V1 E O U 

a Ll O. a+J ul 

i. N 7 

s.E 

T T 

L•r 

T t 

a 

•E > 

>ro 

• +J 

uax a 

G7 E 

u v 

N 

ro E o a.0 a u ro G~ +~ u x aD o x T 

~ 

d O 

C 1-C 

N U 

C C 

N C 

C O 

O r• 

C 

U7 

ro'O 

O. • 4- L 

00 O L 

O. IA i0 

C T ~ 

V1 

v 

r.G • 

r• U O+J 

G! G1 

~ S. 

O1 L 

.II m 

O t1 

E O 

G1 

'O L. 

Q U 

Z i-~ N O 

Q~0 

Z'O C 

+J 

u 

~~ O Z C 

'O +•~ O 

ro 

4- a 

O 

4- N 'a 3 

G7 4-~ 

tA U•r 

Q N 

4- T A 

O 

4- C 

d a T u o o t v o ro o ro u o o a 

4- 

vi~ u 

ro ac 

C7 Tc u Tro u r- - ~ 

CJ d•O s E ~ a+ 

. V - 

Aa~ x a 

1/f 

Aar ~a 

d 2 i-~ N 

y d - y ~ 

+~ 3 

r- 

+~ L 

4..~ 

'O 

Cl T 

U O Uf 

AI i ro 

+~ N a..~ G. A-> 

ro R1 G•r 

i-~ VI 01 

t0 L 

v 

y 

Lc > 3 c 

~ ¢•r ~ Cl 

> 

c 

> 

• C 

s+a -o c d 

u s 

>++ ¢> 

• u C z•r 

>+J u 

• u c 

> 

s 

c Z i~ 01 +~ 

O U.0 +~ 

+~ •r 

u 

+~ •r 

u 

~> Cl •r ;.i 

~ o u 

O! +~ c 

i-> > ~ 0 +, 

u'O Of c u 

N 7•r 

uV 

a c m 

•E 

ro v m a >++•r > a•r ro a a r ro ro a ~ 

v7 

.G 

~ •r Cl 

rt1 d u L 

Yl 

'C ro 

W 

a ro 

N u N u 

r- ~ 0 T/ 

L~ L ro 

O L. Ol 'n C y 

}t W 10 G1 

C ro.N i c 

G! r• 

Ul >f 

ro x 

N r• 

tlf T d ro x R7 

GN 

M 01 V1 

o IA ro 

r 2 GJ 

Md > 

O O Ol 

N U N Of v/ 

Uf Vl C 10 

R1 N ro N+z 

N U•r 

Uf - X co Gl to 

d Gl U GJ •r 

U L 7 Vd O 

L L. 

N O uf 

L L O 

ro 01 

L•O L 

Q r• 

.0 Z 

Gl E 61 .0 U 

L. O L U O 

Ol E'E 

L O 

c U'O 4- ~ U'O 

L 

u •O U 

L 

C7•r u 

2 O r 

O1 3 

11- r 

U i U TJ 

f-Ci U ~ Z L 

U L

'i V ~ L'O 10 C C1 E S O rCr U 1~ 

n L L 

-a 

i 

O 5- 

O 

Vi 

O 

O 

w 

C 

Cf 0— O N 4- N N Vl In Vl 

7 C u O a b 

~ 00 O O 3v O A a cro ro 

O 

~ 

c+ o 

X ro L 

°~ 

~ L uf s s 

~ ~, 

~ i0 L 

°~ 

~ L 

 ~ 

+~ L L 

° a 

i~ t0 

W 7 O. OC L ~ CO •O L CO LO .0 OC L L CO 'O 

'C C~ W'O M M C.0 IN OO C.i ~ F- 00 . U CO OD U I, 

a 

c0 

4-' m 

aEi o 

L L F-N 

.j ++ v) ]e 

ro E Z Z ~-+ ~••' Z t•+ rr i••+ 

S 

Ln 

O a N tn GO 1~ O O I~ O O 00 O CO O N t11 Cp Cp 00 

Z a 000 OeMO O MO O N O N 000 O O 

c L 

O +1 

N C 

O C)m N O 00 O O O O O O G 00 O N OD Oo 00 00 

U E G Q1 Co tD 

M Ol LL9 

1- 00 O 

~ Co t0 

1- CO O 

d• 00 t0 

N 

Ln Ol 

tO N ~ tff Ol 

01 t0 tD t0 

M LL7 tn 1.n 

tD 

Ltf 

O Ol rl r1 lf') ^ e-1 tn •• rl f+f ri M r~ t-i ~ •-I ri 

u 1 O~ rl 

01 

ri 

9-93 
~ 

~ 

o, 

~ rtf v rn c rl ui 4-3 d ro 

C1 C L N +J O U OJ c G1 r 

c 

47 

m 

i~ 

E t O (1) _ E 

C9 •O t17 4J 3 N 

4- r• I~ C Gl G • IA 

d O Q1 O•r Gl Yl 

~ 

O 

o. 

v 

ro L 

Vf iJ VN r 4- 

~ O 

U ~ 4•7 41 ~ d ro m N ro ro 

L 

4J 

~ 

a~ 

c 

' 4- c G7 

O •r p 

~ --N 

]C 

I /OA O C c C•Q Z 

O ro Gl L O O ro 01 

ai = ro c d +~ w o>ro ci 

U U •€ •C O S.ro L (a r- U 

~ Ol N u 7 

(1) 

 L Gl +) •r c 

~ vi Of'O N 4J rl ul € > >W +3 N 

YJ 

C1 

`+ 

+~ 

c L 

7 r 7 

Gl O o O i~ 

L T dJ O. L ro 

c O C 

O L. C1 

u 

uf 

m u .- ro tA 0.+~ X.0 U1 u p. > d 

a a) 

4- 

E O 4-. •r O. 

N•r c N v a) 

C>L+J O•C U Oa 

c 

4-3 

c 

4- 

ae 

O x X 

ro d 

Gl •r 7 Nl .Fa 

.c +) ++ O A•o 

r 01 

++ +J 

E 

~ 

O VIZ+~ u aJ au•r rov ro - u 27 tq c• ro L a €•r r• 

v > > •n r d • ~ 30 •= r a~i 

>

iro' ~°n . 

ln 

W 

L 

N 

Ol O 

r- tt) ul 

61 uN N 

~ n ro L rl C'O 

c 

CI U •r 

.0 

u 

¢ a v ~ ~ro' aroi ~,u i ~ °+%+ o c c c Zf 

Z O GJ O. ~ L.0 O d+1R7 G7 O•r 7 _ 

W ts U•FJ C. ro•r uf •O N r 

(7 ro C) tD N N x Ul r• ~ ro•r 7 O 

>- 

>C 

GJ € 

f-. O M 

~ € Gl r• 7 Gl 

CJ 1-~ Gl €€ 

Gl .L ~ 

L U C 01 

Nl 

O U L aQ 'O UI >•r I u ro dJ vl V) 

C OI .0 i~ O! O Gl •N N G! L.0 7 ro 

z o u ro ~ nz n. ~-~na c ro 34- rn 

~ II 

f 

~c s L) 

Ct C N 

W 'O - G1 O 7 L •r O ~ 'O 

J G O N 7 L O1 C/ 

C] G1 i9 r- 4- 7 C+3 +J d.0 'C! 

6 

~ 

L O Gl 4- •r 7 C 4•7•r 

u0i O o ~ aLi c.0 EE -cL.) O O•r }) c c C. O Of•r 1A r 

C.+> O x ro L •E 

L- L u = L 

•E a O O dJ Gl • tn 'O € 

W ~ n 7 C.0 L? 

'Q 

O 

Gl eP 

N 

O x rl r- (0 d-) N 'E +) v O 111 

u c `n co 

r u ++ L O 

° ° 'n m z 

o .. ~ 

ttJ 

++ 

- u L 

ar 

aCi Oci ~ 4~ -

o c_ 

~ € 

~ iQn II Y 

S r-i O 

•o •C 

.0 L 

C1 (l 

O o. € € 

•r a 

O O 

+~ C 

+.) rl ri M C O 

C L L +) 

NC i L 

O G1 O O O O .0 

c € rn a ~i a~o ro ~ 

o rn ri ri Ln d ro U 1 X: CU CO .G 

9-94 

~ 
single enzyme. More importantly, the relatively long time for recovery suggests 

that cell injury, rather than enzyme destruction, has occurred. Benzo(a)pyrene 

hydroxylase is the first major enzyme in the activation of benzo(a)pyrene and 

several other polycyclic hydrocarbons to an active carcinogen. However, 

additional enzymes not studied after 0 3 exposure are involved in the activation, which makes full interpretation of the effect of 0 3 on this metabolism 

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 9.3.3.2; 9.3.3.3), 

acute exposure to a high 0 3 level (3920 pg/m 3 , 2 ppm; 8 hr) reduced NADPH 

cytochrome c reductase activity (DeLucia et al., 1972, 1975a). After a continuous or 8 hr/day exposure of rats for 7 days, the activity of NADPH cytochrome c reductase increased linearly in a concentration-related fashion (392, 

980, and 1568 pg/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 pg/m 3 (0.8 ppm) 

was an i ncrease i n acti vi ty 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 

exposure for 8 hr/day for 7 days to 1568 pg/m 3 (0.8 ppm) of 0 3 (DeLucia et al., 

1975a). However, monkey data were not reported in any detail. 

9.3.3.5Lactate 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 3 on these enzymes are described in Table 9-7. No lung lavage studies have 

been reported; whole-lung homogenates were used. Therefore, it is not possible 

to determine whether the observed increases reflect a leakage into lung fluids 

and a compensatory resynthesis in tissue or cellular, changes (See Section 

9.3.1), such as an increase in type 2 cells and alveolar macrophages and polymorphonuclear leukocytes rich in lysosomal hydrolases. In some instances, correlation with plasma values was sought. They are described briefly here; more 

detail is given in Section 9.4.3. 

Lactate dehydrogenase is an intracellular enzyme that consists of two 

subunits combined as a tetramer. Various combinations of the two basic subunits 

change the electrophoretic pattern of LDH so that its various isoenzymes can 

9-95 
ro 

4J 

a 

c 

ro E 

a~ 

r ro 

u~o n 

ro Ol U r-1 

~ 

ro s 

ro 

+-) ar 

~ 

E al r• a 

N^ 

roo, U r-i 

Y 

z 

tn 

UJ 

Y 

U, 

LLJ 

~ 

N 

J 

6 

~ 

0 C 

} 

~ 

G 

W 

Q htg..) 

J 

f+ 

a+ 

W 

J 

~ 

GJ 

ro N 

+1 N 10 N 

atA L ~ 

O u •r 

ar •,- 

O O u 

d ro 

•l.l > f0 L y 

G! N 

cav ~ O •r 

O O C C L 

~ 

U 

b~ rn 

Op 

C u C H ro •r 

N N 

~ T 

7 V 

c 

4-) ^ C L 

O O U 4- 

v (u 

~ 0 1 

L ~ 

N 'O 

~ ro 

ro N 

L N 

r= 4-) ~ 

r > > 

~ ro u 

d G. N 

bzla-• ' 

G 

r m v 

cx ro 

L 

 cm 

¢ L 

m Z u 

V1 N ~ 

7 '0

c 

•1•3 ^ 

C L 

O O 

U 4- 

I~ H 

~ m z 

I n O 

O O 

N 00 

~ ~i.-i 6 

~if Y 

O 

O 

O 

tD 

Ln 

rt 

d u 

c ro L 

YO.. C/ d: 

N d .~ 

a• H 

N I 

v 

~..i 

u 

C1 

W i 47 

v 

N 

> L m N 

.G 0 

~ 

dro ~ 

o •° +°.~ X b i W L a 7 

~ 

a 

ro ~a 

e 

a~ ~a 

4! O L t 

ro d) 

a) 

_ 

O.- G 

~ df •9 

o 41 N C 

O C ~ 

u s °

r-I 

m 

~ 

m 

4-3 ai 

O 

.0 

U 

41 m 

m 

41 

r N 4- I 

4- 

O O I 

.io.r-~i c~ 

ro ° s m O~ L 

~ C ; W II 

a•.- 

ro 

> 

r o € ' a+a9oro 

O t-1 i }a 

C L O•> 

r 

CL 

 W O j 

N L c€ I roar•rc 

u d 4-) O C •r .r ri H •O > rl 

o ~o 

~v 

c 

~u ~ 

G L 

00 

C.l V•• 

1--1 ]C 

? 

~r ~ 

4m rr 

ro 

i-3 m 

O 

L 

U 

d-3 m m 

T ~ 

c a 

O a 

.; O 

u+' 

ro ~ 

N O. 

~ d 

T N 

•- ~ 

O O 

c ~ 

7 C 

O C 

N U 

Cro7 L 

f.t 41 

C VH ro 

L L 

O O 

4- 

N N 

4.co S.- ~ I UVO -COf- I 

h ]C 

~

m z 

~ 

~ 

~ 

in 

aJ ai 

3 

O L 

U 

T CI Y 

N pC 

z 0 

i 

O 

O)•~ 

7 iJ > r Vl 

C L7 C.1 Of n' C 

+? 51o r N J C ^ C N 

u N•r a N i roa 0 a) 

J J O r a 

v c •rJ ae m 

Nro •r ~ T 

~ d C/ c ro N 

Gl O O .0 C 

U 10 41 U O 

C L 7 O N 

HU a Z•r 

L 

O 

4- 

~ Uf 

L eT0 - 

.c v 

O n 

H 

Y m z 

~ 

~ 

~ m 

v 

~ 

r- n •r O1 

C ri 

~ 

~ 

N V 7 d 

o N 

• L Gro! ro L 

> C 

4- •r 

O N N d C1 N +1 ro 

r o •~ L 

• p 

ro L 

u ro 

4- O 

f.l O 

N ~ 

7 N 7 T 

7 •Q 7 ~ 

c c 

a-+ ~ +3 ^ c Lc L. O O 00 

U 4- U VH 

]C m z 

a 

a a 

~ 

a c ro 

O ^ 

L Q1 

U rl 

I 

4-) 

ro m 

I C O 

4 2 O 

> W 

x +~ O•> +) 

c ~^ 

r .G •O 

ro~~ 

5-aUi E 

C V- tff 

H 10 CI' 

~ T 

0v 

c 

4-1 ^ C L 

O O 

U VH 

~ m 

z 

O 

O 

~ m lD 

Ln 

r-4 

N 

~ 

V 

O 

E 7 

N 

N 

t0 

4J 

O 

a 

v a 

L 

W 4- 

~ 

a 

i {.1 

N 

C 

Y ~ 

m z a 0 ^ r 

L E 

0 ~ 

N O 

ro a 

Z 

~ 

•L 4-y 

4J 4- N ~ E .G 0 

~ ro o L u ++ 

H G/ ]G C 

II II 

I /••1 tn Y 

Q m 

S Z 

~ V 

I O O s t 

+J +J 

a ai 

E E 

C O q! •r 

E +~ a ro i L 

~ a N •r 

ro .nU 

O I O O O 

j 

 O O O I O O 

cn 

00

10 

I M ~~ 1~ r-I ~rl I r-1 rl I 

. . 
be detected. Chow and Tappel (1973) found an increase in LDH activity in the 

homogenate of rat lungs exposed to 1568 pg/m 3 (0.8 ppm) of 03 continuously for 

7 days. Lower levels (196 pg/m3 , 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 influence the response. In following up this finding, Chow et al. (1977) studied 

LDH activity and isoenzyme pattern (relative ratios of different LDH isoenzymes) 

in the lungs, plasma, and erythrocytes of 0 3-exposed rats (980 or 1,568 pg/m 3 , 

0.5 or 0.8 ppm) and monkeys (1568 pg/m 3 , 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 pg/m 3

(0.8 or 0.5 ppm), but no changes in the plasma or erythrocytes were 

detected. The isoenzyme pattern of LDH following 03 exposure was more complex, with the LDH-5 fraction significantly decreased in lungs and plasma of 

rats exposed to 1,568 pg/m3 (0.8 ppm). The LDH-4 fraction in lungs and plasma 

and the LDH-3 fraction in lungs were increased. No changes were discernible 

in rats exposed to 980 pg/m 3 (0.5 ppm) of 0 3 . The changes in LDH isoenzyme 

pattern appeared to be due to a relative increase in the LDH isoenzymes containing the H(heart type) subunits.. Although the increase in LDH suggests cytotoxicity after 03 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 03 exposure. Further 

studies of the fundamental distribution of LDH in lung cell -types are needed 

to clarify this point. 

Lysosomal enzymes have been found to increase in the lungs of animals 

exposed to 0 3 at concentrations of 1,372 ± 294 pg/m 3 (0.70 ± 0.15 ppm) for 5 

days and 1,548 ± 274 pg/m 3 (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 influence the effects. These increased activities were attributed to the infiltration of the iung 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 hacl been exposed to 

1,372 to 1,568 pg/m 3 (0.7 to 0.8 ppm) of 0 3 continuously for 7 days. Increases 

in p-glucuronidase activity were not observed. The histochemical and cytochemical localization suggested that 03 exposure results in damage to the lung's 

9-97 
lysosomal membranes. Castleman et al. (1973b) also found that the bronchiolar 

epithelium in infiltrated areas had lower NADPH- and NADH diaphorase activities and higher ATPase activities than similar epithelium of control lungs. 

They discussed in greater detail the enzymatic distribution within the lung 

and suggested that some of the pyridine nucleotide-dependent reactions could 

represent an enzymatic protective mechanism operating locally in the centriacinar regions of 0 3-exposed lungs. Chow et al. (1974) also observed an 

increase in lysozyme activity (lung homogenate) in rats exposed continuously 

to 1568 pg/m3 (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 

rats to 392 or 980 pg/m 3 (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 observed by some after 0 3 exposure to morphological changes has not been demonstrated. 

9.3.3.6Protein 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 9-8. 

Hesterberg and Last (1981) found that increased collagen synthesis caused 

by continuous exposure to 0 3 at 1568, 2352, and 2940 pg/m 3 (0.8, 1.2, and 1.5 

ppm) for 7 days could be inhibited by concurrent treatment with methylprednisolone (1 to 50 mg/kg/day). 

Hussain et al. (1976a,b) showed that lung prolyl hydroxylase activity and 

hydroxyproline content increased on exposure of rats to 980 and 1568 pg/m 3 

(0.5 and 0.8 ppm) of 03 for 7 days. At 392 pg/m3 (0.2 ppm), 0 3 produced a 

statistically insignificant increase.inprolyl hydroxylase activity. Prolyl 

hydroxylase is the enzyme that catalyzes the conversion of proline to hydroxyproline in collagen. This conversion is essential for collagen to form the 

fibrous conformation necessary for its structural function. Hydroxyproline is 

an indirect measure of collagen content. When rats were exposed to 980 pg/m 3 

(0.5 ppm) of 0 3 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 3 exposure. When rats were exposed 

to 1568 Ng/m3 (0.8 ppm) of 0 3 , 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 

. m 
d u 

C 

d i 

d N~- 

d 

Q: 

N 

d 

• U 

d 

c N 

N 

N 

LLJ 

S 

Z 

> 

N 

Z 

H 

W I ~ 

~ 

O. I 

Z 

J 

Z 

O 

W 

z 0 

N 0 

O 

N 

U 

~ 

W 

W 

1 

~ 

01 

w J 

Q 

H 

~ 

d ~ r 

i O 

7 C U 

V1 O O 

O •r .1•~ 

~.+> O 

w~ o. 

V 

a-) 

U 

d 

w 

4- 

d 

9 

d 

> 

d 

w 

.n 

O 

N 

~ 0 

4J 

d 

ca 

r r 

rn r, 

ro 

41 

d 

+~ 

ro 

J 

~ 

z 

d 

v s 

d 7 

d ; U+ +1 

ro~ a ro 

L o xa ui i d 

v v- ro 

~ YpV 

d 3 d 

Url dV 

d0 ~~ ~ 

ro a ro 

y 

1lf i Y Vl '. ++o;c 

Cli 

O. O ~ ro 

X•r O 5-

da~ i,+1 

i O. d 

d d 3c 

L uf d O 

u~uc

+ju 

i •r i s 

~ +d•i'03E 

~

o d o a 

suw -a 

n U L N ~ ao Tu io 

I O Orn•-+a_T 

I Q O L Y O 

i w 

o T W. V 

A O 

'0 Q1 

L Y 

co rl 

m ro N t0 

N r 7 Ol 

= ra 

ro z 

i 

41 E 

d O. C ro L t0 

a>,~ •O 

o ~nwxoao 

C.EO 5-Z XN 

Cf O.v n a i ~ 10 

C t0 uf C Yl 

\ O r CL d 

CCD 

 CM 

S.1d r 

 t

}1

0 O ~ L 

a +J Q~1 = a•o c 

d d +) U O ~•~-1 'r 

L V- f0 C r 

U V- O +., + ~ •— 

c d"O c V 3•r 

O Z 'O 4d~ - M 1n 

+J Z 0 C w t0 

c d d d V1 

c tm d O 3 

d•rN ~ d r d 

d ~ C 

co 

 fp E r 

O r d ~ 

1 u•r O LO O 

C 04J U u C i 

+1 0G E r ~ X 

s%~~ 

C x CO d ~ a d O u L L L 

U i•r O 41 7 

o ~oa> >,a ~ 

u L.CQ uf i m 

i 

O 

N-• 

O 

C uf 

O V 

U r 

~ 

O N 

I

co (n tcnM 

ro 4J 

C 

d 

E O 

d O 

7 1-~ 

~ E d 

z 

C E 

O O. 

r d 

4-1 

C L O +1 

N C 

(:D ann 

 E 

U 1 

a z 

N d c0 GO Ln 

O O O O O 

cn 

M ~ ~ ~ ~ 

 Y 

m 

N Cp O •cf 00 - 

O C N O O 

NcoO N 00 

' M r~-iM ~ r

LO

i 

f••• N 

N tn OO 

O O O 

NO GO 

rn (71 ~ 

N-IttD00 

6666 

Nd'cm m rnaorto 

M r -4 a) 

9-99 

~ v 

N C 

•r ro 

ro m 

Y+)r 

arn 

v ro r+ 

C J 

ro ~ yl 

+~ o ~n 

Vl CG O 

ro C1 i 

J ri Q 

i) 

ro z 

C C C 

O O O I 

4) i-) i-) 

d d d 

uai aui a

u

i 

Vl V! V1 

C C C 

+d., c. .°J~ +d.+ 

n o. a 

V O D U _a 4J _T _T 

rnro rn rn 

0.+) O O 

C 

i~ ~ 

rd 

L X 

+J aj 

to 

d 

m ai m m 

L U L L 

u ro U u 

Gd 4-1 C .c+ 

H 

T 'II 

L. 

O O L 

i V- O 

~ 

4- Vl 4-1 

4- ~ n > 

7 L 

in 3 7 •O 

C O C rl C 

4J 

L'p }1 C O E N 

CO M U r-1 t~ cM tO.0 S 

M 

co rn rt 

ro 

~ 

d 

ro +~ 

0 

t.~ 

~ 

z 

v 

r0 

N 

O 

4J 

ro 

!E 

41 M. 

ro n. 

d N 

'0 iJ 

ro ro • 

c vdi 

Df d 

ro s 

U 

O •C 

U r . 

~ o. 

c. m m 

L ~ 

cro 

N d 

>1tD 7 V v N 

LO 3 X 

~C ~ q-• 

'a N O 

L ~ T 

to3 ro0 

ro 

+~ 

d 

s 

m rn ro c 

41 M . 

/0 00 L Q1 

m r4 

d 

O 

o ~a z cc 

L 

rn 

1fM 7 a+ +~ o 7 i L > N L 

^C~i~ t~.7 d• ~ r N tV0 ro 4d- > 

C ~ d ~ NC rorow,r > o am >r• +> >, 

Ln i 4*Z E ro.0 a) ro E U•' M rv! d x dV 

0E O d E A b . y) 

LN T 

C d •O O 'O ~ T L i L O TO U! 

y' vOi C.aX C O 7 L d r.N 

Of X N, 9- >' CUl ' ro d C_TO 04- I L 4- r~F •r 00 G. d. 0 o d s T u maro co 

v d ~,at0irov ri z 

d ro s d t, L 1A vu V1 Uf. • 

. d N•r d C~d T L i ro '6 i d 

rci oc w r c-• Lni m~-c+ r 

fh N 

'o ITd 7 'O IC 

r 

C i 

U 4•Oo, r rn rr 

ro 

4J 

d 

4J 

m J 

I 

4J 

ro z 

'A 

N C 0 0 

.0 +~ 

4-• L 

4J 

~ C 

r U 

•N C 

i~ U 

c L 

N (D 

C 

d ~ 

rn 

r Yf 

O r u 3 

4- 

O u 

4-) 9 

s ro 

~ r• . 

V1 d N 

i R3 O u c 4- d (A 

++ O L 

Y 

3 

M 

i 

O 

I N 

I rr 

n T 

O~ 

C O • ~ 

O O u +- 

.0 
n 

~ 

O 7 

c 

~ 

c 0 

U 

V 

N 

W_ 

S 

~ 

~ 

N 

x 

W 1'•' O 

x C. 

2 

JI 

Z 

O 

W 

Z 

O 

O 

LL. O 

H 

C~ 

W 

W 

W 

~ 

rn 

J 

M 

C) 

~ 

ro 

41 ar 

a.) 

N 

J 

~ 

~ 

O In fL') tn !n 

o 1-i N .. o a 

+ o S 

= v 

tn 

N = 1 

Ci cY O cD O 

~N t~ ~ r

o

i 

'N •M 

N O~l N 

N N 

.r a 

S S Z 

v v 

2 

C N O I C pp 

tn

C O W O 

W r 1 

9-100 

zt 

0 

Q1 

00 

Ln 

N ri 

m 

~a 

c 

m 

~ •o ai o 

L L 

a7 N tn 00 

r-1 N ~

C a 

O O 

~ a 

ar m C L 

O +~ 

N C 

~ i0,7 E 

C 

tOD I1 

W 

O 

O CYM Q1 

a 

C• 

ro 

e 1 

a> 

+~ N 

~ 

Y 

Z 

~ a 

\ 9 

t ri 

c0 M 

4J 

d 

41 

U 

C 

a~ 

o~ 

ro 

O 

U 

C 

ar 

N m 

v i 

u 

N 

d u 

c 

ro 

L 

m 4- 

EU 

C 

d 

~ U 

a 

h 

N 

N 

V 

w 

Vro 

~ CI 

? L 

ro 

N 

a 

O 

'O c ar m r7 C t01 

W L G 

~ 

a 

7 

t; L d 

.~ I C 

y ~ y 

L 

C7 

a 

c 

m 

a) o N [0 

J r•1 

41 

m 

C 

W O 

41 

C 

d 41 

c 

c°~ 

N 

C 

i 7 

N 

X O 

L y 

O +1 

L O 

~ Q 

c q~ 

m 

C N 

•m ai 4J rn 

L tC0 

nU 

~ O 

x d 

N 

61 N i Qf 

c ~ 

M r'• 

o T I V- ~O v 

N 

CD 

O 00 

C ~ 

C 4J 

V 7 

m C 

N 

N m tt7 C 

mL ~Q O! 

L

tm 

u 3 00 m ~- 

v ca oe o ~ 41 r Q 

V O N w 61 

m O 

 ra 

~ M m 

L UL b L 

O V7 • 

r" N N N 41 ~ N (1)M 

LS+~ +~~ 

vm 

G1 VYaJ Nr 

u 3 ~ C N C C N 

U •N 01 . Of ~GJ N 

L +1S .- a 

Ua 

 CL 

 UU+) 

rO7 N ~ N p L 

~ O7mr1 mN N 

L S N 

m0o C m C•...0 

U U •r- N •r- ~ C 

O ~ O rl O'II V~1 

L. 

0 

L 

O ~ 4- 4• • N 

N .01 

~ ~ Q 0 

~ 

c 

o 

~ C tn 

C •O C }~ 

U f- C07 M 

~ ro 

~ 

r~•I 

m 

41 

ar 

4-+ 

N 

I

m 

J 

I m 

4"O1 — •- C m L •~ 

0~i E ? ct, 

o O L N u I 

N c ocii V- }J 

N•e- O iJ 

~ V 3 

S O O N L 

v+ ~2 

I 

ro~s s%; 

O C N E C N 

v~ 

O 10 N~ 

m • -m 

W p ~ 

/~ 4 d NQ~ ^ O N 

tn 

++.Z.'1tl c ~ -- ui 

u L 

VO- V! N 

ro V N C 

ro V- •~ •'- 

C1 N N 

E V N 

. 01 4-1 y ar ai 

}f .0 .0 

v •- m .- 

Gf N 

C 41 47 +7 N 4- 

V ~f ~ m 7 U O f r N O G1 L N 

4- 

•rc C 

1J 9- CI 

r- L 4- a 

C G7 N 

Ql Ol Df 

•-mm 

+J ro Ol E N r-• 7 Lo a~c 

N 

0 0 o c~ Z Vz u u rr m o+a 

L 

40- N 

N N N 1~0 'p 

c ° c M 

o O O ~ 

V 9- CU I, 

a 

a~ ~ 

m 

a~ ai 

~ 

N 

m 

J 

/•~ 

C ~ 

7 7 

z3 m 

G7 

Y 

~ m 

avm 

r- L 

ro a 

~

C 0 

C L 

~_ 3 • 

O O. N u 7 m 

r L° s Of u 

GI Ifl ~ 

ro o., 

N Or1 L C 

C +1 V 

~ C 

+~ iO .iv 

C 

d E ~ 

~ aro 

•'- d' Of 

~ tD L 

w O @ 

m 4- N V 

L s Oc E O o 

40-N ~ i m L ~ i-- ~ c •-..0 ~o m ar ro N •i- C _N 1~ +1 L O•v0 O

~ +O

a VOl N.- 

C t

>

0 GCI ~ 

 tn 

 O a t~0 m X O L 

+1 y+N mI m d L O! N G) C i~ •O >f'O O.+> >f ij O O•r- m Cl ••- u'7 m C 

U Cl E l2~ •O Gl L 3 fM -0•.- 

41 

C E d 

S 

Z 2 
++aic +~ ov ++a°i~ +~o~ 

C..ir• Cv ro C.~ir Cv ro 

Cl 

u 

cai 

s. 

Gl 

w N O..' 

O 

• V d 

c N I 

-~r 

m 01 

rl 

io 

dJ 

N 

L 

G1 

~ 

4~ 

C 

~o 

~ 

N 

16 

ro 

N 1~ 

ro z 

a 

~ 

N 

T 

rf 

ro 

41 

a~ 

3 O 

L 

U 

4-) m 

a: 

4-; 

N ro J 

~ 

ro 

rn L 

G1 a 

asi 

IN 00 

2 r

C"

i 

ro 

~ 

•O 

.c 

• L 

u N N 

'O 

-P 

O 

c 

0 

z 

N 

~ 

V m W 

NN 

.fl 

G1 

> d 

~ 0 

~ c d ro r- s o ~ C V 

N o O O •r dJ 

W i O_ 

~ 

~ 

L 

ro ~ 

c 

N 

E C 4) O 

L L 

7 41 

rt E d Z 

c E 

oa •r a +1 

m ro 

C L 

N C 

O V 1E 3

O Of 

u Z 

~ 

C! 

C 

N 

LLJ 

O V ~ 

H 

N 

S ' 

z I > , N 

Z 

F-1 W 

FO ' 

a 

C.7 z 

~ 

J 

z 0 

z 

C N 

O 

O 

N 

H U W 

LL 

w 

C6 

01 

W 

m 

~ 

~ 

N C 

d i 

+•+ 3 

TN 

N ~ 

c T 

ar m 

O fC 

L 

aro 

c ~ ro y 

G •r 

ro 41 N 

04- 

u O 

c +~ 7 r• 

r• ~ 

N 

y i Zf 

A •~~ 

i N O 

C ~ Gal 

t-i L L 

C 

~ a 

f0 N 

i0 V C 

G N 

•r y 

L 

rf ~ 

~ c. 

~ 

c L 

Od) 

N 4- 

,N Ol 

L 61 4J L 

G +1 

N ~ 

c cGo 

G! N 

~ 

s~~ 

av ~ 

v .- to 

~ ai en 

a 

n wa 

L 

ro 4J 

~ r• 

tD ~ 

a~ 

a~ 

~ N 

> L 

°u ° d - 

L m 

N ~ Q 

•r ~o cr 

N N 

Liy 

I ~ aC 

I X 

1 • 

• 4) d Y1 }1 L y 

L L 4..1 

°• (7) ? 

N ro N 

ro G d L y 

L 4) 4~ 

C A i 

d •. 

U L 

C C O 

 co 

Ero rn 

C 

r0- C 7 

a~ a 

a ca a d N p L 

r N Gl 

•N +1 . 

L C r- VO! 

N O G s0 

Cr •r 61 

I NL i0 C 

C i•~ 

Cl C 01 •r 

m u o a r C 41 r O O C 

O u N d 

U > 

G C QI 4- •r O L 

o,oi a 

N ~11 ~ i 

ro ro.- a 

L O! a Nf 

+1 u i~ G. 

aca~ x Z •r E N 

Y 

a c m ~ 

O

to

i 1

-t0 CO CO CO N tn 

0 0 0 0.4 ri 

~Np d' 00 Oo W N O 

rl rN-1 

U'j rl rl N N 

~ ~ ~ 

cli 

 ~ 00 

cli rl r~i r~

l N ~ 

rn 

N 3 

> ~ 

Cl 1 •N C 

oc~ r 

E r L G! 

c~ o ro 

tp > Q i 

p14) v- c 

0 o r 

f0 

a-~ (D C +1 

ro o c c +~ 

•FJ i•) i) O 

c O N f0 

+J 

c 

Gl 

iJ 

ar a c> > o c 

G V V 

COI, ~ tn N (L) a.. 

aci o ~ ro ro 

rnc.c ++ 

,-arn roar u o 

O+~ u N 

v c° °u ~ u ++ 

Ovi Y'O N L d N iOA 

ro 3 a) +1 Orn of 4- t0 

i t0 m aui 

i 

t r• - 

O 

a) L 

C+~ V- 

~--~ Q V d C O m u 

O Z ti 

•• L tO•r N N O NN 

o ro E ro 

w 

4- v- C•o o o a~+ 5- 

` 

+O+ °' ~ >, `O 

1-

~e T 3 0 ~c o s +~ u- ro io •o ; 

y w c ; c ~ 

4-

. •B ° \ ~ 4J ~ a 

L R44 M 

3 3 

.N a.) ~LY 

C •C m!,tO O O O U w U rl N W C d i--~ 7 v f0 L I, Id 

•v 

a 0 

7 

.y 

~ 0 

4J 

O 

O. 

~ d L 

4- 

7 

a 

ro 

L 

41 7 

C1 

G 

]~L 

m 

z 

a L 41 

E 

41 

t 

a 

~ 

~ 

II ~ 

~ o 

L F~ 

GI 

E •N 

i-~ d•~ 

N O ro O. 

I v •O 

d 

V L 

•L V414-

G! 7 

6 a 0 

~ ro 

O L v ++ 

t~•• N 

_ Y C 

a~ u 

N ]~C 

qzm 

2 

V ~ 

O O 

L L 

N 41 

d N 

E E 

~a c 

c o y .r 

a ro 

L i 

7 ~ 

N • 

tO ~ ro 

U 

~ 

I ~ T 

~ v G 

n 

C f. 

U YO ~ 

C 

~ ^ 

C L 

U V•• 

~ T 

c~ 

~ 

C L 

oo C..7 VN N 

~ a 

~ v 

~ n 

C L 

U V°-• 

9-101 
through day 7 of exposure. Incorporation of radiolabeled amino acids into 

collagen and noncollagenous protein rose to a plateau value at about 3 (collagenous) or 4 to 7(noncollagenous) days after exposure. Synthesis of collagen 

was about 1.6 times greater than that of noncollagenous proteins during the 

first few days of exposure; no major differences were apparent by 7 days of 

 exposure. After the 7-day exposure ended, about 10 days were required for 

recovery to initial values of prolyi 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 1, 2, or 3 weeks at levels ranging from 980 to 3920 pg/m3 

(0.5 to 2 ppm). A general correlation was found between fibrosis detected 

histologically and the quantitative changes in collagen synthesis in minces of 

03-exposed rat lungs. The stimulation of collagen biosynthesis was essentially the same, regardless of whether the rats had been exposed for 1, 2 or 3 

weeks; it was linearly related to the 0 3 concentration to which the rats were 

exposed. 

Protein deficiency and food restriction do not have a major influence on 

the effects of 0 3 on lung hydroxyproline, lung elastin, or apparent rates for 

lung collagen synthesis and elastin accumulation (Myers et al., 1984). In 

this study weanling or young adult rats were exposed continuously to 1254 pg/m3 

(0.64 ppm) 03 for 7 days. It appears that 03 caused an increase in apparent 

lung collagen and protein synthetic rates and no major change in elastin 

accumulation, but the results of statistical analyses were not reported. 

Continuous exposure of mice for 7 days to 882 pg/m 3 (0.45 ppm) of 03 

caused an increase in collagen synthesis after 5 or 7, but not 2 days of exposure (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. 

9-102 
Bhatnagar,et al. (1983) also"studied rats exposed continuously for 90 

days to 1568 pg/m3 (0.8 ppm) of 03. Prolyl hydroxylase activity continued to 

increase through 7 days of exposure. By 20, 50, and 90 days of exposure, no 

significant effects were observed. 

Last and his-colleagues performed a series of studies to evaluate the 

effects of subchronic exposure to 03 on-rats. In one experiment (Last et al., 

1984b), young adult (60-65 days old) rats were exposed continuously for 90 days 

to 1254 pg/m3 (0.64 ppm) or 1882 pg/m3 (0.96 ppm) 0 3 ; the higher level increased 

lung collagen content, while the lower level only caused a marginal (p <0.1) 

increase. Rats were exposed to these same concentrations in an intermittent 

regimen consisting of 5 days of exposure (8 hr/day), 9 days of air, 5 days of 

exposure, 9 days of air, etc., for a total of 35 days of 0 3 and 54 days of air 

within the 90-day experiment. Both concentrations of 0 3 caused an equivalent 

increase in collagen content. The magnitudes of the effects after continuous 

or intermittent exposure were not statistically different. 

Weanling rats (28 days old) were examined after a 6-wk exposure (8 hr/day, 

7 days/wk) to 1254 pg/m 3 (0.64 ppm) or 1882 pg/m3 (0.96 ppm) 03 (Last et al., 

1984b). Immediately after exposure, only those animals exposed to 1882 pg/m 3 

(0.96 ppm) 0 3 exhibited an increase in lung collagen content. However, 6 wk 

postexposure, animals exposed to both the high and low concentrations of 0 3 

had increases in, collagen content. It appears that the coilagen content 

increased during this postexposure period, but no statistical comparisons were 

reported. 

Young adult rats exposed continuously for 3 weeks to 1882 pg/m 3 (0.96 ppm) 

03 had an increase in lung collagen (Last et al., 1984b). Rats exposed to 

2352 pg/m3 (1.2 ppm) 03 for 1 wk and examined 2 wk later had an increase in 

collagen equivalent to that of the 3-wk exposure group (1882 pg/m 3 , 0.96 ppm). 

Weanling rats were also studied after intermittent exposure to 1882 pg/m 3 

(0.96 ppm) 0 3 (Last.et a1.,.1984b).. The r,egimen.-was 3 days of exposure for 

8 hr/day, followed by 4 days of air; this unit was repeated 6 times. No 

significant changes in collagen content occurred. 

Lung collagen af juvenile cynomolgus monkeys (6-7 mo old at start of 

exposure), exposed to 1254 pg/m 3 (0.64 ppm) 03 for 8 hr/day, 7 days/wk for 

1 yr (361 days), was studied by Last et al. (1984b). Collagen content was 

increased. Collagen type ratios were determined, and there were no apparent 

shifts in collagen types; however, the authors report that given the su.rgical 

variation, small shifts in collagen types would not be likely to be detected. 

9-103 
Rats were exposed to 980 pg/m3 (0.5 ppm) continuously for up to 180 days 

and examined at various times during exposure and 2 months after exposure 

ceased (Last and Greenberg, 1980). The total protein content of the lungs 

increased during exposure, with the greatest increase occurring after 88 days 

of exposure. By 53 days postexposure, values had returned to control levels. 

Hydroxyproline content of the lungs also increased following 3, 30, 50, or 88, 

but not 180, days of exposure. No such effect was observed at the 53-day 

postexposure examination. The rates of protein and hydroxyproline synthesis 

were also measured. Protein synthesis was not affected significantly. Although 

the authors mentioned that hydroxyproline synthesis rates "appeared to be 

greater," statistical significance was not discussed and values appeared to be 

only slightly increased, considering the variability of the data. In 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 alveolar 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 3 (0.2, 0.8, or 2.0 ppm) of 03 (Costa et 

al., 1983). At the two lower concentrations, rats exhibited an equivalent 

decrease in hydroxyproline. At the highest concentration, an increase was 

observed. Similar findings were made for elastin levels. 

Most reports, such as those described above, are on the effects of 0 3 on 

collagen synthesis. Very little is known about the effects of 03 on collagen 

turnover (i.e., the integration of synthesis and degradation). Curran et al. 

(1984) found thatin vitroexposure to high levels of 0 3 (19600 pg/m3 , 10 ppm 

for 1-4 hr) caused degradation of collagen. A lower level, 490 pg/m 3 (0.25 ppm) 

did not cause degradation, but the collagen became more susceptible to proteolytic degradation. 

Last (1983) and Last et al. (1983) investigated the interaction between 

03 and aerosols of ammonium sulfate [(NH 4) 2504] and sulfuric acid (H 2 SO4 ) in 

rats. Rats were exposed continuously for 7 days to four concentrations of 0 3 

ranging from 980 pg/m3 to 2940 pg/m3 (0.5 to 1.5 ppm) 03 . According to the 

authors, these levels were determined by the KI method and should be multiplied 

by 0.8 to compare to UV photometric methods (i.e., 0.4 to 1.2 ppm). The 

authors' actual measured values are noted here. The 0 3 exposure resulted in a 

linear, concentration-related increase in collagen synthesis rate. A 7-day 

continuous exposure to 5 mg/m3 (NH4 ) 2504 (0.8-1.0 pm mass median aerodynamic 

9-104 
diameter, MMAD) had no effect. However, when mixtures of 0 3 from 1568 to 

2940 pg/m3 (0.8 to 1.2 ppm) and 5 mg/m3 (NH4 ) 2SO4 were used, the collagen 

synthesis rate increased to about 180% over the rates of 0 3 for the higher 0 3 

levels (apparently 2352 and 2940 pg/m 3 , 1.2 and 1.5 ppm). 

Mixtures of 03 and H2SO4 were also reported (Last et al., 1983). Rats 

were exposed to 980 pg/m3 (0.5 ppm) 03 or this 0 3 level in combination with 

1000 pg/m3 H2SO4 (0.38 pm, MMAD) continuously for 3 to 50 days. As expected, 

0 3 exposure increased collagen synthesis rates at the three times of examination (3, 30, and 50 days). The mixture of 0 3 and H2SO4 caused greater effects. 

Examination of the slope ratio of regression lines indicated that H 2 SO4 in the 

mixture caused a 220 percent enhancement. The authors state that H 2SO4 alone 

had no effects. 

The statistical procedures applied in these collagen studies (Last et al., 

1983) were questioned by Krupnick and Frank (1984) in a letter to the editor. 

The general criticism was that too few statistical tests were applied to test 

the hypothesis of synergism and that too few pollutant data points were used 

in the design to develop robust regression lines. The authors (Last et al. 

1983) responded that they did apply most of the statistical tests and found 

significant differences between regressions comparing 0 3 only to 03 plus 

aerosols, but the journal would not accede to publishing these analyses. 

The proximal acinar regions of rats from the above-meritioned collagen 

studies, exposed for 7 days to 2352 pg/m 3 (1.2 ppm) 0 3 alone or in combination 

with 5000 pg/m3 (NH4 ) 2SO4 , were also evaluated. Ammonium sulfate alone caused 

no morphological or morphometric effects. Ozone exposure resulted in a thickening of the interstitium and an influx of inflammatory cells. The mixture of 

0 3 and (NH4 ) 2504 caused the same response plus an apparent deposition of 

fibrous material. These lesions were then examined morphometrically. In the 

0 3 and 03 plus (NH4 ) 2SO4 groups, there were no changes in the volume ratio of 

the lesion per lung or the volume ratio of the extracellular connective tissue 

per lesion._ In examining volume density of different cell types in the lung 

lesions, both groups had an increased percentage of fibroblasts and smooth 

muscle. When the number of cells of each type present per area of lesion was 

calculated, there was a 340 percent increase in the number of fibroblasts in 

the 03 plus (NH4 ) 2SO4 group compared to the 0 3 group. These findings correlate 

well with the biochemical effects. 

These studies were expanded by Last et al. (1984a) to better evaluate the 

influence of length of exposure. Ail exposures were continuous to 0 3 , ( NH4 ) 2SO4 , 

9-105 
or a mixture of the two; the ammonium sulfate concentration was about 5000 pg/m3 

(0.5 pm MMAD), the precise concentration depending upon the experiment. After 

3 days of exposure to either 1254 pg/m3 (0.64 ppm) 03 or (NH4 ) 2SO4 , no significant 

effects on collagen syntbesis of rats were observed; the mixture of 0 3 and 

(NH4) 2SO4 more than doubled the collagen synthesis rate over controls. The 

pulmonary lesions (i.e., aggregations of interstitial inflammatory cells in 

terminal bronchioles) of the proximal acinus in these rats were examined 

histologically and morphometrically. Ammonium sulfate alone caused no effects. 

In the 03 and 03 plus (NH4) 2SO4 groups, there was no fibrosis, but thickenin,g 

by edema and inflammatory cell infiltrate was observed. Morphometrically, 

(NH4) 2SO4 caused no effects. However, 03 and 03 plus (NH4) 2SO4 increased 

total cell numbers in lesions, with the mixture producing a significantly 

greater increase over 0 3 alone. This increase was principally due to an 

increase in the numbers of macrophages, monocytes, and fibroblasts. The 

increase in the number of fibroblasts is consistent with the biochemical 

findings. 

Similar measurements were made on rats exposed continuously for 7 days to 

1882 pg/m3 (0.96 ppm) 0 3 , 5000 pg/m3 (NH4) 2SO4 , or a mixture of the two (Last 

et al., 1984a). Control data were not presented and all statistical comparisons 

reported were in reference to the 0 3 alone group. Ammonium sulfate exposure 

resulted in fewer. numbers of cells than 0 3 alone. For macrophage and monocyte 

numbers, 03 and (NH4) 2SO4 were apparently additive. For fibroblast and total 

cell numbers, 03 and (NH4) 2SO4 were apparently synergistic. 

The frequency of occurrence of lesions was also examined in the 3-day and 

the 7-day studies described above (Last et al., 1984a). There was no difference 

in the frequency of lesion occurrence between 0 3 and 03 plus (NH4 ) 2SO4 . The 

individual lesions were larger in the 0 3 plus (NH4 ) 2504 group; the (NH4 ) 2SO4 

group had almost no lesions. Using collagen staining procedures, the 0 3 plus 

(NH4) 2SO4 group had a greater volume of collagen than did lesions in the 0 3 

alone group. The authors summarized these studies (Last et al., 1983; Last 

et al., 1984a) by stating that (NH4 ) 2504 increases the severity of 0 3 effects 

at lesion sites without increasing the number of lesions; and the cellular 

changes correlate with biochemical and histological indications of potential 

later fibrosis. From all these studies it is apparent that synergism occurs. 

However, it further appears that the synergism is dependent on the concentration 

of 03 . For instance, Last et al. (1983) demonstrated that 5000 pg/m 3 (NH4 ) 2SO4 

9-106 
increased collagen synthesis rates in rats exposed to 2352 pg/m 3 or 2940 pg/m3 

(1.2 or 1.5 ppm) 03 , but not i n those exposed to 1568 pg/m 3 (0.8 ppm) 03 . 

Protein synthesis (incorporation of radiolabeled leucine) was increased 

in rats after 3 days of continuous exposure to 1568 pg/m 3 (0.8 ppm) of 0 3 

(Chow et al., 1976b). Recovery had occurred by 6 days postexposure. No 

adaptation was observed, because when animals were re-exposed to the same 0 3 

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 amino acids. Rats were exposed 

continuously for 7 days to 1568 pg/m 3 (0.8 ppm) of 0 3 . No statistics were reported. 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 incorporation into blood or alveolar macrophages. Hence, the observed increases were 

due to lung tissue protein synthesis, and not a concurrent influx of alveolar 

macrophages or serum into the lungs. 

The production of mucous glycoproteins and their secretion by tracheal 

explants have been reviewed by Last and Kaizu (1980). Mucous glycoprotein 

synthesis and secretion were measured by the rate of incorporation of 3 H-glucosamine into mucous glycoproteins and their subsequent secretion into supernatant 

fluid of the tracheal explant culture medium. This method has been found to 

be a reproducible index of mucous production, and these authors maintain that 

this measurement ex vivo following exposure in vivo is representative of 

injuries occurring in vivo. When rats were exposed to 1568 pg/m 3 (0.8 ppm) of 

0 3 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, 

and 392 pg/m3 (0.6, 0.4, 0.2 ppm) of 0 3 . Glycoprotein secretion decreased 

only at the higher concentration. Tracheal explants from Bonnet monkeys 

exposed to 0, 980, or 1568 pg/m3 (0, 0.5 or 0.8 ppm) 0 3 for 7 days appeared to 

have increased rates of secretion of mucus (Last and Kaizu, 1980). However, 

few monkeys were used and statistical analysis was not reported. 

9-107 
A combination exposure to 0 3 (980 pg/m3 ; 0.5 ppm) and sulfuric acid 

(H2SO4 ) aerosol (1.1 mg/m3 ) caused complex effects on mucous secretions in 

rats (Last and Kaizu, 1980; Last and Cross; 1978). A 3-day exposure to 

980 pg/m3(0.5 ppm) of 03decreased mucous, secretion rates, bu -t H2SO4had no 

effect. In rats exposed to the combination of H 2SO4 aerosol and 0 3 , mucous 

secretion significantly increased. After 14 days of continuous exposure, the 

rats receiving a combined exposure to both H2SO4 aerosol and 0 3 had elevated 

values (132 percent) over the control group of animals. Because mucous secretion 

and synthesis are intimately involved in diminishing the exposure of underlying 

cells to 03 and removing adventitiously inhaled particles, alterations in the 

mucous secretory rate may have significant biological importAnce. Experiments 

reported to date do not cl early i ndi cate what human heal th effects may be 

likely, nor their importance. 

9.3.3.7 Lipid Metabolism and Content of the Lung. If 03 initiates peroxidation 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 3 exposure are difficult to determine in 

the absence of rigid dietary control. Studies on lipid metabolism and content 

of the lung are summarized in Table 9-9. Generally, the unsaturated fatty 

acid content decreased in rats exposed to 980 pg/m 3 (0.5 ppm) of 03 for up to 

6 weeks (Roehm et al., 1972). 

Peroxidation of polyunsaturated fatty acids produces pentane and ethane 

to be exhaled in the breath o;' 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 i ncreased when rats were defi ci ent i n vi tami n E and exposed to 

1960 Ng/m3 (1 ppm) of 03 for 60 min. The provision of 11 (minimum vitamin 

requirement) or 40 IU (supplemented level) of'vitamin E acetate per kg of diet 

resulted in a decrease in expired ethane and pentane after 03 exposure. 

Dumelin et al. (1978a) also measured breath ethane and pentane in Bonnet 

monkeys exposed to 0, 980, or 1568 pg/m3 (0, 0.5, or 0.8 ppm) of 03 for 7, 28, 

or 90 days. They failed to detect any additional ethane or pentane in the 

breath of these monkeys and attributed the lack of such additional evolution 

to be due to the high level of vitamin E provided in the food. 

. ~ . 
G.7 

Z 

~. 

J 

W 

S 

FW 0 

F•- 

Z 

w 

~ 

z 

0 

U 

c 

Q 

~ 

N 

M 

O 

m 

~ 

w E 

C 

d !, 

J 

Z 

O 

~ 

~ 

0 

o_ 

w 

LLJ 

Z 0 

N 

O 

LL 

O 

1•- 

U 

W 

L. 

W 

w 

Qi 

rn 

W '- 

J 

m 

¢ 

H 

II 

c 

m rG1 O 

L C U 

O O O 

N •r i~ 

G A L 

X i G 

W O 

II 

ro +1 

C 

G! 

GE! O 

L L 

7 N 

N Gl 

~ E 

E 

N 

U 

C 

d 

d 

w 

d 

C 

N 

N 

• U 

G1 

a 

N 

41 

u 

Gl 

4•- 

G1 

a 

N 

~ d 

~ 

O 

~ 

n I 

rn 

r♦ 

ro 

4-) 

N 

E 

v O 

C 

~ 

~0 

~ 

u 

ro 

~ 

d 

C 

r m 

aw 

7 01 

C1 ri 

T 6! 

Y 

O 

g ' 

GT1 M 

OS ^ T 

m ,•~ 

O d • 

Q • 

1 

•~ ro 

Y d 

~ 

a 

.G 

(0 

z 

T 

i~ 

+J 

m 

4- 

4- 

O 

O 

+~ 

f0 C 

L •r 

O L 

d ~ 

L •~ o u 

c~ 

II ~ 

ai c 

N r t6I 

V •r ' 

N U 

i C cO 

+= E o ci • Q 

4J 

G7 r0 

CL 

O +J 

NC 

o w u E c 0 cn 

U O. 

+~ 1 

•r O 

E C 

b~ 

aa 

c m ro 

U 

U •~ 

C - 

O O 

II 

r C 

L •r 

tQ 0) 

L. N 

m ra 

c L . 

r U N 

.mII a 

N U 

SO. 'O V 

U •r •r 

r_ rorw 

O Y '- 3 

N ~O 

7 

O L 

O O 

~ 

+J d• 

O •• 

U N 

> 

~ 

m m m 

0 00 

O O GO 0 0to 

rn rn uO 

r+ 

ia 

4J 

N 

C 

r- .p 

e~ 

~ Ol 

C1 r-I 

~ 

10 

a: 

C d O G! 

r a L N aJ 1-1 ro 

•• II •r ri y 

++c3 ic 

a_ro wuo 

o d •r 

a C . II 4J 

O C C U +J•r o o - ~ 

C i~ •r .r- }1 a 

N U +) +J d O 

r -7 U M •r i. 

•U II 7 41 O CL 

r _O II C 

M- - L O G! O1 CJ 

GJ a LE Y C 

aaN ~ ro 

wc a~ aWc m c nc ai 

c +J M 7 •r n 

•eaci.L N~ a 

m 0. 61 W•~ 1C 

4J a a c > m 

 •> vi vOi •E OC - 

rmmm— m 

~ L Lv O ~ ~- +

3, C7U>d'O) 

~ C 

E i 

O L UD v 

o--/ 

Y 

m 

Z 

G 

Z 

O O 

O O 

~o ca 

rn ri rn rt 

W a N o 

i0 G~

1 N 

++ •.- r- c a ai 

N > 

a r• 

C ~ L 

y RI L 

C E 

m.rs 

0~1 ro •3 

c 

c•ra 

o a) 

c ~ 

Fj O C W 

U •r Cl 

w +~ E c 

4- U O/ •r 

w O - E 

m oc°".+m+ O f.. = •r 

z a N > 

i b 010 

w 

O 

arn 

m 

O L 

~ O 

L 0~ 

00 N 

II N 

d 

• L 

U 

N 

N 

a 

~ 

0 

C 

11 

G 

z 

U 

• L 

+J 

d 

E 

b 

O 

11 

t••i 

i 

~ d 

E 

4~ 

0 

G. 

~ 

u 

~ 

ci 

a 

~ v 

O 

E 

~ 

• N 

N 

m 

~ 

d 

II 

N 

i 

a 

w 

~ .n 

m L' 

4J 

a 

C 

II 

oC 

m 

2 

II 

O 

L 

~ 

N 

E 

4J 

c ai 

E N i 

N 

m 

ar 

z 

m 

9-109 
Kyei-Aboagye et al. (1973) found that the synthesis of lung surfactant in 

rabbits, as measured by dipalmitoyl lecithin synthesis, was inhibited by 

exposure to 1960 pg/m3(1 ppm) of 03 f o r'4 . hr. Pulmonary lavage showed' an ' 

increase in radiolabeled lecithins. The authors proposed that 0 3 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 3 exposure. However, although changes in lipid composition of lavage fluid occur, the changes apparently do not alter the surface tension lowering properties of the fluid, as shown by Gardner et al. 

(1971) and Huber et al. (1971) when using high levels of 0 3 (> 9800 pg/m 3 ; 

5 ppm). 

9.3.3.8 Lung Permeability. Table 9-10 summarizes studies of the effects 

on lung permeability of exposures to different concentrations of 0 3 . 

The lung possesses Several active-transport mechanisms for removal of 

substances from the airways to the capillary circulation. These removal mechanisms have been demonstrated to be carrier-mediated and,specific for certain 

ions. Williams et al. (1980) studied the effect of 0 3 on active transport of 

phenol red in the lungs of rats exposed to 1176 to 4116 pg/m3 (0.6 to 2.1 ppm) 

of 03 continuously for 24 hr. Ozone inhibited the carrier-mediated transport 

of intratracheally instilled phenol red from the lung to the circulation and 

increased the nonspecific diffusion of phenol red from the lung. These changes 

in ion permeability may also explain in part the effects of 0 3 on the respiratory response of animals to bronchoconstrictors (Lee et al., 1977; Abraham et 

al., 1980). 

As another index of increased lung permeability following 0 3 exposure, 

the appearance of albumin and immunoglobins in airway secretions has been 

examined. Reasor et al. (1979) found that dogs breathing 1960 to 2940 {ag/m 3 

(1.0 to 1.5 ppm) of 0 3 had increased albumin and immunoglobjn G content of 

their airway secretions. Alpert et al. (1971a), using rats exposed for 6 hr to 

490 to 4900 pg/m (0.25 to 2.5 ppm) 0 3 3 , also found increased albumin in lung 

lavage in animals exposed to 980 pg/m 3 (0.5 ppm) or more. 

In a series of experiments, Hu et al. (1982) exposed guinea pigs to 196, 

510, 1000, or 1960 pg/m3(0.1, 0.26, 0.51, or 1.0 ppm) of 03for 72 hr and 

found increased lavage fluid protein content sampled immediately after exposure 

to concentrations >510 _ ~ag/m (0.26 ppm) as compared with controls. Ozone- 3 

exposed guinea pigs had no accumulation of proteins when the exposure time was 

9-110 
al 

U 

c 

d 

L 

N 4- 

v 

C 

N 

N 

u 

d 

n 

t/f 

r F- H 

J 

H 

m 

W 

~ 

W 

1 

z 

J ~ 

Z 0 

w 

Z C 

N O 

UO 

.N 

~ 

U 

W L,

LL 

!JJ 

O 

r-1 

~ 

W . J 

m 

¢ 

H 

~ 

v 

41 

U 

~ 

4•- 

al 

~ 

N 

i 

G1 

N 

a 

O 

' D 

C 

a ror 

Z O 7 C U 

N O O 

O •.- +- 

C. o 

X t0L 

W L C. 

7 

~ 

a 

ro +-) 

c 

E 'O 

G! O i L 7 .1J 

m ~ 

N, 

c E 

O n 

Yn 

C i 

O 4J 

N C 

C 

U 7. 

i 

i L 

t 

V 

t0 N 

H Y Y 

~ Z Z 

~ O 

ro 4J 

d 

a

41i 

G/ 

> 

ro 

'Q 0 ci > 

N O 

ma 

N ~O 

Z 

C7 O C C 

~. ro 

LO 

N 

L9 

 O 

 U 

c0 r••~ l0 rl ' 

00 r/N 

O O O O 

dOmi~~ 1-Ln M rl 

r-1 r-i r-I r-I 

riNMCr 

r O r Oo 

•~ o~ 

3 +-+ 

~ 

ro 

z 

V 

G1 

4J (a 

"Q 

G1 

E 

i 

G1 

• s 

Z 

ro 

U 

~FO 

N 

N 

O 

E 

~o 

~ v ro 

O o L 

U •r .{-) 

re E N 

Y 7 C 

11 •N II 

N 4~ Y 

~ n Z ' 

O O 

~ L 

~ 4-) 

E E 

.yJ C 

C O 

E +) 

o a ~ N r 

4) U 

'o a 

v 

ro 

i 

w 

N-• 

~ 

a 

~ 

L 

4J 

7 G1 

C 

Y 

m 

Z 

>; L 

•!•1 

N 

E 

~ 

O 

L 

n 

7 

~ 

I 

~ 

~ 

N 

u 

c 

C1 

U (

`ni 

E 

Oi 

4J 

•E al 

N E 

L O 

U iJ O 

d L N n 

L ~! 

n ~ 

N 11 ro 

Qi > 

~ 

I 

1.1.1 V 

U O 

i E 

d 7 

dJ 

CI N 

E N 

ro 4J 4J 

N O 

ro n 

f ~ 

N U i 

L w 

E N a 

O •O 

~ L ro 

r

a r` 

rn 

¢ r-1 

4J 

ro m 

E n 

n 

Ln 

0 

4J 

0 

C O 

~ 

ro 

7 

E 

u 

ro 

c 

a 

4J 

L 

n 

L 

ro 

b 

a.+ ai 

cu L 

'O r 

c o 

ac 

ad 

N L 

"O n 

c Z 

O O N 4J ro +J 

L Z 

C 0. 

w N 

u G 

C ~6 

O L 

U fJ 

ro 

~ al 

i O 

N 01 

a~ rn 

m 

0 

c 

ro 

~ 

.~ 

C 

r-V 

c 

a 

O 

07 

O C 

E 

E 

~ 

~ 

c 

E 7 

a 

'p C al o tll •r 

m +~ 

G1 d 

L Z 

C N 

H N 

L 

L 

N 

H 

~ 

i O t C) 

r-1 r•i 

O O 

cm 

r

cn

i N 

N 

~ 

~ 

m 

N 

d 

~ 

x 

f0 

O1 C 

•.- 01 

~ •~ 

c~ n 

+) 

O 

1 C 

C 

ro 

4J 

Ol 

~ 

E G 

Z U O. •^ v 

7 

C 

n d ~ 

O r y lO 7 i~-1 

•GJ n E•r +Jxro> 

- N N Z 

ot v od on c 

C.L > ~ Q d N ro 

O 1 C 

N N 

L i 

L Gl 

O rl 

r ^ L N ~ GJ ~ 4~•• 4-' 

4-L741 iN ro roa 

N N u N 7 Z 

L07rn ~ G. aCi Gl o aii ~ N oi Rf t0 x> > o.+J a7 a! O 

• t>O >> Ow ~ N~ro O >CJ X 

~•~ C 

tn 

~ F- +a ~ n 

o ro cn «~ al O o. c a 

C N 

r o 

o +~ o m a n N ••O rt1 C O L _ N +f - 

Gl E S.. 'O n Gl L.0 

> E Cl •rd•.- 4-3 -o C 

Ln ~ G7 Z 

f-1 +] l~ • - 

n 

i. 

•O G. ro U d~ rtf O. ^ 7 '_ N O/ n f- c 

N t d U G7 4F 

3 ^ N 

•C B O d G1 C. O. 

G1 L 

N O f0 l0 f0 

N N tA •U 

L . ri •r4- 

Ol 

01 N n X 

ro ro d 

G1 

i t.f) 

C~ i (1) 0 ro L ~ 4- C 

~•-~ n l o a z . J u.o O 1--1 0 

N L 

L tD V t 

L •Q 

\ O 

N 

t~ 

N L rl 

L 

L 

C+p O 

O 

O 

•• Lf) d i i N i 

L L Z L 

 M - O 

M 4- M M 

IU ~ 

tC ri Op tD 1-1 

rl N i1! O r-i N uY 19 

OOOr-1 G 00 O 

tO O OC f'M 00 O 

r

01

-I~ ~~ CM ~~[f T ~ 

9-111 
reduced from 72 to 3 hr, unless the time of lavage was delayed for 10 to 15 hr 

following exposure. The protein content of the lavage fluid determined 10 to 

15 hr following'a 3-hr exposure increased in a concentration-related manner 

from 500 to 1470 pg/m3 (0.256 to 0.75 ppm). Again 196 pg/m 3 (0.1.ppm) had no 

effects. The lavage fluid protein content of guinea pigs exposed to 353 pg/m 3 

(0.18 ppm) of 0 3 for 8 hr per day for 5 or 10 consecutive days was not different from air controls. No effect-of vitamin C deficiency could be found on 

the accumulation of the lavage fluid protein in guinea pigs exposed to 196 to 

1470 pg/m3 (0.10 to 0.75 ppm) 0 3 for 3 hr (Hu et al., 1982). In contrast, 

vitamin C-deficient guinea pigs have increased sensitivity to NO 2 (Selgrade et 

al., 1981). Polyacrylamide gel electrophoresis of lavage fluid proteins from 

animals exposed for 3 hr to 196 to 1470 pg/m 3 (0.1 to 0.75 ppm) 0 3 showed the 

appearance of extra protein bands which co-migrated with serum proteins and of 

increased intensity of bands that also occur in air-exposed controls. This 

led the authors to conclude that the main source of the increased protein was 

serum. 

Prostaglandins are intermediates in tissue edema resulting from a wide 

variety of inechanisms of injury. Increased lung permeability and edema produced by 03 might also be mediated by prostaglandins. Non-steroidal anti-inflammatory drugs (aspirin and indomethacin) at appropriate doses inhibit lung 

edema in rats from exposure to 7890 pg/m3 (4 ppm) of 03 for 4 hr (Giri et al., 

1975). Prostaglandins F 2a and E2 were markedly increased in plasma and lung 

lavage of rats exposed to 7840 pg/m 3 (4 ppm) for up to 8 hr (Giri et al. , 

1980). Ozonolysis of arachidonic acid in 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/m 3 (3 ppm) 0 3 

inhibits uncompetitively rat lung prostagl•andin cyclooxygenase (Menzel et al., 

1976). 

Earlier reports that prostaglandin synthesis inhibitors exacerbated 

03-produced edema (Dixon and Mountain, 1965; Matzen, 1957a,b), as did methyl 

prednisolone (Alpert et al., 1971a), tend to confuse the interpretation of the 

role of prostaglandin in 0 3-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 3 -produced edema, their exact role is 

still unexplained. 

9-112 
9.3.3.9 Proposed Molecular Mechanisms of Effects. Experts - generally agree 

that the toxicity of 0 3 depends on its oxidative properties. The precise 

mechanism of 0 3 '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. Environmental Protection Agency (1978); National Air Pollution Control Administration 

(1970); North Atlantic Treaty Organization (1974); National Research Council 

(1977); Shakman (1974); Menzel (1970, 1976); Nasr (1967); Cross et al. (1976); 

Pryor et al. (1983); and Mudd and Freeman (1977). From these reviews and 

recent research detailed here or in previous biochemistry sections, two hypotheses are favored and may in fact be related. 

9.3.3.9.1 Oxidation of polyunsaturated lipids. The first hypothesis is that 

03 initiates peroxidation of polyunsaturated fatty acids (PUFA) to peroxides, 

which produces toxicity through changes in the properties of cell membranes. 

Ozone addition to ethylene groups of PUFA can take place in membranes yet give 

rise to water-soluble products that can find their way to the cytosol. Aldehydes, peroxides-, and hydroxyl radicals formed-by peroxidat-ion all can react 

with proteins. In addition to the direct oxidation of amino acids by 0 3 , 

secondary reaction products from 0 3-initiated PUFA peroxidation can also 

oxidize amino acids or react with proteins to alter the function of the proteins. 

Since large numbers of proteins are embedded with lipids in membranes and rely 

on the associated lipids to maintain the tertiary structure of the protein, 

alterations in the lipid surrounding the protein can result in structural 

9-113 
changes of the membrane-embedded protein. At present, methods are not available to differentiate effects on membrane proteins from effects on membrane 

lipids. 

Some of the strongest evi dence that the toxi c reacti on of 0 3 can be 

associated with the PUFA of inembranes 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 3-induced increase 

in enzyme activities of the glutathione peroxidase system (Section 9.3.3.2) 

and those involved in oxygen consumption (Section 9.3.3.3). More details are 

provided in Table 9-5. Morphological effects due to 0 3 exposure are also 

1 essened by di etary vi tami n E(Secti on 9.3.1.4.1.1). A1 though thi s i s not 

the strongest evidence, vitamin E supplementation also prevented 0 3-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 03 in animals. They also support indirectly lipid peroxidation as 

a toxic lesion in animals. HoWever, although the influence of dietary vitamin 

E is clear, its relation to vitamin E levels in the lung, where presumably 

most lipid peroxidation would occur, is poorly understood. Rats of various 

ages (5, 10 and 90 days old and 2 yrs old) were fed normal diets; and 90-dayold 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 protective effects were conducted with diets having far less than 200 mg/kg diet.) 

Rats were exposed to 1764 pg/m3 (0.9 ppm) 0 3 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 3-induced responses in the lung are.independent of the vitamin E content of the lung. Independent interpretation of this 

study is not possible, since very minimal descriptions of responses were provided 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 mo-rphological responses of rats to 0 3 . 

9-114 
9.3.3.9.2 Oxidation of sulfhydryl or amine groups. The second hypothesis is 

that ozone exerts its toxicity by the oxidation of low-molecular-weight compounds 

containing thiol, amine, aldehyde, and alcohol functional groups and by oxidation of proteins. Mudd and Freeman (1977) present a summary of the arguments 

for oxidation of thiols, amines, and proteins as the primary mechanism of 0 3 

toxicity based upon in vitro exposure data. Amino acids are readily oxidized 

by 0 3 (Mudd et al., 1969; Mudd and Freeman, 1977; Previero et al., 1964). In 

the following descending order of rate, 0 3 oxidizes the amino acids cysteine > 

methionine > tryptophan > tyrosine > histidine > cystine > phenylalanine. The 

remai ni ng common ami no aci ds are not oxi di zed by 0 3 , Thi ol s are the most 

readily oxidized functional groups of proteins and peptides (Mudd et al., 

1969; Menzel, 1971). Tryptophan in proteins is also oxidized in vitro by 0 3 

as shown by studies of avidin, the biotin-binding protein. Oxidation of 

tyrosine in egg albumin by 0 3 occurs in vitro, converting the 0 3-oxidized egg 

albumin to a form immunologicaliy distinct from native egg albumin (Scheel et 

al., 1959). Ozone inactivated human alpha-l-protease inhibitor in vitro (Johnson, 

1980). When treated with 0 3 , alpha-l-protease inhibitor lost its ability to 

inhibit trypsin, chymotrypsin, and elastase. 

Meiners et al. (1977) found that 0 3 reacted in vitro with tryptophan, 

5-hydroxytryptophan, 5 - hydroxytryptamine, and 5-hydroxyindolacetic acid. One 

mole of 03 was rapidly consumed by each mole of indole compound. Oxidation of 

tryptophan by 03 also generates hydrogen peroxide. Hydrogen peroxide is a 

toxic substance in itself and initiates peroxidation of lipids (McCord and 

Fridovich, 1978). Other active 02 species such as HO• and 0 2- 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 

0 3 (viz., 1960 to 7840 pg/m3 , 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 ievel 

(1960 pg/m3 , 1 ppm) 0 3 exposures. The enzymes decreased.include those catalyzing 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 03 exposure. Respiratory control of mitochondria is lost, 

9-115 
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 glycerides. Mudd and Freeman contend, 

however, that proteins are far more easily oxidized by 03 than are lipids. 

Indirect evidence in support of the idea that amines in particular are oxidized 

preferentially by 0 3 is the protective effect of p-aminobenzoic acid (Goldstein 

et al., 1972a). Rats injected with p-aminobenzoic acid were partially 

protected from t-he mortality due to high concentrationss of 03 . Presumably, 

the added p-aminobenzoic acid is oxidized by 0 3 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 

result in membrane stabilization, which could account for the protective 

effects of these compounds preventing edema and inflammation. Acetylcholinesterase found on red blood cells is protected from inhibition by in vitro 

exposure to 78,400 pg/m3 (40 ppm) 03 through p-aminobenzoic acid treatment 

(Goldstein et al., 1972a). 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. 

9.3.3.9.3 Formation of toxic compounds through reaction with polyunsaturated 

1 ipi ds. The effects of 0 3 coul d be due to the el aborati on of products of 

peroxidation as well as peroxidation of the membrane itself. Menzel et al. 

(1973) injected 10 pg to 10 pg 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 Privet -t (1972) injected 

methyl linoleate ozonide into rats or gave the compound orally; in each case 

acute pulmonary edema resulted. The pulmonary edema and changes in fatty acid 

9-116 
m 

composition of the serum and lung lipids were similar to those occurring after 

03 exposure (Roehm et al., 1971a,b, 1972; Menzel et al., 1976). 

Peroxidation of lung lipids could lead to cytotoxic products. Phosphatidyl choline liposomes (spheres formed by emulsifying phosphatidyl choline in 

water) were lysed on exposure to 03 (Teige et al., 1974). Liposomes exposed 

to 03 were more active than 0 3 alone i-n lysing red blood cells. The products 

of ozono1ysis of phosphatidyl choline could be stable, yet toxic, intermediates. 

9.3.3.9.4 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., ROH, 

RCHO or RCO2H). 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 3 to PUFA, free radicals may be generated 

(Pryor et al., 1983), and these free radicals may be the ultimate toxicants. 

Because the metabolism of PUFA peroxides in the lungs is intimately 

linked with the metabolism of thiol compounds (such as glutathione, GSH), 

direct oxidation of thiols by 0 3 (hypothesis B) may link the two major hypotheses A and B with hypothesis C, formation of toxic products. Ozone depletion 

of GSH could render the peroxide detoxification mechanism ineffective. However, 

at levels of 0 3

. below 1960 pg/m3 (1 ppm), increases in glutathione have been 

obs,erved (Plopper et a1., 1979; Fukase'et al., 1975; Moore et a1., 1980; 

Mustafa et al., 1982). The rat lung is sensitive to the increase of glutathione 

peroxidase, glutathione reductase, and glucose - 6-phosphate dehydrogenase 

activities at levels as low as 196 pg/m 3 (0.1 ppm) 0 3 continuously for 7 days 

(P1 opper 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 acyte exposures to >1960 pg/m 3 (1 ppm) 

0 3 , decreases are observed in ttiese enzyme activities and glutathione levels. 

Other species (mi'ce and monkeys) exhibit similar effects at different concentrations (Table 9-5). These changes at the lower levels of 0 3 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 inetabolic activity would 

be expected to cause an overall increase in the metabolic activity of the 

9-117 
1ung. This was substantiated by Mustafa (1975) and DeLucia et al. (1975a), 

who found that the 02 consumption per mitochondrion was not increased in 

03-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 03-exposed rats were due to the demands of the glutathione peroxidase 

system for reducing equivalents in the form of NADPH. Cross et al. (1976) and 

DeLucia (1975a,b) reported that 0 3 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 

radicals formed by the enzyme cytochrome P-450 or already produced hydroperoxides. 

Chow and Tappel (1973) suggest that peroxides formed via lipid peroxidation 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 cell.s 

(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 03 in vitro. 

They found also that 125 I from radiolabeled 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 0 3 toxicity. Fairchild and 

Graham (1963) found that thyroidectomy, thiourea, and antithyroid drugs protected animals from lethal exposures to 0 3 and nitrogen dioxide. Fairchild and 

9-118 
Graham ascribed the mortality following 0 3 to pulmonary edema. Wong and 

Hochstein (1981) suggested that 0 3 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 3 sensitivity. 

9.3.3.9.5 Summary. The actual toxic mechanism of 0 3 may involve a mixture 

of all of these chemical mechanisms because of the interrelationships between 

the peroxide detoxification mechanisms and glutathione (See Section 9.3.3.2) 

and the complexity of the products produced from ozonation of PUFA. A single 

chemical reaction may not be adequate to explain 0 3. toxicity. The relative 

importance of any one reaction, oxidation of proteins, PUFA, or small molecular 

weight compounds, will depend upon a number of factors such as the presence of 

enzymatic pathways of decomposition of products formed (peroxides), pathways 

for regeneration of thiols, the presence of non-enzymatic means of terminating 

free radical reactions (vitamins E and C), and differences in membrane composition of PUFA (relative ease of attack of 0 3 ), for example. 

9.3.4 Effects on Host Defense Mechanisms 

The mammalian respiratory tract has a number of closely coordinated pulmonary.defense mechanisms that, when functioning normally, provide protection 

from the adverse effects of a wide variety of inhaled microbes and other particles. A variety of sensitive and reliable methods have been used to assess 

the effects of 0 3 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 0 3 on host defenses. Other reviews have recently been written that 

provide valuable references to the complexity of the host defense system and 

the effects of environmental chemicals such as 0 3 on its integrity (Gardner, 

1981;'Ehrlich, 1980; Gardner and Ehrlich, 1983; Goldstein, 1984). 

This- section describes the existing data base and, where appropriate, 

provides an interpretation of the data, including an assessment of the different 

microbial defense parameters used, their sensitivity in detecting abnormalities, 

and the importance of the abnormalities with regard to the pathogenesis of 

infectious disease in the exposed host. This section also discusses the 

9-119 
various components of host defenses, such as the mucociliary escalator and the 

alveolar macrophages, which clear the lung of both viable and nonviable particles, and integrated mechanisms, which are studied by investigating the host's 

response to experimentally induced pulmonary infections. The immune system, 

which defends the overall host against both infectious and neoplastic diseases, 

is also discussed. 

9.3.4.1 Mucociliary Clearance. The mucociliary transport system is one of 

the lung's primary defense mechanisms against inhaled particles. It protects 

the conducting airways by trapping and quickly removing material that has been 

deposited on the mucociliary escalator. The effectiveness of mucociliary 

clearance can be determined by measuring such biological activities as the 

rate of transport of deposited particles; the frequency of ciliary beating; 

structural integrity of the ciliated cells; and the size, number, and distribution of mucus-secreting cells. Once this defense mechanism has been altered, 

a buildup of both nonviable and possibly viable inhaled substances can occur 

on the epithelium and may jeopardize the health of the host, depending on the 

nature of the uncleared substance. As an example, regardless of the severity 

of the influenza infection, the virus concentrates initially in the lining of 

the airways. The virus then spreads to alveolar cells of the lung parenchyma. 

It is suspected that the macrophage might be the site of replicati,on of this 

virus (Yilyma et al., 1979; Nayak et al., 1964; Raut et al., 1975). 

A number of studies with various animal species have reported morphological damage to the cells of the tracheobronchial tree from acute and subchronic exposure to 490 to 1960 pg/m 3 (0.20 to 1.0 ppm) of 0 3 . (See Section 

9.3.1.) The cilia were either completely absent or had become noticeably 

shorter or blunt. By removing these animals to a clean-air environment, the 

structurally damaged cilia regenerated and appeared normal. Based on such 

morphological observations, related effects such as ciliostasis, increased 

mucus secretions, and a slowing of mucociliary transport rates might be expected. However, no measurable changes in ciliary beating activity have been 

reported due to 0 3 exposure alone. Assay of isolated tracheal. rings from 

hamsters immediately after a 3-hr exposure to 196 pg/m 3 (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 pg/m 3 

H2SO4 (0.30 pm volume median diameter), a significant reduction in ciliary 

9-120 
beating frequency occurred. The magnitude of this effect was, however, significantly less than that observed due to the effect of H2SO4 expbsure alone. 

In either case, the animals completely recovered within 72 hr when allowed to 

remain in a clean-air environment. These authors found only a slight decrease 

in beating frequency with a simultaneous exposure to 196 pg/m 3 (0.1 ppm) of 0, 

and 847 pg/m3 H2SO4 (Grose et al., 1982). These data indicate that 0 3 appears 

to partially pr.otect against the effects of H2 SO4 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 glycoproteins (Last and 

Cross, 1978) resulting from exposure to these chemicals. 

Studies cited in the previous• criteria document (U.S. Environmental 

Protecti on Agency, 1978) gave evi de.nce on the effect of 0 3 on the host' s 

ability to physically remove deposited particles (Table 9-11), The slowing 

of mucus transport in both rat and rabbit trachea as a result of 0 3 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 3 on the defense mechanism of the mouse lung was to diminish 

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 0 3 or 

before a 4-hr exposure. Concentrations of 0 3 were 1180, 1370, 1570, or 

1960 pg/m3 (0.6, 0.7, 0.8, or 1.0 ppm). The physical clearance and bactericidal capabilities of the lung were then measured 4 to 5 hr after bacterial 

exposure. Exposure 17 hr before infection caused a significant reduction in 

bactericidal activity beginning at 1960 pg/m 3 (L 0 ppm) of 0 3 . When mice were 

exposed to 03 for 4 hr after being infected, there was a significant decrease 

in bactericidal activity for each 0 3 concentration, and with increasing 0 3 

concentration, there was a progressive decrease in bactericidal activity. The 

investigators proposed that because mucociliary ciearance was unaffected by 

subsequent 03 exposure, the bactericidal effect was due to dysfunction of the 

alveolar macrophage. Warshauer et al. (1974) reported tha-t a deficiency in 

vitamin E would further reduce the lungs' bactericidal activity. 

Friberg et al. (1972) studied the effect of a 16-hr/day exposure to 980 

pg/m 3(0.5 ppm) of 0 3 on the lung clearance rate of radiolabeled monodisperse 

polystyrene and iron particles in the rabbit and of bacteria in the guinea 

9-121 
W 

U Z 

C 

6 W 

J 

U 

0 

d 

Z 0 

rr 

H 

Vf 0 

W 

LLI

O 

S 

Z 

x 

c~ 

S 

z 

W 

W 

LLJ 

C 

N 0 

s 

z 0 

W 

Z 

N 

0 

LL. 

0 FNv W lL. 

W 

ri 

°1 

W J 

m 

H 

.n 

~ 

cli 

~ 

r-4 

~ 

O n L H 00 01 I- 00 

r

cn•i 

°1 C ~ ~ 

~ "4 I"1 

1

-i •• U ~ ~ ro ^ . ' u 

~ cm o ro ~ ro m m +J 

L i0 13 ya N 40-• i•7 fC0 C N ~ d C~l N C 

0/ CJ 01 L r C C i~ Gl L 

L~ i~ b •~•Oi 

vOf 4- a a ro o 

~ o o 

aci •~0 s. s .L° o C7 U C7 Y lL la. 6- Q U. 

°f 

N 61 L •O. 

ro 4J ro 

•Gl N 

1071 N 

1011 a C Gl N 

fA ~ ro 

•7 L O 

Tc C C7 Nf ~ 

ro a ro 

•i' ,U 

+J c ~ O IVO VOi ~ N ++ . E 

Lm ~ G. 

a L L b a 

+~ c. 

U _ O 

u c .n m i a°i ° °1 

.N s°1 0 

L Rl y V a' a 

• 

~ cd Ol 5- ~ ~ ro C 1~0 

L 

~ 

G! 'Q w N 

T 

U 

cc 

~f •O N 

N 

O ~ }1 U c N a ~ 

u N fi H 

c"i c c c o m i° c ° n ai o 

c~ o.•~ 

ar c. c 

40-• -+~ •° L L+1 

Nro o ~ 4- Y- .n 01 

v~- Ol of 

Gl f0 N ~ Gl 

.c 

c C.1 

•~ G1 Gl 

u 

• ~ .G 

O ro > ro Q1 L O 

~ a ~O ~ 4~•~ N 

~> N +j o 

> s>' 

a°i c c "°~ 

~'c 

°cc ro o 

u

L o °ror' 

ro 10 ~ 

~ ro o 

~ > t0 ro

~ 0 Gl •'- L E a a ro 

a 

4- 4..1 ~ r IO 

~ {.t d U 47 C Ol c 

O •U 

L r U 

N+3 = U Ol f0 U Gl •U r. U t0 C/ . 

u N 

c 

e0 

fy 

Cl O 

fp ~ ..- 

i. C/ 3 c'o ~ c O f.7 - N ~ F- ' N f.. L ro Q! V- c o Ol O t0 

tO 

{.) ro q- c r ro U+~ y 4- o o r 7 L 

N G~1 a 

GCl ~ ro 

O U Gl ro ro w 

•F~ ~ 

•tj N O 

+j +~ u 

+L.1 +1 V ro o • U N f. N o C) c v L V G1 ~- 

a 

io ~ o 

+7 

~ s. o 

N 

> 

N 

ro••• 4- C. a) 

07 ro 

G) 4- U•.- •ro ~~" +' +Oi c f~p Rt t0.. 7 

4-

ar 

U 

c 7 a~i a•r>- o 

O ~ C N N 7~ 

U D C1 L 

L .0 U 

6a1 

z r,av 

Rf •rac a 

d C 

o•.- 

C•~ 

rr > O 

z•- 

Gl 

~ 

Gl O 

zz n ro o 

u U O 

O 

~ 

L 

L >> Y" 

L n' 'O L t cu

.r 

7 O L \ 1-) dJ L 

O 1p L L .L L d• LQ E L L U 

W O M M d' 'd' r-I N 

nri a 

4-3 

L •fl 7 

f[J 

~ 

N '0 

L N M ~ U Z y Z Z U ~ 

a ZE 

0 0 0 

1 

O G. 1n t19 L2'! In 

roro - o 0 0 0. i o 0 0 0 0 

cL 

N c dp O GI cn tp LO n 

C ~ 

LO '••i 

rM-1 ° M 1 

~ S Ol ~-- 1 

N 

n CO 

n 4o °1 n r-1 1- ri ~ Cl 00 Gl c1 °l n 

9-122 
~ N 

7 

C 

+1 

C 

O U v 

W U 

C 

Q 

W J 

t~ 

CI 

Z 

6 

z 

0 

Ft/f 

d 

w c 

~ 

Z 

2 V ' 

W 

Z 

W 

w 

w 

C 

N 

x z 

0 

W 

Z 

O 

O 

lJ.. 

O 

H 

LLI 

V 

6L. 

4. w 

~ 

r-1 

G1 

J 

m 

cr 

1~ 

~ 

u ar w 

w 

W 

'O 

d 

> L 

ar 

I .0 

0 

O 

U 

4J 

O S. 

s n 7 17 Vl C - oaro 

W O 

+1 

i0 

L 

7 

~ 

~ 

C N 'O 

N L 

i +l 

v~i E 

ro 

a 

~ 

E 

O C 

d m C i 

N C O V 

C E O U = 

ar 

U 

C 

N 

i N 

V~- 

~ 

N 

U 

G1 

N 

a 

N 

n 

r

a

i ~h ~ 

rl rl 00 ~ 

. .. Qr•Il cn C - 1.4 

• • ro ,O 

N G1 ~ 0 N O 

a L iOl Cl C N •E . 

d 

R1 

c.o o i ui. •ui c..7 3 m m 

a~ 

O 

O. 

'O 

L 

IV 

g K S C E ~ .O 

to i 

~ 

7 

O 

C 

II 

H H 

Gl ]C 

N fG 

~ 

U Z 

O N U 

3 ro v ~- o +Lo s>. '

C c0 L`Oj) r~ y W O O. 4J 

~ .L.'O~ c ~ O O 

~^ ~ S. c 7, af0 U L 

°a ~ 7 a Os ~ c ro °' 

s

i ~ 

v i ai4-

~ 4- + u aroi ~ 

"

I U r O ro f0 u L ro u u 

G1 V r 

Et! 

t0 > 

b 

~ 

U~tl L. 

C r GJ 

Of 

C 

>> 

S. 

> d iJ 

+3 a1 ++ -- o o.c 7 ro •" al 

rt7 7 L > V •U ~> ~ r 

r 

V E 

. a'p 3 ~ Q7 •L C U 

m w 

C L. O 

wO r 

ai 

41 

ai re 

t L 

ro 

C! 

L) 

O 

d•- 

U C 

O a ~ d U u ++ v 

U VN ~1 

x ~, o ra at a+ •~ 3 ai > 

~ y y U•r .G L. ~ 3 

L L ro 

E C•- 

GU •E Of l c L C! •rU- ~ ~ 

•L 

'O 

W~ rt3 U i C! 'O Ol CJ 

b ~ 

01 

f

>

0 

7 

.. ~a +- u i ro a

tq

i •" +°~' ~ 3 CI 

O°~ ++iro a °ai o us c a~ aEi o 

zuro o ~ ~ incorn m c 

u

i 

N ~ A 

t0 N 

G N 

C7 to ? 

I

4-

O •i. uf L 11 0 

OJ >f. L U 

L U 'O eh L L L = S. 

~ 

U 

~ a r~ d• en ~r 

~ •e 

~ II 

a S 

•L 

V! L 

V ~ 

a1 E 

Y C FJ 

Z ~ 

~ u~ 

N 

• 

~ 4J 

~ ~ t E 

rl r'I y ~ . ei 

E Y 

L II 7 

w 1-- 

r^i : ^e'M M i~ T ~e+1 S ~ 

r-4 r-1 r-1 ~i ~ ••1 N ~0 

9-123 
pig. The results from the guinea pig studies showed a reduced clearance of 

viable bacteria. The rabbit's lung cl,earance rate was not affected by 0 3 . 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 3 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 monodisperse polystyrene latex spheres were followed after 0 3 exposure. A 4-hr 

exposure to 1568 pg/m 3 (0.8 ppm) of 03 significantly slowed the early (tracheobronchial) 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 (SO4 ) 2 , H2SO4 , (NH4 ) 2SO4 ] 

gave clearance rates very similar to those for 0 3 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 3 

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 

pg/m3 (1.2 ppm) of 03 . This exposure caused a substantial delay in rapid 

(mucociliary) clearance in the rat. However, if the animals were exposed 3 

days earlier to 1568 pg/m 3 (0.8 ppm) of 03 for 4 hr, the pre-exposure eliminated 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 rechallenging with a higher concentration of 0 3 appeared to afford complete protection at 3 days. After a 13-day interval between the pre-exposure and the 

challenges, this adaptation or tolerance was lost. 

9-124 
These results were confirmed when Kenoyer et al. (1981) repeated these 

studies, with three different concentrations of 0 3 , 784, 1568, and 1960 pgim3 

(0.4, 0.8, and 1.0 ppm). At each of the three concentrations, a delay was observed in early (0 to 50 hr postdeposition of particles) clearance, and an 

acceleration was seen in long-term (50 to 300 hr postdeposition) clearance, as 

compared to controls. Concentration-response curves showed that clearance was 

affected more by the higher concentrations of 0 3 . 

The velocity of the tracheal mucus of sheep was not significantly altered 

from a baseline value of 14.1 mm/min after a 2-hr exposure to 980 pg/m 3 (0.5 ppm) 

of 03 (Abraham et al.., 1980). The authors state that 1960 pg/m 3 (1 ppm) of 0 3 

for 2 hr did significantly reduce, both immediately and 2 hr postexposure, the 

tracheal mucus velocity. 

9.3.4.2 Alveolar Macrophages. Within the gaseous exchange region of the 

lung, the first line of defense against microorganisms and nonviable insoluble 

particles is the resident population of alveolar macrophages. These cells are 

responsible for a variety of important activities, including detoxification 

and removal of inhaled particles, maintenance of pulmonary sterility, and 

interaction with lymphoid cells for immunological protection. In addition, 

macrophages act as scavengers by removing cellular debris. To adequately 

fulfill their purpose, these defense cells must maintain active mobi1ity, a 

high degree of phagocytic activity, an integrated membrane structure, and a 

well-developed and functioning enzyme system. Table 9-12 il1ustrates the 

effects of 0 3 on the alveolar macrophage (AM). 

Under normal conditions, the number of free AMs located in the alveoli is 

relatively constant when measured by lavage (Brain et al., 1977, 1978). 

Initially, 0 3 , through its cytolytic action that is probably mediated through 

its action on the cell membrane, significantly reduces the total number of 

these defense cells immediately after exposure (Coffin and Gardner, 1972b). 

The host responds with an immediate influx of cells to aid the lung in 

combating this assault. Little is known about the mechanisms of action that 

stimulate this migration or about the fate of these immigrant cells. The 

source of these new cells may be either (1) the influx of interstitial macrophages, (2) the proliferation of interstitial macrophagic precursors with 

subsequent migration of the progeny into the air space, (3) migration of blood 

monocytes, or (4) division of free AMs. The rapid increase in the number of 

macrophages is evidently a biphasic response, arising from an early phase 

9,125 
H 

Z 

O 

~ 

W 

J 

Q 

W 

0 a: Qc~ 

S 

~ 

N 

~ 

W 

X 

W .

cn~' W 

u.. 

6 

c~n 

s 

4 i W_ Z 

0 

W O 

tN 

13 4W. W W 

W 

-1 

1-2 

a 

n 

~ n H ~ to ~ 

^O1 'i 

~-

a%i o r1 

r°. n n 

00 

l0 aii 'i ^ r-I 

: 

t~ - rn rn 

~ ~ 

rn Orn c - Cm 

a ~a _~ 

in io n r- w - .~ ro 

0 L. ro ~ a ~ i+~ • f. 0.7 ro ro f0 fd 

~ai to 

wa•• 

~ai t 41 

c fO o ++ 4.+ 4-3 +~ c c .N 

C L ~ t~„ c 

y ~ 

a a a N ro ~ a 

C a 3 L iJ 41 ^ L Cal C C ~ Y 

a N N a a V- 4- 'O • C L N c a S. L. 3 G. 'O 4- 4- fo 

ca 

f0 7++ c3 o cn 0 7 O 1n 00 

cw 

o 

ca 

_^ 

ni 

d .0 •r}7. ~ 4-3 ++ 41 4-3 +J a 

a. .'°a e+j a 

N 

(a 

C 

~ 

u a c 

f~0 Ca.i 

v,. 

~ 

a 

N ~ ~ ~ o 0 ~ 

> c 

~ ~ 

a 

~ ~ c a o 

CJ ~ to

C N C ro C ~ ay a 

~ •~ e ta

R tL.l 

~ 

I 

:*b .0 z 

V1 D 

a f~0 

N e- dJ U ~ N 

1O N C ~ ~ 

'' q. 

~ ro 

IA N 

~fC 

f

W

0. a ~ L YI C C. ~ 4- ro ~ O. G .0 O. r-~ . ~ pl •~ fa. 

a 

~ 'C 

~4t 

S. O +a.> >, 

a 

o~u 

~O) } 

V-• S. a:~ u O 

~ 

Q/ 

~^ 

V- — . 

o 

L 

ro ayi 

~ - 

v- i 1 

ro 

~o 

p 

+, ro 

C 

~ 

o 

N 

~tl a'a t1 i- a 4.~i 

o a r~o m ~ >" 

~, 'c o 

>, 

d~ w ro ~.~+ u o 

L°- ~ui ~ 

>. ~O N 3 a 

c 

a 

y 

i Vl .O 

~01 c 

~~~ 

tloS T7 e~ 

ro ~0 •~ c 

+j C i •O 'O Cl 'C7 'O iE ro o•.- ~ i N C a— N o a N N rp1 a N a N Vl ai m N.c a N a> N 10 a N c.roo toc roa i m roz ma. tv (ac ro a - a Fi a E CL a a Z a O a a ro a 

c iA ro 6i o ii N 17 d.l di n V v L. 

cii G cii 

J•.C- y.~ N V O a r0 N. G7 O C E q q tJ 

~-Ci 

a 

a O O 

N~ L C ~ ~ . 

a•rI 

ro G ~ ~ 

C. C 

t~ 0 > i1 •>I•>I 

f~0 

+1 

 >I 

~ N 

\ >> 

O tp O t Zf L CI I LV 

ro N cn c0 I~ e'7 v 00 1, M [7 M N V N M 

L 

'O 

.O 

ro 4- 

mv C

Lft

! .c ]C qN ]C ]C ]C ]G W 

~C 

]~G 

C=.7 

A 

~ 

c tn O 

O~ rl O N N t19 ttY t!~ t19 L9 t^p 

a ro a ori o 00 0 0 0 00 

L9 0 ~ 

0! o 

C i 

O ~1 

N C 

0 d 

U 

C rz o ll 

tl CJ tDO N O O O O O O m O O CO 

~ Ql l0 Ol Ql CO W CU W OO rl Oo l0 tf9 rl ~ t'7 st 01 Ol Ol 01 

G1 r-1 ~ ~r-i H 

V ~ 
ai 

C 

i 

w 01 

~ 

~ V N 7 

C 

i-~ 

C 

O 

V ..+ 

~ 

Z 

O 

LLJ 

,•-~ 

Q C 

H 

J 

LLJ 

$ 

¢ 

2 

C 

F 

N 

z N 

1•-• 

Z 

¢ 

2 

u w 

~ 

W 

W 

w 

c 

I-- 

0 

x 

z 

CD 

LLJ 

Z 

N 

O 

LL I N I 

N 

U 

W 

LL LL w 

~ 

1 

rn 

w 

J m 

H 

O 

u 

~ 

L L 

7 C N 

X rCtl w 

C 

O 

ro L 

v' 

L 

ro +-1 

d 'O 

N t 

~ 

ro E 

~ 

O 6 +~ C 

C L 

N C O G7 

c E 

u 

z 

V 

~ 

u 

+ w 

G1 

~ 

0) > 

i 

d 

m 

O 

N 

N • U 

Gl 

G. 

Vf 

01 

n 

Q1 

~ 

c 

ro 

4J 

~ 

L 

U 

V 

c 

ro 

N 

~ 

i 

3 L 

U N 

N 

Y 

C ' 0 

z 

N 

00 

Ol 

r-1 

N 

N 

i 

3 C 

U 

N 

V 

C 

ro 

c 

ro 

E 

~ 

~ • s 

L 

v 

iJ 

~ 

0 

m ~ '•1 

ro 

m 

~ Of 

c 

.0 

N 

Y 

a 

a 

ro 

z 

~ 

ao 

~ 

t0 

4J ai 

C 

~ ¢ci 

E 

iY to m 

N 

a ~ O 

a rCD m 

a 

O 

S. 

U 

ro 

E 

rn 

C . 

•r -0 

~ ro Of L 

E ~ 4- E ° u 

a ~ 

7 r 

cv 

ro~ uai c 

oro 

I O 

ro 

a 

~ 

~ 

co 

O 

~ 

~ 

w 

w 

~ 

U 

O 

N 

L I a' 

'Q d w m ro 

s. 

U 

c 

M 

A ro 

~ 

N 

~ 

M FN 

~ 

co 

O 

co Ln un r•1 

c 0 L 

G1 

4- 

L a0+ 

~ 

a U 

v' 

~ 

O. 

O 4J 

~ . ro 

~ o1 

ml ~ 

c ii ~ 

o r ~ 

L 

L 

M 

Z w x 

U 

4 Ln 

rn~ 

r+ orn 

9-7 

N 

d 

ro L 

~ o 

~ u 

r (tl E 

f6 0 

~ 

0 cc rn ­1 

.27 

c as) 0.0 

~ L. - oP ~ 

E ro 

LIL iJ U 

d 

•> ~ 

Al~ 

vv4-) 

~ •00 E 

C 4) +1 

i L _ 

~ 

~ 

~o ~ 

~ 

ro 

+~ a 

E 

L ro L 

a 

H 

0J 

Uf 

7 0 E 

C 

O 

N 

~ r 

L) ° aroi 

~ L L 

L •

C-• b 

~ 41 a 

4- 7 C 

L a O 

G! L 

c ai v°1- 

~ Q1 L ro d O L+3 

S_ O >Iro o r s 

c E cr 

C ro ~ U ° oc~ir 

+J d 7 47 

d w ro n.t 

°1+L~ m ~ 

z 3 4-3ai n 

w 

ro V 

~ 

c 

Z 

C1 

O 

m 

m uO r-I 

~ 

~ 

m 

ro 

+J d 

C 

f0 

L 

O 

O 

m 

~ 

~ 

rt 

1A . 

cv 

n 

S. OOl 

E N 

4-4

4-)

- 

o m 

i 6 

G1

. 

 r 

7 L C 7 

cw 

ar +°~ 

L 

u~ ' -0 

a cu 

O ~ 

s ro rn •° 

wrI N 

ro V 

O 

rn 

0 

z 

C6 

O 

~ 

~ 

~ m 

~ ~ ~ Ol 

t6 ~ 

d ro 

uf G! 

L N 

E •+~- 

¢ 3 

Y 

m 

C 

O 

4-) u 

~ v 

~ 

O. 

to 

V 

ro 

L 

c 0 ._ 

ro 

d 

v 

0 i N 

N 

V 

Cl 

Vl 

ro m 

v a) 0 

L L 

M 

i-°~ 

L. 

L 

:. 

Y 

z 

.iJ 

t0 Lt1 

ri M I -4M ~ 

a 

00 

n 

~ 

ro 

iJ 

ar 

c .~ +3 

ul a 

O ta 

4-) (c 

C 

G) O 

Lo 

Y ~ 

.0 to L c. 

o a 

G. N 

G i U 1VO 0 

~ ... 

ro L. 

d C1 

H 41 

ro0 ~ . 

CU 

a c 

•a ~ o 

7 .F1 

~A 

u N cm 

ac ~ ~L ro 

4- .F1 •r 0.5 w +0+ Y1 ~ u .°~ aa 

L 

Ln 

Z 

t19 

N 

0 0 

~ 

r-1 

co 

01 

r-1 

C 

.~ 

4J 

'O 

0 

U 

v 

c 

ro 

L 

E 

Nd 44 

ro 

z 

~ 0 u U 

cc 

I Ovi 0. O 

i L 

C V0 

w 

Of V L 

~ °u . r o 3 

tn 12 L W 

rroro fO 1.3 L •L NO +°.) ro csi 

~~ E ar 

C! L 

OE s%^ o 

-o 4- >, L O r 

Ni O N c au o. 

E E u 7 

~ ror 

c cr u 

L 

Ln 

, z 

LL9 IN 

0 0 

m 

O1 ~ 

a 0 

~ • N 

to 

i~ 

O 

G 

a 

GI 

L 

G1 

w 

W 

7 a 

ro 

L 

4J 

7 

N 

C 

11 

Y m 

z 

~ 

E Of 

~ L 

G O 

p, L 

u 

~ E 

u ~ 

~ 0 

> 

u ro t0,7 L 

d c C E 

~ C. 

~ ~ 11 sci s: ~' ~7 ~ 

C7 Yf O 0. 

L n E C1 

°' a •' a Yl E V/ U 

vi 

a1 ~ 10 Y 

O 4J O 

nu a 

2 u W ro 

U 

v 11 4- o 

36. a o L i a U rO L 

d a %- E v 4J ~ >, 

aJ E c o O L1 c y 11 11 

11 $ Y Z °z v z a 

u 

L 

'O +y~ ~ 'Q 

L E L 1

0» 

V O 4J O 

E 7 E Vl 

}J u C O 

C 

~1yO 

E Y 41 ~N 

to3 II .0 • 01 

tn 

c0 N r a 

toz a u¢ 
apparently correlated to a local cellular response and a later phase of interstitial 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 3 

can result in a macrophage influx in several animal species. Exposure of mice 

to 1058 Ng/m3 (0.54 ppm) of 03 23 hr/day for a maximum of 34 days resulted in 

an increased number of macrophages within the proximal alveolar ducts (Zitnik 

et al., 1978). These cells were highly vacuolated and contained many secondary 

4 

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 to 1568 pg/m 3 (0.8 ppm) 

of 03 for 7 days, but only a fourfold increase after exposure for 90 days 

(Brummer et al., 1977; Boorman et al., 1977). In similar studies, other 

authors found that exposure of both monkeys and rats to concentrations as low 

as 392 pg/m3 (0.2 ppm) of 03 for 8 hr/day on 7 consecutive days resulted in an 

accumulation of macrophages in the lungs of these exposed animals (Castleman 

et al., 1977; Dungworth, 1976; Stephens et al., 1976). The data from these 

studies suggest that these two species of animals are approximately equal in 

susceptibility to the short-term effects of 0 3 . 

Thus, the total available data would indicate that, after short periods 

of 03 insult, there is a significant reduction in the number of free macrophages 

available for pulmonary defense, and that these macrophages are more fragile, 

are less phagocytic, and have decreased enzymatic activity (Dowell et al., 

1970; Coffin et al., 1968; Coffin and Gardner, 1972b; Hurst et al., 1970). 

However, histological studies have reported that with longer exposure periods, 

there is an influx and accumulation of macrophages within the airways. Such a 

marked accumulation of macrophages within alveoli may appear to be a reasonable 

response to the immediate insult, but it has been speculated that the consequences of this mass recruitment may also be instrumental in the development 

of future pulmonary disease due to the release of proteolytic enzymes by the 

AMs (Brain, 1980; Menzel et al., 1983). 

A number of integrated steps are involved in phagocytosis processes, the 

first being the ability of the macrophage to migrate to the foreign substance 

on stimulus. McAllen et al. (1981) studied the effects of 1960 pg/m 3 (1.0 

ppm) of 03 for 4 hr on the migration rate of AMs. Migration was measured by 

9-128 
determining the area macrophages could clear of gold-colloid particles that 

had been previously precipitated onto cover sl.ips. In this study, it was not 

clear whether the gold was actually ingested or merely adhered to the outer 

surface of the cell. Nevertheless, the cells from 0 3-exposed rats appeared 

less mobile, in that they migrated 50 percent less than the sham-exposed 

group. 

A decrease in the ability of macrophages to phagocytize bacteria after 

exposure to concentrations as low as 980 pg/m 3 (0.5 ppm) of 0 3 for 3 hr was 

demonstrated by Coffin et al. (1968) using. rabbits. However, Christman and 

Schwartz (1982) may have demonstrated that with longer exposure periods, the 

effects may be different (i.e., the phagocytic rate may increase). In this 

study, rats were exposed to 1568 pg/m 3 (0.8 ppm) of 0 3 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 enhancement correlated well with a significant increase in cell spreading 

of AMs from exposed rats as compared to controls. If longer-term 0 3 exposure 

enhances macrophage function and causes a migration of macrophages into the 

lung, the comparisons of function of these new cells with controls may not be 

valid, because these new cells are biochemically younger. Another significant 

problem with this study is that the cells examined were a selected population 

because only the cells that adhered to the glass surface were available for 

study. The cells that did not adhere were removed by washing. In this study, 

only 51 percent of the collected cells adhered.after 3 days of 0 3 exposure, 

compared to 85 percent of the controls. -Aithough the effects of 0 3 on cell 

attachment have not been studied directly, there is evidence that 0 3 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 3 exposure might never have been 

tested, because they were discarded. 

Gol dstei n et al .(1977) studi ed 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 

exposure to 980 or 1960 pg/m3 (0.5 or 1.0 ppm) of 0 3 . A decrease in concanavalinA agglutinability of trypsinized red blood cells obtained from rats exposed 

for 2 hr to 1960 pg/m3 (1 ppm) was also noted. Hadley et al. (1977) investigated 

AM membrane receptors from rabbits exposed to 980 pg/m 3 (0.5 ppm) of 0 3 for 

3 hr. Following 0 3 exposure, lectin-treated AMs have increased rosette formation with rabbit red blood cells. The authors hypothesized that the 0 3-induced 

response indicates alterations of macrophage membrane receptors for the wheat 

4 

germ agglutinin that may lead to changes in the recognitive ability of the 

cell. 

Ehrlich et al. (1979) studied the effects of 0 3 and NO 2 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 pg/m 3 (0.1 ppm) of 0 3 and 

0.5 ppm of NO2 (3 hr/day, 5 days/week) did viability in macrophages decrease 

significantly. In vitro phagocytic activity was also not affected by a 1-month 

exposure to this level of pollutants, but after 2- and 3-month exposures the 

percentage of macrophages that had phagocytic activity decreased significantly. 

It has been reported that the acellular fluid that lines the lungs also 

plays an important role in defense of the lung through its interaction with 

pulmonary macrophages (Gardner et al., 1971; Gardner and Graham, 1977). These 

studies demonstrated that the protective components of this acellular fluid 

can be inactivated by a 2.5-hr exposure to 0 3 levels as low as 196 pg/m 3 

(0.1 ppm). When normal AM's are placed in fluid lavaged from 0 3-exposed 

animals, they showed an increase in lysis (10 percent over control). A similar 

effect was seen when normal AM's were placed in protective fluid that had been 

exposed in yitro to 03 . The data indicate that some of the effects of 0 3 on 

lung cells may be mediated through this lung lining fluid. Schwartz and 

Christman (1979) provided evidence that normal lung lining material enhanced 

macrophage migration, but the macrophages obtained from rhesus monkeys after 

exposure for 7 days to 1568 pg/m3 (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 0 3-exposed macrophages did enhance the migration, but 

it was still significantly less than controls. 

Macrophages are rich in lysosomal enzymes. Because these enzymes are 

crucial in the functioning of the macrophage, perturbation of the metabolic 

9-130 
or enzymatic mechanisms of these cells may have important consequences on the 

abilities of the lung to defend itself against disease. Enzymes that have 

been identified include acid phosphatase, acid ribonuclease, beta galactosidase, 

beta glucuronidase, cytochrome oxidase, lipase, lysozyme, and protease. Ozone 

decreased significantly the activity levels of lysozyme, P-glucuronidase, and 

acid phosphatase in macrophages after a 3-hr exposure of rabbits to concentrations as low as 490 pg/m 3 (0.25 ppm) of 03 (Hurst et al., 1970). Such enzymatic 

reductions were also observed in AMs exposed in vitro (Hurst and Coffin, 1971). 

The ability of 0 3 to alter macrophages' enzyme activity was also studied by 

means of unilateral lung exposure of rabbits (Alpert et aT., 1971b). A significant reduction in these same three intracellular enzymes was found to be 

specific to the lung that breathed 0 3 rather than a generalized systemic response. These effects were concentration-related, beginning at 980 pg/m3 (0.5 

ppm) of 03 . The extracellular release of such enzymes may occur either as a 

result of direct cytotoxic damage and leakage of intracelluiar contents, or 

they may be selectively released without any cell injury. Hurst and Coffin (1971) 

showed that the reduction in intracellular lysosomal enzyme activity observed 

after in vitro exposure coincides with the release of the enzyme into the surrounding medium. In these studies, the sum of the intra- plus extracellular 

enzyme activity did not equal the total activity, indicating that the pollutant 

itself can inactivate the hydrolytic enzyme as well as alter the cell membrane. Recently, Witz et al. (1983) and Amoruso et al. (1981) reported that 

in vivo 03 exposure affected the production of superoxide anion radicals (0 2 ) 

by rat AMs. This oxygen radical is important in antibacterial activity. 

Exposure to concentrations above 3136 pg/m 3 (1.6 ppm) of 0 3 for 2 hr appears 

to result in a progressive decrease in 02 production. No statistical evaluation 

of the data was performed. The type of inembrane damage as well as the mechanisms by which this damage is incurred are not well understood. It is not 

known .whether the 0 3-induced inhibition of 02..,production arises from the 

direct oxidative damage of the membrane enzyme involved in the metabolism of 

02 to 0 29 or whether it is a result of oxidative degradation of inembrane 

lipids that may serve a cofactor function. 

Shingu et al. (1980) reported the effects of 0 3 on the ability of two 

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 pg/m 3 (1.0 ppm ) of 0 3 for 3 hr exhibited a depression in interferon production. Interferon production by 

9-131 
tonsillar lymphocytes was not significantly depressed by 9800 pg/m 3 (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 i.nf­tiops. In neither of the studies were statistical analyses of the 

data reported. Ibrahim et al. (1976) also exposed mice to 0 3 and illustrated 

that 1568 pg/m3 (0.8 ppm) of 03 for a period of 11 days inhibited the invitro 

ability of tracheal epithelial cells to produce interferon, but no effect was 

observed with alveolar macrophages. 

9.3.4.3Interaction with Infectious Agents . In general, the consequences of 

any toxic response depend on the particular cell or organ affected, the severity 

of the damage, and the capability of the impaired cells or tissue to recover 

from the assault. , Do small decrements in the functioning of these various 

host defense mechanisms compromise the host so that it is unable to defend 

itself against a wide variety of opportunistic pathogens? It has been suggested from epidemiological data that exposure to ambient levels of oxidants can 

enhance the development of respiratory infection in humans (Dohan et al., 

1962; Thompson et al., 1970). Measurement of the competency of the host's 

antimicrobial mechanisms can best be tested by challenging both the toxicexposed animals and the clean-air exposed control animals to an aerosol of 

viable microorganisms. If the test substance, such as 0 3 , had any adverse 

influence on the efficiency of any of the host's many protective mechanisms 

(i.e., physical clearance via the mucociliary escalator, biocidal activity 

mediated through macrophages, and associated cellular and humoral immunological 

events) that would normally function in defense against a microbe, the microbe, 

in its attempt to survive, would take advantage of these weaknesses. A detailed 

description of the infectivity model" commonly used for 03 studies has been 

published elsewhere (Coffin and Gardner, 1972b; Ehrlich et al., 1979; Gardner, 

1982a). Briefly, animals are randomly selected to be exposed to either clean 

air or 03 . After the exposure ends, the animals from both chambers are combined 

and exposed to an aerosol of viable microorganisms. The vast majority of 

these studies have been conducted with Streptococcussp. At the termination 

of this 15- to 20-min exposure, the animals are housed in clean air, and the 

rate of mortality in the two groups is determined during a 15-day holding 

period. In this system, the concentrations of 03 used do not cause any mortality. The mortality in the control group (clean air plus exposure to the 

microorganism) is approximately 10 to 20 percent and reflects the natural 

9-132 
resistance of the host to the infectious agent. The difference in mortality 

between the 0 3-exposure group and the controls is concentration-related (Gardner, 

1982a). No studies have yet been conducted to determine the,lowest possible 

number of viable microbes that when deposited in the lung will cause a pulmonary infection in the host exposed to 03 . Miller et al. (1978a) examined the 

cumulative data from nearly 3,000 control mice used in these infectivity 

studies and determined that within the range of 200 to 4,000 colony-forming 

units per lung, there is no correlation between number of bacteria deposited 

in the lung and the resulting increase in percent mortality. It must be noted 

that in these studies, the control animals are capable of maintaining pulmonary 

sterility by inactivating the microbes that are deposited in the lung. 

If the test agent does not impair the host's defense mechanisms, there is 

a rapid inactivation of inhaled microorganisms that have been deposited in the 

respiratory system. However, if the chemical exposure alters the ability of 

these defense cells to function, i.e., rate of bacterial killing, the number 

of microbes in the lungs could increase (Coffin and Gardner, 1972b; Miller et 

al., 1978; Gardner, 1982a). This acceleration in bacterial growth has been 

attributed to the pollutant's alteration of the capability of the lung to 

destroy the inhaled bacteria, thus permitting those with pathogenic potential 

to multiply and produce respiratory pneumonia. With this accelerating growth, 

there is an invasion of the blood, and death has been predicted from a positive 

blood culture (Coffin and Gardner, 1972b). 

Coffin et al. (1967) treated mice with 0 3 (0.08 ppm and higher levels for 

3 hr) arid sub.sequently exposed them to an aerosol of infectious Streptococcus 

sp. In this study, 0 3 increased the animals' susceptibility to infection, 

resulting in a significant increase in mortality rate in the 0 3 -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-1 mice and 

Streptococcus sp., Miiler et al. (1978) studied the effects of a 3-hr exposure 

to 03 at 196 pg/m3 (0.1 ppm) in which the bacterial aerosol was administered 

either immediately or 2, 4, or 6 hr after cessation of the 0 3 exposure. For 

these postexposure challenges, only the 2-hr time resulted in a significant 

increase in mortality (6.7 percent) over controls. However, when the animals 

were infected with streptococci during the actual 0 3 exposure, a significant 

increase in mortality of 21 percent was observed. When this latter experimental 

regimen was used, exposure to 157 pg/m3 (0.08 ppm) of 0 3 resulted in a significant increase in mortality of 5.4 percent. 

9-133. 
The differences in results among these studies may have been due to a 

variation in the sensitivity of the method of 03 monitoring, a difference in 

mouse stra ~', n chan es in the atho enicit of the bact ' g p g ~ y eri a, o`r' 'di fferences i n 

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 3-treatment group were significantly less 

effective in combating the infectious agent to the extent that, even at low 

concentrations, there were significant increases in mortality over control,s. 

As the 03 concentration increased, mortality increased. In some studies, 

additive effects were reported. These effects, increases in respiratory 

infection, are supporteci by many mechanistic studies discussed in this 

section. They indicate that 0 3 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 ttudies have continued to 

confirm these previous findings and improve the existing data base (Table 

9-13). 

When mice were exposed 4 hr to 392 to 1372 pg/m3 (0.2 to 0.7 ppm) of 0 3 

and then challenged with virulent Klebsiella pneumoniae, a significant increase i n mortal i ty was noted at 785 pg/m 3(0.4 ppin) 03'(Bergers et al ., 

1983). Groups of 30 mice inhaled approximately 30, 100, and 300 bacteria/ 

mouse. At 392 Ng/m (0.2 ppm) of 0 3 3 , the 03group showed' an i ncrease i n 

mortality, but it was not significantly different from controls. At 785 pg/m 3 

(0.4 ppm) of 03 a nearly threefold lowering of the bacterial LD 50 value computed from the three challenge doses of bacteria was found for the 0 3-exposed 

group, indicating a significant increase in mortality. In the same study, the 

authors al so found that 1372 pg/m 3(0.7 ppm) of 0 3for 3 hr 'resul ted i n a 

decreased ability of the lung to clear (bactericidal) inhaled staphylococci. 

9-134 
n 

t0 

(n 

ro 41 a) 

C 

4= 

0 

U 

4) 

7 

0 

tn 

a 

7 u u 0 u 

O +J , 

L ' 

4J N 

O ~ 

4-11 

ro 

L 

O e .c 

C1 

m 

u 

u 

I •~ 

ro 

oo 

n 

rt 

Gl 

tn 

i 

~ 

f 

61 

7 

0 a 

O 

01 

c 

L 

~ v 

Co 

~ 

}1 /\ 

O 0 

E uf 

r N ~ 

U 

m uo I 

cu cii 

i' 

. r_ O y S.. 

C v 

U ~ 

w ~ 

a 

~ c o 

O7 O. 

N OXJ 

n n r

4ml 

ro 

41 Gl 

L 

U 

i 

w 

N 

7 0 a 

yl 

U u 

O 

u 0 

~ SO.. 

41 

N 

0 41 4, ~%1 

0 

E 

~ 

Gl 

m 

i u 

H 

L 

M 

C1 

Z 

O 

O 

~ 

ri 

i 

L 

M - 

W 

S 

V 

r-1 

O 

O 

O 

O 

t0 

rl 1~ 

Ln 

L 

L . 

M 

W 

2 

U 

r-I 

O 

t0 rNi 

O 

co rm

i 

ib 

+) ai 

cm 

4: 

ca 

M 

N 

7 

E 

° 

~ ~0 N 

E w 

C O 

% E 

N41' 

N ~ aJ 

L Cul 

W - 

L 

L 

M 

Z 

S U 

M 

O ~ -1 

O 

~ 

lf•f 

t0 

~ 

0 n 

~ 

L 

~ 0 

m 

a 

C 

m 

C 

w 

W 0 

U 

Cl 

tn 

7 

Z 

C 0 

i u 

G! 

4- 

c 

~ 

4-7 

.c r: 

C. O t07 0 

~ u 

OO 

Vl 4.1 'a 

U +J C 1N 

a 

r-1 

~ 

r-I 

ro 

61 

W ro 

E 

L 

H 

G1 

~ 

Z 

N 

L 

d 

~ 

4-) 

orn C 

~ 

N'. • N'. 

0 

~ 

U 

i 

a 

.0 

4-) 

E 

~ • L 

G1 ~ u ro a O Z

> 

rG! 

ro 

4) L 

U 

c 

1•H 

~ 

~ 

-f 

m 

4J 

v 

'O 0 O 

~ 

L 

N 4•,1 

cc 

ro 

O 

c 

u u 

L ro u ~ 

o a 

4>)' a 

d L vl 

Gl C. , > 

L 3 

~ c 

o .N u 

C v0- 

cu 

E rC 

u~ 

L C 

N E 

Z G. 

Y 

3 

~ 

S 

2 

6.i 

~ 

O 

~ N 

H 

N I 

Z 

W 

C7 

Q 

t/f ~ 0 

H 

CU 

W 

LL 

Z 

x 

F3 

tN Z 

O t-i 

V 

z 

w 

F~- 

z 

N 

N F N 

1-•1 

Q 

x 

w 

~ 

w 

N 

1.Zt.t 

W 

Cf 

N 0 

S 

a 

O 

W 

Z 

N 

O 

1L 

O 

U 

W 

LL 

W 

M 

r-1 

~ Ol 

W 

J 

m 

H 

C! v cai 

S. 

4- 

d 

N 

U 

01 a tN 

~ 

v 

4) W 

4-

N 

b a! > 

i 

O1 

N 

a 

O 

O U 

O 

4J 0 

L a 

7 II 

V1_ C o m 

W O 

~ ro L 

~ 

'O 

ro 

C N 'O 

N L L 1.~ 7 N 

N ~ 

ro 

N 

f 

O E +~ G 

v ro C i 

N C 

O OJ 

C Z 

u Of 

1 

M 

~ 

rr 

m 

+) ro 

C 

L 

Q 

01 

7 

s 

c 

O 

4J 

U 

W 

C 

ro ~ L 

~ 

ro .~ 

O 

41 

> 

.n .~ 

aw 

u 1A 

In

u 

in O 

y ~ I 

t0 ai v L L 

C N 

M \I 

S. 

W 

~ a ro 

L 'O Oen 

NN ri 

_ 

~ 

O 

t0 r

(n

i 

M 

Cf3 t~~i 

ro 

a-) G1 

w 

f. 

m 

L 

m 

ul 61 

~ 

~ 

ro •C 

C E 

7 

• O C 

4- ~ 

=a 

4~ r LOY 

E ~ .C- E 

•G7 N d at0i ai -41 

L f0 O 

.c+J 

r bJ M 

++ 3 c 

U Cf /01l 

v= aci a-' - U 

ai ow L4- 

N U W 

i L 

d 

I 

M 

cz 

,^ 

O 

N 

O 

N n 

ri 

N 

M 

N 

O 

~ 

ro 

+-) G1 

E ro L 

L 

Q 

C 

Ol 

Gl 

~ 

ro 

C 

E 

7 

N 

C C. 

ro 

L 

N 

ro 

.G 

4- . 

O ~ 

~ ~ 

> O 

in ro 4- L 

o 

u a 

C +J, 

! i L ro' 

C O. w .~ 

L 

O vro 

L fT0 

t ~ 

M N 

G1 

z 

M 

O 

~ 

tCf 

l0 rM-~ 

ro 

++ 

®l 

w 

O 

e 

N 

H 

N 

7 

O 

L 

E 

7 

i a a-) 

ro ~ 

O u 

iC' 

O Fa 

G! u C ro .N 

N y 

L 

O ~ N 

U ~ 40- U 

V- L 

G! G7 

L 

z 4-' 

L 

-Ie C 

ro~ E 

i tT6 N I~ r t2Y 4.. 

CI 

Z 

N 

rr °er Ol 

N 

N O 

a% 

ro 

41 N 

++ ~ 

O 

U 

3 

d 

7 

f 

O1 c ~ .T N 

L L 

dJ ; 

W y 0 

o ro 

U y 

4- 

10 

c c 

L ~ 

•> L 

a ~ 

aro 

p •r 

3 0 C 

0, 

~ 

7 N 

o: O L 

a 

3 

~ 

N 

~ 

uO 

O 

O 

co 01 

i 

Gl 

.J N 

E 

U 

- C ~ o 3 

m 

cmo~ 

N LT• 

i.) v 

E 

O 

0 

L 

a 

> ~ 

!! 

7 

~ 

ci 

U 

c cOJ 

N e .~ 

~E m L 

u 

01 

ro L a N 

ro rn 

il 

Z 

2 

C..1 

~ 

d a • L 

u Vl 

•0 

+J' 

O 

C ! ~ 

CI 

Z 

V 

O 

L 

~ 

E 

~ C 

IS 

L 

~ 

'0 

~ 

L 

L 

M 

O 

Z 

O) 

O 1~ 

O 

d ~O nr-1 

N n 

rr 

Y 

>\ 3 

LeT0 O 

2O M tA F 

Z 

W 

S 

v 

0 

i--i 

0 

N 

~ 

9-135 
This finding is consistent with that of Goldstein et al. (1971b) and confirms 

previous studies reported by Miller and Ehrlich (1958) and Ehrlich (1963). 

Sherwood et al. (1984) established a chronic pulmonary infection in rats 

by inoculating agar beads containing viable Pseudomonas aeruginosa (PAO-381) 

and then exposed these infected animals for 4 wks to 1254 pg/m 3 (0.64 ppm) 0 3 . 

The exposure to ozone did not affect the pulmonary antibacterial defense 

systems--i.e., no increase in number of organisms cultured from the lung--but 

it did cause significant anatomical damages. The lungs of rats exposed to 0 3 

were larger and heavier when compared with controls, and had an increased 

number of macrophages in their terminal bronchioles. The authors state that 

the reason why these results are different than those described above for the 

"infectivity model" is that in these later studies, the infective organisms 

are given by inhalation and are therefore deposited throughout the lung and 

the pollutant was able to interfere with the initial phase of the host-parasite 

interaction. In this chronic study, the infection was isolated to the distal 

area of the lung and it was in its later stage of development when the animals 

received the 03 exposure. Thus, the timing of the exposure to 0 3 may be a 

significant factor in the impairment of the lung's antibacterial defenses. 

Exposure to ambient levels of 0 3 (0.5 ppm) for 2 to 4 wks has been shown 

to alter the pathogenesis of respiratory infection of mice with influenza Avirus (Wolcott et al., 1982). The 0 3-exposed animals showed a reduction in 

severity of the disease (less mortality) and an increase in survival time. 

The reduction of disease severity appears to be dependent on the continuous 

presence of the 0 3 during the infEctious process. This effect did not correlate with virus, interferon, or neutralizing antibody titers recovered from 

the lung, or with neutralizing antibody titers in the-sera. The reduced 

disease severity in the 0 3-exposed animals appears to be due to significant 

alterations in the distribution of viral antigens within the pulmonary tissues, 

i.e., less widespread infection of the lung. 

Chiappino and Vigiani (1982) also reported that 0 3 potentiated pulmonary 

infection in rats. In this study, the investigators wanted.to know in what 

way 03 modified the reactions to silica in specific pathogen-free (SPF) rats. 

Silica-treated animals were exposed to 1960 pg/m (1.0 ppm) 0 3 3 (8 hr/day, 5 

days a week for up to 1 year) and housed in either an SPF environment or in a 

conventional animal house, thus being exposed to the bacterial flora normally 

present there. The SPF-maintained, 0 3-exposed rats showed a complete absence 

9-136 
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 3 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 3 exposure have 

also been tested in sheep. In these studies, sheep were infected by an inoculation of Pasteurella haemolytica either 2 days before being exposed to 588 

pg/m3 (0.3 ppm) of 0 3 or 2 days after the 0 3 exposure. In both cases, the 0 3 

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 3 insult followed the exposure to the bacteria (Abraham et al., 

1982). Unfortunately, only a small number of animals were used in each 0 3 

treatment (n = 3). 

Thomas et al. (1981b) studied the effects of single and multiple exposures to 0 3 on the susceptibility of mice to experimental tuberculosis. 

Multiple exposures to 1960 pg/m 3 (1.0 ppm) of 0 3 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 

03 did not produce any significant effects. In an earlier study, Thienes et 

al. (1965) reported that exposure to 2940 pg/m 3 (1.5 ppm) of 0 3 4 hr/day, 5 

days/week for 2 months also did not alter the resistance of mice to M. 

tuberculosis H37RV, but he did not measure lung titers. 

Table 9-14 summarizes a number of studies that used mixtures of pollutants 

in their exposure regimes. Ehrlich et al. (1979) and Ehrlich (1983) expanded 

the earlier 3-hr-exposure studies to determine the effects of longer periods 

of exposure to 0 3 and NO2 mixtures. In the earlier studies (Ehrlich et al., 

1977), they reported that exposures to mixtures containing 196 to 980 pg/m 3 

(0.1 to 0.5 ppm) of-03 and 2920 to 9400 pg/m 3 (1.5 to 5 ppm) of NO2 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 

up to 6 months to mixtures of 196 pg/m3 (0.1 ppm) of 0 3 and 940 pg/m3 (0.5 

ppm) of NO 2 and challenged with bacterial aerosol. The 1- or 2-month exposure 

9-137 
t/f 

LU 

R' 

~ 

S 

S N 

pq 

~ 

W 

s 

W 

W _ 

tL. 

LU 

Ct 

N 

~ 

2 

O 

W 

Z 

C 

N 

O 

LL. 0 

e W 

LL. 

W 

a' 

r'1 

1 

01 

W 

...1 

m 

F~- 

00 1"1 GO ~ 

~ 

T 

~ 

C

0

1 

~ 1 ~ ri 

m U M r- O ~ 

c ro ao ro io ao ei 

L i3 O 

rn 

1-•1 i0 +3 i0 4J 

rn 

rl 

ro 

W 

w w w 

~ m 

. Gl 

Q1 

L r-1 L 

4-3~ 

Cl L 

+~ 

y L L 

C U U 

1~11 

Ol 

1~11 

U U 

~ Q1 ~ •r 'O •C 

W rOi W C7 G7 C9 W W Q 

N 0) 

C! G1 61 GI GI +) C1 CI G1 

•u 

N 

7 

N 

7 

N 

7 

Vl 

7 

N 

E 

N 

7 

N 

7 

I/N 

7 

a !N i 

N N i r-• iJ iJ 

ro- v'- 

'•• ~-1 u 

u 4- C Vvi Gl 

ovva r L u N 

4.1 •r 

Mi 'C 

tq 

61 

O 

u 

r O 

•C •r {.> •C 

L 

4- 7 O 

N •u Ol C 

+1 •r 4-7 O 

'O 

•Ol ro 

i 

7 

o 

+~ 

01f/ C 

•r U r0 ~ - 

Of 0 +1 

X 

+~ O1 

1+~ 

u i'O Gl a 

Gl Gl Cl m N 

N GJ U •N O `~'r > . 

i 4- u o rt7 L 

L 

C N 

>~ Gl •E 

L 

d.~ 

C C4- 

r 

r•C 

C•r 

r.1••1 r- L 

_r N 

C) 

W tOL+~ 

.G N U 

U! 

1J 

N L 

7 6> +3 

N 

'O >f Ql 

10 

'O Gl 

r L.) 

.G •r 

r'p u 

.O CJ C 

~ 

~ 0 •r 

...'p y•r C N 

u 

fN 

'C N L 

d o ~ 

C 

10 

O }l 0J i•r 

i) ° N 

a 

~ 

f0 3 • Z N tp 

 ro s 

Gl C GJ L E GJ C 

N o N 7 N m 01 

qVN a N 

7 x o 

i~ 

7 >> 

+> 

7 ro 1O 

>> 0/ 

7 L c 

> C 

G1 

a Gl R7 

N L. N 

ro•r (0 +) Y O Cl 

G! .1•~ 01 x 7+~ • 

d 

~ 

f0 Cl a 

(~ o~ 

+~ tA L 

'O Nf to O N•r rC G! 

r N 

U U•- 

N c u 

L u L•r N N+.~ 

~~ u Ol u'E 

~ •2 al L r• ro O•r c4F C r• m 

Oi 

> 

N.- ao a ai D. •O 

o a 

N u ai a o ar ci a Ol N N a N •O c ai r C r-i C a O 

• 

r r r 

~F ~ • L L'O L o ro Cl •• o>> ~ F1 N • c 

Gi 

N 

O~ 01 

+7 r G 

0 

>> 

~ 

a 

7 o 

N C L 

O c V7 

N•r N 

G1 ro 

LL 

N•r ro 

L~ 

C O . c c•r f0 

f0+j M O O 

L 

O 

x 7•r 

•

E E N 

~.+ ++ 

u•r 

Gl > 

Ol 

u 

N 

O•r a 

G 

X 0! N 

O S 

C. c 

X O L 

u O. 

N o 

V i 

+J u ar 

c CI C1 

O 9- 

u o•- •r d v 

• >> ro i~ N c 

4- +~ O • (0 10 

L L. 

0 6/ Gl 

4- •r 

4- i~ 

~ 

N 

Cl N C 

ro o 

0l •r pl 

+3 > 

u 

i•~ Cd 

E C r.F•1 Md d 

~ •c •i--~ o•r L 

+J +J 1J G1 U Cl r• u O c E tO •r >> O)•r 'O L u¢ 

4- 4- 

Of N rt7 

61 

N 4- 

•o 

d ul 

io S. ro ' 

• u 

io 7 

• •O >> 

ro 

u i 

~,.- 

•O 

.a ar L 

•V1 •r N G/ •r .r c 

L 

7 4~ +3 

> C N N 

~O C 

GJ Ol 

+) C i., 

C•r 01 .1~ 

+~ G! +l 

C S. 

• 10 

4- r 

C t0 C 

i0 c ro +~ 7> O >, a.n O L C t~ 

tN u u 

O d d 

i~ •r 

r t L 

41 L u 

7+

, 

 +~ G/ 

C! > 

7 iJ •r 

r O 

•C N 

O u 

M E•r 

r Q! •r d-~ t0 

L U i- C! 3 

Xv~-v~- v ~ c ° m~ dw a~i ro c`

J

i °f> ++ ~ ~ ~~.~ ~~ c= 

W N Cl ¢ 3 -i tN u Z Gl tn u(0 tn f0 ¢ a C 2 u) R1 L Of t0 6. 

O 

U 

C 

L 4-N fTE N a L Y O 

7 17 L 10 C ~\ M " 

u! C } } +J •O O •Q N O 

d ro i L L i L i L O L E L b, 

M M M MN M N M M r~ 

tD 

LO 04- 

+J 

L 

7 

•O • 

14 

C 

ar a 

s°- +°.+ ' m (i 

~ ~ 

c-) c.) v c-) U U c> > 

~ 

c q 

10 V7 

_ •F •F + O t O t O t O ~• i• i• + v ~ 

O 

N 

o70 

NN 

e70 C 

N 

M N 

h 

M N 

N 

c+1 e7 

N 

e+JO 

N 

oJ0 

NS 

MO Z C. OZ OZN O= 02 OS OZ OZ Ot/7v 

~ 

O 

OE C! tn 

4~ a 

O O rl ri r-i ri rl rl 

Gl gO O O O O O O O O 

O •{1 

N C 

O Ci 

U n tD O M 

C E Ol O OD 

O 

U O) 

S 

000 

O7 tD 

M 

rl ~ O 

1 1 C 

T 

tD a' 

Cl 1 

rl ~ 

t00 

Q1 O 

rl °l 

t00 

01 C1 

rl ~ 

t00 

Ol ~' 

rl 01 

t00 

01 s}' 

ri Ql 

tD 00 

Q1 O st 

rl 

G rl ri N M r01 

ri 

9-138 
~ 

V 

N 

7 

C 

~ 

C 

O 

v 

N 

z 

~ 

X 

x 

V• 

r-I 

1 

Ol W J 

m ¢ 

~ 

ro 00 

~ rn ~ 

a 

ai 

u ui - 

C 

01 W 

N 

O 

L i m 

G/ 

. Y- C 

U +-) 

6~ V d 

C • N 

4.1 

C 

ro C 

O ~ N O r ^ N m O01 

C.7 ri 

t0 

J 

L 

d 

G1 N 

U N 

o. o m ro N S C' 6f 

c 

• d N 

++ E 

O ~ C L Vml • 

~ C•r• O G. • ~ L N 

uf 

+J 

> •rro•.-4J O •O ••- 

uaro 

'O c 

a0 

aui i u am- ~' s;' c ~ io 

V.- aa m+1 Of O m 'O 

G) ur d C d ~ LC 

v ro m l.l a o c r- L u G O 

47 '- O Ol E C N i.~ > i Y- •U u O•r- N 

•rN•rro L CJ y V 

N L L N Cf CI 7 L N r y d L L r 

c C ~ 

+1 7 

x 

•L 

O > U C7 N 

tv~i LO~~ 

E m t ^ •E O 

C 

X 

L +~ • •• > ~ O d 

r'O m U o L Gl b E 

ro ar a L ro v+J Gl v= 41 41 O+1 

•-u u 

•N Gl 3 

~ Ol u 

•Ci. y N u 

O 0) II 

O Nw X4- a 1 m L N d W GI 

L 

+J - E 

O 

U 

O 

i•~ Q 

41 

L i 

N 

t~0 

L C 

Q 

- 6! C. 

i 

O O i. V > ro 

 IA C 

4- Gl r > > 

.D O ~ ~ V~- L 

L aii `° aii a 

r-~

1 II C 

-. oG fO 

t 

v 

L. 

a lroi 

" tL o L u u oG N - = o 

. +1 

~ ro m x rl .n e}' O al c+f 

u ci L c N ~ a) ¢ 

a u i 

N C 

ro 

~ 

C U 

c •E N O 

E O 

7 C 

O/ L 

i ++ > > 

C 

E • 7 al 

N E 

¢ ai 4J L 7 

~ u ~ 

3c ai rn 

N t0 

ro E 

+ r m 

i1 M p 

a t C O O 

+ 

N 4J 

~ C 07 ~ O L 

+ + v~i + a v~i i I I O N MO M N M 61 NZ II $ LL _ _ 

U d 

O f~ L ~Oq 

O E rl N ~ 

G i O .- a 

+•+ a 

r1 tf7 CO E N 

c 

ar ro 

C L. 

o 

O - 

0 0 +J o 

Ocj • 

N C 

O C7 

c{1~ 

00 eM E m N• - 

U 

C E 

1~ rl 

~ ~ 

O 

O 

i > 

~ C7 

O 

U Of 

tD O 

rf cC 

00 

GO M 

0000 

I , O O O 

N i 

m ~ 

1 N t, 

M 

01 1 

~ 

tP7 t[7 tt1 tn 

r-i M c•'f M 

G! O 

ro aQ 

9-139 
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 3 and NO2 mixture 

after the infectious challenge, a significant increase in mortality rate could 

be detected 1 month earlier. The clearance rate of inhaled viable streptococci 

from the lungs also became significantly slower after the 3-month exposure to 

this oxidant mixture. 

In more complex exposure studies, mice were exposed to a background 

concentration of 188 pg/m 3 (0.1 ppm) of NO 2 for 24 hr/day, 7 days/week with a 

superimposed 3-hr daily peak (5 days/week)'containing a mixture of 196 pg/m 3 

(0.1 ppm) of 03 and 940 pg/m 3 (0.5 ppm) of NO2 . Mortality rates from streptococcal infection were not altered by 1- and 2-month exposures, but a marked, 

although only marginally significant (p <•0.1), increase was seen after a 

6-month exposure. 

The same laboratory (Aranyi et al., 1983) recently reported a study in 

which mice were exposed 5 hr daily, 5 days/week up to 103 days to 0 3 and a 

mixture of 0 31 SO~ , and (NH4) 2SO4 . The 3oncentrations were 196 pg/m 3 (0.1 ppm) 

of 031 13.2 mg/m of S02 , and 1.04 mg/m of (NH4 ) 2SO4 . 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 03 was the major constituent of the mixture affecting the 

host's susceptibility to infection. These investigators also measured a 

number of specific host defenses to determine 7f host response to the mixtures 

was significantly different from that of the controls or of the 0 3-only groups. 

Neither the total number, differential, nor the ATP levels of macrophages 

differed from controls in either group, but the complex mixture did produce a 

significant increase in bactericidal activity over the 03 -alone and the control , animals. 

Previous studies (Gardner et al., 1977) indicated.that a sequentia.l.exposure to 196 Ng/m3 (0.1 ppm) 03 followed by 1000 pg/m3 H2SO4 significantly 

increased streptococcal pneumonia-induced mortality rates in mice. Ozone and 

H2SO4 had an additive effect when exposed in this sequence. However, the 

reverse sequence did not affect incidence of mortality. Sulfuric acid alone 

caused no significant effect. Grose et al. (1982) expanded these studies and 

showed that a 3-hr exposure to the mixture of 196 pg/m 3 (0.1 ppm) of 0 3 and 

9-140 
483 or 241 pg/m 3 H2SO4 also significantly increased the percent mortality, as 

compared to control. 

The effects of exercise on the response to low levels of 0 3 were also 

studied by using the infectivity model. Mice were exposed to 196 pg/m3 (0.1 ppm) 

of 0 3 and 588 pg/m 3 (0.3 ppm) of 03 for 3 hr while exercising. Each exposure 

level yieided mortality rates that were significantly higher than those observed 

in the 0 3 group that was not exercised (Illing et al., 1980). Such activity 

could change pulmonary dosimetry, thus increasing the amount of 0 3 reaching 

the respiratory system. Thus, such studies clearly demonstrate that the 

activity level of the exposed subjects is an important concomitant variable 

influencing the determination of the lowest effective concentration of the 

pollutant. 

9.3.4.4 Immunology. In addition to the above nonspecific, nonselective 

mechanisms of pulmonary defense, the respiratory system also is provided with 

specific, immunologic mechanisms, which can be activated by inhaled antigens. 

There are two types of immune mechanisms: ' antibody (humoral)-mediated and 

cell-mediated. Both serve to protect the respiratory tract against inhaled 

pathogens. Much less information is available on how 0 3 reacts with these 

immunological defenses than is known about the macrophage system (see Table 

9-15). Most studies have involved the systemic immune system which, to a 

degree, is compartmentalized from the pulmonary immune system. 

The effects of 2900 pg/m 3 (1.48 ppm) of 0 3 for 3 hr on cell-mediated 

immune response were studied by Thomas et al. (1981b), who determined the 

cutaneous delayed hypersensitivity reaction to purified protein derivative 

(PPD), expressed as the diameter of erythemas. In the guinea pigs infected 

with inhaled Mycobacterium tuberculosis, the cutaneous sensitivity to PPD was 

significantly affected by 0 3 . The diameters of the erythemas from the 0 3-exposed 

animals were significantly smaller during the 4 to 7 weeks after the infectious 

challenge, indicating depressed cell-mediated immune response. Exposure to 

980 pg/m 3 (0.5 ppm) of 0 3 had no effect. 

The systemic immu.ne system was studied by Aranyi et al. (1983), who observed the blastogenic response of splenic lymphocytes to mitogens and alloantigens and plaque-forming cells' response to sheep red blood cells after a 

chronic exposure to 196 pg/m3 (0.1 ppm) of 0 3 5-hr/day, 5 days/week for 90 days. 

No alteration in the response to alloantigens or the B-cell mitogen lipopolysaccharide (LPS) was noted, but a statistically significant suppression in 

9-141 

i 
} 

O 

O 

O 

z 

~ 

N 

Z 

S 

~ w 

z 

w 

LLW 

w 

G 

i -• 

N 

O 

S 

2 

O 

W 

Z 0 

0 

lJ.. 

O 13 

w 

LL 

W 

Lr; rl 

01 

W 

m 

F 

0 

co 

ro 

rO1i t 

00 

~ 01 00 a L 00 

O ~ r^ r-i ~ 

C~ N ~ Q 

O r-1 ^ ro ^ rl c 

ai 

+~ m `0 

t0 

v `0 ~ L ,O Ol +) 

O 

G aJ O 

~ }~ O O p~ 

d.' O 

•Gl 'O •rC- 

(U 

^ Y G 

•b ~ N i O E n n ~ 

¢ CD O c.7 - I— c) r-i tL . • II 

Of 

QO~ 

rr 

N 

u a> m v 

a°i ai a c G1 N {N N 

O. 

, C N N 

N O . O O 

•7 O O i 7 

~ E z cD E f m a ~ O 

E 07 

+1 C N ~ 

rn rn 

Y aJ v 

•c vOi 

Uai a 3 +~ . 

N w ° ~ 

+m+ sO7. ~ai ro +°+ m cn 

' ayi a i aCi 3 

C~ U c°~ ~ 

a' .^ 

c o~ 

z°o C7 O •r 

b 

~ 'O 

+a N > 

N nJ 

+•~ 

N +i.~ 

G. i. 

aN 

N o ro¢.- • ~cri 

c 

c •r • c 7 `A ai 

°u L' o L 

.ncro 

r• 

+~ o U +.+ OUf c c°J L 

ro ~ orn 

C~ 

~E~ o 

E OO

f +i~+ 

c vro r ro 

m ~ •JC T~ a j~ 4- r O C w dJ •r 

Olr— > 

•r fp C 

E O 

O ij 

U N 

O C+.> 

::. 

> O C7 C C ^ E tr 7•r 

'O O. 

N+J 

V- d•r 

Cl S +J ' i 

O +J Cl 07 Ql 

E•r •r II O 

G. 

i Gl S J d •~ i s N V N O U 

~ ~ 3 

R1 

~ 

r 41 1y ~ GJ QI N G/ ~ C C 

N 

a 

G. U) T 

n^ Cf V 

dl y 

L ra C 

E 7 U 

E~0 'O I ~ 01 

R7 O 

}o •r 

+1 7 

• c C E 

N 

U N O 7 C O O U• 7 i— • r N N,N 61 Cl T C O. 

. N Cf ~-1 .0 

0)•r O. •r 4- 

C 10 

C7 U 

t1'1 f0 

N N 

N 

•N fL 

'O t 

G. C+1 

QJ J 

U 

C ~ E E 

T O Cl 

C.0 rtl 

r~}- r 

U i L 

O U 

y L 

i G. 

N G1 

. 0. C 

N 

m.r r- C1 Q +~ 'o 4- G c. -a a•r G i V E r• 

d U 

o 

4 3N 

Cl c.0 

N•r 

N ~ O y-O c 

4- r U•r 

•7 r 

•C 

1 C O_•r 

E E 

fp b 

Cl N C 

N p• N 

d O_ i L O C N r GN! G! r D) f0 Gl C] d 67 tp L r ~ p L y •RJ 

Of U C G. Y N C GJ C 

d

~+~ O. E C. Ur0 

•E R1 

N Q J tr U•r p O+J ,-E+ r+E C i— V L f0 U C 

w u 

rg td 

U lI i a 

~ w N ¢ 1

L 

C 

to Y 7 ~ O O 

Jf N I~ N 11 U 

a c ~~,b ~v 

C. O .0 'O a L ~ M'O w • C cl' L S C 

W O u'f ~A ~ d• U M M M r-I r~-I ~"~ 4J 

4-) O 

cis ~ 

L OO 

~ '° ro ~ • L E U G1 

b N L + ) .o ° aci v 

d ° N ~ w ° . G ~

E cu O 2 ra ~ U Z 2 II ~ 

m 

° ~ 

o = 

z rs. 

u~ ao cp ' i o •~ -o v r"1 L ~ C ^ ^ O E~ rl ~ ttI tn u~'f Co E N o ro a o 0 0 0 0„ o c 

c i ++ o 

Ne o m E m o ai 

C 

v ~ a ~r-1 o ~.~ 

P N 

OO

i 7 N 

U 3. lD O O O 

10ff 

t00 N,ap 

rn 

oi rn 

ari ro.¢ 

9-142 
blastogenesis to the T-cell mitogens (PHA'and Con-A) was detected. The authors 

suggested that the cellular mechanisms for recognition and proliferation to 

LPS and alloantigens were intact, but 03 might have been interfering with the 

cellular response to PHA and Con-A through alterations in cell surface receptors 

or the binding of specific mitogens to them. Ozone exposure enhances peritoneal 

macrophage cytostasis to tumor target celis. There was no effect on the 

ability of splenic lymphocytes to produce antibodies against injected antigen 

(red blood cells). 

The effects of 0 3 on the humoral i mmune response was ai so studi ed by 

Fujimaki et al. (1984). BALB/c mice that were exposed continuously to 1568 pg/m 3 

(0.8 ppm) had an increase in lung weight after 3, 7, and 14 days of exposure. 

Spleen weights were decreased only after 1 and 3 days of exposure and then 

returned to normal. The thymus weight was decreased at a11 time periods 

tested, i.e., 1, 3, 7, and 14 days. Similar mice exposed to 0.4 ppm failed to 

show any marked changes in the organ weights. At 0.8 ppm 03 , exposure depressed 

the antibody response to sheep red blood cells (T-cell dependent antigen), but 

not at the antibody response to the T-cell independent antigen. These changes 

were correlated with the changes of thymus weights. The authors also concluded 

that 0 3 affected mainly the T-cell population rather than the B-cell population. 

Campbell and Hilsenroth (1976) used a toxoid immunization-toxin challenge 

approach to determine if continuous exposure to 1150 pg/m 3 (0.59 ppm) of 0 3 

for 36 days impaired resistance to a toxin stress. Mice were immunized with 

tetanus toxoid on the fifth day of 0 3 exposure and challenged with the tetanus 

toxin on day 27. Compared with controls, the 0 3-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 3 can alter the functioning of pulmonary 

macrophages, Goldstein et al. (1978a) tried to counteract this effect by using 

a known immunologic stimulant, Levamisole, which protects rodents against 

systemic infections of staphylococci and streptococci. The purpose was to 

determine if this drug might repair this dysfunction and improve the bactericidal activity of the macrophage. In this study, two concentrations were 

tested, 2940 and 980 pg/m3 (0.5 and 1.5 ppm) of 0 3 for 4 hr. In no case did 

Levamisole improve the bactericidal activity of the 0 3-exposed macrophages. 

The cells still failed to respond normally. 

9-143 
The possibility that 0 3 may be responsible for the enhancement of allergic 

sensitization has important implications for human health effects. Gershwin 

et al. (1981) reported that 0 3 (0.8 and 0.5 ppm for 4 days) exposure caused a 

thirty-fourfold increase in the number of IgE-containing cells in the lungs of 

mice that had been previously exposed to aerosolized ovalbumin. In general, 

the number of IgE-containing cells correlated positively with levels of 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). The number of IgM and IgG containing cells did not 

increase. These authors showed that a significant increase in anaphylactic 

sensitivity occurred when antigen-stimulated and 0 3 -exposed animals were 

compared to controls (Osebold et al., 1980). Significantly greater numbers of 

animals were allergic in experimental groups when 0 3 exposure ranged from 0.8 

ppm to 0.5 ppm for 3 days. The effects observed were most pronounced when the 

allergen (ovalbumin) was administered by I.V. injection than by the aerosol 

route. Further studies are needed to determine the threshold level of these 

effects. 

9.3.5Tolerance 

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 interest to toxicologists for years. Tol-erance, 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 Beliles, 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 toxicant 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 3 

exposure (Chapter 10, Section 3). This adaptation might well result from a 

different biologic process than that referred to in the various animal tolerance studies. It is not yet known whether the laboratory animal develops 

9-144 
adaptive responses similar to those seen in humans (i.e., respiratory mechanical functions, symptoms of respiratory irritation, and airway reactivity). 

Thus, to date, the precise distinction or definition of these two terms, 

tolerance and adaptation, are not yet fully understood. There are also 

limited data regarding the ability of cells in these "adapted" respiratory 

systems to once again return to a pre-exposure condition following termination 

of the exposure. However, Plopper et al. (1978) found that after 6 days in 

clean air, the rat's lungs were almost recovered from the damage caused by 

3 days of exposure to 0.8 ppm 0 3 and that these "new recovered" cells have 

approximately the same degree of susceptibility to a re-exposure of 0 3 as 

their pre-exposed counterparts, i.e., fully susceptible to re-exposure. These 

morphological findings confirm similar conclusions based on concomitant 

biochemical studies of Chow et al. (1976b). 

In animal oxidant toxicity studies, the term tolerance classically is 

defined as• the phenomenon wherein a previous exposure to a nonlethal 

concentration of 0 3 will provide some protection against a subsequent exposure 

to a concentration of 03 expected to be lethai. The degree of tolerance 

depends considerably on the duration of the exposure and the concentration. 

Tolerance occurs rapidly and can persist for several weeks (Mustafa and 

Tierney, 1978). The term tolerance should not be considered to indicate 

complete or absolute protection, because continuing injury does occur and can 

eventually lead to nonreversible morphological changes. This protective 

phenomenon seen with oxidants was originally described by Laqueur and Magnus 

(1921) in cats undergoing exposure to phosgene. 

In the typical experiment, animals are pre-exposed to a lower concentration of 0 3 and then challenged at a later time to a higher concentration. As 

early as 1956, Stokinger et al. presented data clearly i,ndicating that an 

animal could also become tolerant to the lethal effects of,0 3 . Such toierance 

has also been reported by many investigators, including Matzen (1957a), 

Mendenhall and Stokinger (1959), Henschler (1960), and Fairchild (1967). The 

observation of this tolerance phenomenon in experimentally exposed animals has 

led to the speculation that it may also be a mechanism for - protecting environmentally exposed humans. Tolerance to 0 3 also provides cross-protection 

against the pulmonary effects of other chemical agents, such as NO 2 , ketene, 

phosgene, and hydrogen peroxide (Stokinger and Coffin, 1968) and recently to 

hyperoxia (Jackson and Frank, 1984). 

9-145 
The previous criteria document for 0 3 and other photochemical oxidants 

cited various studies that examined 0 3 tolerance and presented some evidence 

indicating possible mechanisms of action. Review of these earlier data reveals 

that pre-exposure to a certain concentration of 0 3 can protect test animals 

from the acute lethal effects of a second exposure to 0 3 . This protection has 

been attributed to a significant reduction in pulmonary edema in the pre-exposed 

animals. Table 9-16 lists the key studies on 03 tolerance. 

Because 03 has a marked proclivity to reduce the ability of alveolar 

macrophages to function, studies were conducted to determine how the pulmonary 

defense system in tolerant animals compared with naive animals. With the 

bacterial infectivity model• (Section 9.3.4.3), the pre-exposed (tolerant) 

animals were only partially protected from the aerosol infectious challenge 

(Coffin and Gardner, 1972a; Gardner and Graham, 1977). The partial protection 

was evident at 03 concentrations that had been shown to be edemagenic; however, 

at the lowest concentration, 200 pg/m3 (0.10 ppm) of 0 3 , there was no significant 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 prophyaactically aided the animals' defenses against 

the inhaled microorganisms. Because the protection was only fractional and 

did not occur at the lowest level, however, 0 3 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 0 3 exposure did 

induce tolerance against pulmonary edema in the exposed lung; however, there 

was no protection afforded against the cytotoxic effects of 0 3 at the cellular 

level. The cytological toxic injuries measured in this study (including significant reductions in enzymatic activities of macrophages and an increase in 

inflammation, as measured by the presence _of p.olymorp.honuc.],ear leukocyaes) 

showed that there was no protection against these cellular defense mechanisms. 

Frager et al. (1979) studied the possibility of tolerance to 0 3 in mucociliary clearance. Exposure of rats to 1.2 ppm of 0 3 following particle deposition 

caused a substantial delay in mucociliary clearance. The 0 3 effect could be 

eliminated by a pre-exposure to 1600 pg/m 3 (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 0 3 level that lasted for about one 

9-146 
LU 

Z I 

0 

a 

O 

FU 

LU

¢ ' C 

J I 

O 

tj~ 

rl 

~ 

W ' 

m 

Q 

H 

.~ 

ro m n N tD 

n n 

~ 

I` 

~ 

14 

M 

CO 

IC ri I~ 

l0 

.O Ol ri rl 

ai 

u 

m 

L 

r°'+ rn 

rl 

r~i ai

i 

C 

n 

Ql 

rn 

rl N 

N 

i 

v 

f7 

p (tl 

ro 

C:J 

. N 

~ J 

v- N a 

c Y 

m 

Y a 0 

ro 

a 

d' ro N Y Gl v 

T 

c 

IC _ ~ 

Yc . N ro . i 

N 

C 

i 

N 

C 

Y 

i 

i 

O 

C 

N 

Y Y 

i S- 

•O L 

Y i al 

n 

01 

N 

4-- 

4- 

C 

10 d N 

_n n 

(:J C:3 C7 ' U W Q Q 

d Y 

U N 

d) 

7 LT Y 

N 

N 

7 

N 

7 Y L 

c~ ~ ~ C £ ' £ K 

1 C 0 d 

N 

ro E 16 

N L 

L d 

Y a 

y 

i 

N GY1 

L r 

.~C 

a VI a 

Y 

o I 

N N ~ 

u•r 4- 

c 

r Gl 

E s- . ro Y ~ 

Y n 

r E i T O' ro•r 

al o 

u c ~ 

N NE 

n u•r 

10 cV 

ai m 

'O Y 

r o --a a n. o •c o>, 

E ro c0 Y 

"O L N N Ol a 

Gl 

L ~ 

U c 

a N 

N Y 

O) m Y 

d 4- 

i O 

m N 

O+- E O 

O 

n C) L 

Y 

ro 

N ro O 

Y 

N 

y N 

N O 

c•r c 

7 N ro 

n 

T 

ro•r > 

W E C C 

x N•r 

4! O L 

u N c 

G! Id Gl 

Y 

O a 

n 

~ N i N i 

N~ N 

O•r RS •r 

C 

1 a C 

Ql 

r- 

'O 

C 

i Ul 

7 a N 

U 

. N 

X m 

dIOY 

N N > 

Oa 

N ro a N 

Y GlL 

L m 

nO O 

r N N 

O i N 

4- 

w1 C y d N 

Y 16 Y 

i 

C U•r 

7 ai 01 ~ 

U'O Y 

aJ a! ~6 T 

Y 

i y 

C 

o w 

n i ro 

x 7 3 

N i C a 

n O•r 

C N G 

r Y•r 

C i r 

ro O 4-- 

4- N i t6 

4- • N E 

O G d) 

4- 0 7 Y r 

N U 

U a 

. a ai > i>, ai ro 

a•-N E r• T 

v o .n v 

Cl E n o o N a 

T>, 

u N 

m u 

O) o c 01 

c m c 

i W C Y Y O O 

m 

C! •r a(0 

ro Y I 7 

E U E E 

Y'- 

r C ~ o N ~ E~ r• 

N 

N 

' L 

00 

T C 

Y m 

r U L. 

a Y 

•r >~ c 

C N> 

O a i t O 

Y N O rN N 10 T 

a E L L 

v T m Y ~ 

r o L 

I 0 ro 

Y~.a 

i 

n N 

G_ 

d i 

a 1 O a 

rt N N 

10 X u N n ro T o Y C Ol 

o 

O•r i T itl Y 

i 7 N 

N o r O 

(O > 3 

Y•r 

U7 a 

N v I 

u ~ Gl i 

C T E ro 

Y •r L 

G Y . 

E> n Y 

04F• 

Y Y n 

ro O) X 

i N N 

O D U 

Ol i 

U 7 N 

ttJ L C 

i Y N O 

a1 •r a N 

U G) m U 

i •41 y 

N U U 

Y O r N u G1 

r U ~/ 1 

E ai c 

i ttl 

c N N 

~O ttl C 

or c~ ~ o E 

r E 

c n n c c 

rt1 o O•r i0 

3 d c vi 

O i ro•r 

w- N 

O i 

c c ai 

7 ro Y i 

i i 

N N Gl 

L Ol O 

N E n 

D ro~ 

Y L E n 

i r r Y i 

N GJ G) ro N 

i N 

rOl N N 

d! 

~G L 

f-. u n 

L N N 

O•r O 

N 

O N N 

Y Gl 

O•r a C 

> > U 

O N N•r O O•r O N fQ 7 •r O ~ F L 

J EY H roK Z 3 W•r H aaa Y N EY O~ J C 3 Y al Y 

μ- N 

OO i Y a 

L N Ql C O 

Y O Y OJ •r i E i L . i N N i 

01 n ~ Y i 

cx ro ro a 

L 

o 

L L L T ^ro L 

v v n m M LO N M Ha M 

J 

O O ' N 

G E G1 C o ri rl 

~ N n 4Y- Y 

O vro 0 Gl 

r n O O r-1 

O 

N O O O 

N 

N 

I

C7 E i Y •II c\aco 0 

0 01 Y Ol •r 

N 14- Y L I O l0 

o 

N O N O O 

o v ro^ Ul 

o• 

tD tD 

r~i r

~

i 

tO 

r

m

i 

t0 

r

cni m 

00 

~ 

00 i~ t0 

O1 m  r

~

i 

N 

~ . ~ 

I ~ 

° s01 ° a 

L I ~ 

Y N N 

i 

L 

E i i 

L L 

i 

L 

L 

L 

fC 

a 

t.. 

L 

C G. n M M t0 lD 1-4 M M 

41 X 

J N 

N O 

C E I 7 ~ ~ ri 

O n N N 

N nL. 0 

rl 

1 

~ 

O 

1 O v n n 

X 

ri rl N 

L9 

M M M t[7 

G7 o O O O O O O O 

N O O 

N m i 

c E I 3 

l0 

rn 

O 

00 

l0 

rn 

o~ a N rI rn rl 

0~0 

~1p 1 

~ v n X 

d 

Q

w

1 - 

rl 

T 

rl 

Oo

i OD 

-zt 01 

O~o 

tf) LS) 

00 

6f) 

W 

Ql 

9-147 
9 

7 

cu 

C 

.j 

C 

ou 

v 

W Z 

N 

0 

H 

~ 

~ 

O 

!•- 

1D 

r••1 

01 

w 

J 

~ 

~ 0 

rn 

rl 

3 

U 

~ 

~ 

1 

Cl 1 N f0N 

U•r r• 0 n E C1 

C V ro N O L. 

s% E E ~~ C

m 

 3 Cl E Vl •O ~ 'rO •r vN v E o'r ro•r > In rn ci 

+~ E L•r y C i~ 

G/ •• N 4-1 r Gl Vl 

C 04) n N Vl r- 0 

-vai b c al 3.c a) 

In r• 07 E U In 

10 udi L b N

a,

~ ~ 

•O ro.0 r C C N•r 

'O G/ 0) O X 

.-~i'ic ~ aci C r- o" i°. 

In 

0

•-3 'rL L G. 

3 nc m ~r~ivxi 

•O S 4! •6 N N rl C.7 

O 1n r G! L O 

'L U If1 L C) ~ In L 

O) C L 7•p 1-r• d•~ 

nr U U aL 

E 

 C 

+J v s otic•r•- 

C N N i ri C m rt a.+ ro a 

R/ L t0 0UI 07 •O N Ol 

^ aUi T aroi m~ voi aroi z 

C~ r L•O M O f.. d 

C1 C1 f.lro p. U 0

.0 O i~ Cl Ol C/ X C d 

3+1ro•a r-I E a! •r O 

L 

s 

0 

M 

lO 

rn 

r+ 

ro 

41 

ai 

.~ 

q•1 

41 ro 2 

a 

7 

f 

aQ 

o vt0 O 

~ 

O 

C O 

1 ~ 

r 

O W ~ 

O d 

•1•) > 

v '> 

i0-17 

ro w 

O G! 

L E 

X N 'Q 

w G1 In o •r 

E N n 

cnuo 

m L ~ 

_ Q 

.- Itq O 

Q O U 

L 

G E Y C O 

O GL W Cl •r 

N Gf04>' L O v 10 Gl 

r O. 

a7 L ai ~ a+~ a 

o ~ aaco 0 olu- a •r 

N 1R7+1 L 

pv ^ Q 

W 

O 

N 

~ 17 ul 

C L o 

(V nX 

N 

a 

1 

LO 

M 

O I O 

n 

~ 

t0 

a 

L 

rM 

N N 

O rl Ct Z N 

CD 0 

t^A M C1 N 

r-i N Z 

al 

M 

N 

'O .0 L t 

M I ch I rl I '..1 

N 

N 

C 

ro 

M 

0 

CD 

co 

~ 

i 

M 

O 

CD 

~ 

/b 

n 

N 

b 

'O 

M 

aa 

U 

m 

L 

61 w ai 

~ 

N 

N 

U 

G 

N 

N 

1 

U

1 

w 

4) 

m 

L 

Yf 

O 

4- N O L L 

~ C9 iJ 'O 

+•1 O V- O! •r 

Of O roY i cx mm 67 y r p 

J 

N 

I` 

T rt 

ro 

~ ai 

L 

C 

~ 

ro Y, 

N 

.G 

ro 

C 

• O 

10 .0 E O. 

GJ L rN d E E 

O a~ ~ L O O 

C ro n N 

U Ow'•a- cl E O 

1~ r •O 

7 N C! 

ClG.•r N 

L. X f0 

70 10 GJ 

Ina..lY L . 

cT€dN 

x .- a a •r 

61 C L > 

i

O U L. •r 

C V tti OU 

~ C! L Nl ffl 

rO ~ N O/ 

L > ~ O N 

, C! 0/ C! Y C 

ro'0 c aoi °D r d ror d 

'C C Cil L I

m

O 

~ ro-rori O C! O 

•i-+ .°-~ " u v 00 :3 T 

3a- Z C,L 

L 

M 

a 

in 

r- (n 

to . r` ro 

an 

rl +1 

N 

3 3 o O 

.0 L 

U U 

4~ 

C 

N 

C 

O 1 O 

r tp N M t 1 u ro 

iY Gl 7 rtraa 

7 u L r 

~ ro 

Olr• V- E 

OI O•r 

4- C O ~ ^ RI 

IN Vryl CJ i. . 

r N fC0 r~ 

+1 6) - L ro - N E >V Vr•r 

y c O C 

41 L. N i-~ f0 

. ro al al w 41 

C W O C I^ o roro 

C N L 

C Cl O) N 

GJ Y 07Cr 

N ro O 7 O 

ro Y L. r 4- 

so.. ~ TCo 

U O)L•r G 

•00 L N O 

L tOn C! o y 

G7 RS +- •r V -vro.cd 

r L Y L ro'x nro ro 

vEi s°.. o ~ € 

C) .0 r- O 

Q n n Of U 

L 

r 

0 

CO I\ Q1 

r. 

ro 

+~ a 

f-. 

n c. 

a 

4- 

1' 

O 

c ++ ro 

rnn ro ro 

n Q L. 

r 7 

a 

01 • O 

v L X 

r 7 CJ 

N 

C1 O M 

~ X O 

L y 41 

Gl L 

>w 

O O fq 

U4~ 

L y C 

O •r 

N N vl n ro ' 

b cOi T • •o In r In 

7 C N Lcwo ~ 

s Ol r i~ 1•> i-1 - YI C 

a- ~ roo 

Q4-r U 

n 

N 

O T lo 

Ol 

11 

rn 

rt 

ro 

+J 

ar 

v 

b 

I LL 

N 

C 

IO 

iG! = Ua 

C 

ro - 

L. T 

~ b 

M 

Y 41 

a ro 

O. G! 

E U 

OC 

U ~ 

vro 

(n 0- 7 U 

U i 

o 

o •r 

41

O 

L 7 n

~ E roa 

N 

E.~ O 

~ Tr-I 

O 

d_

L 

 •O

N 

 C 

L 

0 

V' 

N I\ O1 

r-1 

m 

~ 

a 

lo 

L 

E 

41 

f 

ro d 

C 

•= 07 

C.7 C 

N 

c 

O 

Y 

In 

O 

CL 

N 

L 

U 

m 

a 

ro ~ 

q.J r 

G! O 

c tL.o 

a~ N 

H ro 

L 

r-I 

N 

C E 1 

O O N 

7 

0

N G 5-O 

O~ dX 

d 

a

— d 

0 L 

O \ C! N 

N OI L O 

pv aX G7 

In 

O 

O O Ol 

t17 

n 

O 

O 

r, 

rl 

O 

O 

O 

~ rl 

O 

O 

O 

t00 r-i 

r-I 

O 

O

to

i 

r-1 

rl 

O 

O

tn

i 

b 

a) 

i 

U 

w 

N 

~ 

4J 

0 

c 

II 

G 

Z 
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 

03 . As the secretion returns to normal, the protection is lost. The authors 

suggested that another possible mechanism for this protection involves the 

ciliated cells and their cilia. In this case, the protection could result 

from either the formation of intermediate cilia (Hilding and Hilding, 1966) or 

the occurrence of some other temporary change in the regenerating ciliated 

cell. 

Evans et al. (1971, 1976b) also measured tolerance by studying the kinetics 

of alveolar cell division in rats during a period of exposure to an elevated 

03 concentration of 980 or 1372 pg/m 3 (0.50 or 0.70 ppm, up to four days) that 

followed initial exposure at a lower concentration of 686 pg/m 3 (0.35 ppm) for 

four days. Tolerance in this case was the ability of type 1 cells to withstand a second exposure without any increase in the number of type 2 cells, 

which would indicate a lack of complete tolerance. Similar to the host defense 

studies cited above, these investigations showed that tolerance to the initial 

concentration of 03 did not ensure complete protection against re - exposure to 

the higher 03 concentration. 

Attempts have been made to explain tolerance by examining the morphological 

changes that occur due to repeated exposures to 0 3 . 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 03 damage as indicated by DNA synthesis. These effects are fully described in Section 9.3.1.2, and they indicate 

that with continuous exposure to 0 3 , the lung attempts to initiate the repair 

of the 03 lesion, resulting in somewhat reduced or less than expected total 

damage. These authors suggest that this is an indication that although the 

damage is continuing, it is at a lower rate, and they refer to this phenomenon 

as adaptation. 

It has been suggested that the tolerance to edema seen in animal studies 

can be explained through the indirect evidence that more resistant cells, such 

as type 2 cells, may repiace the more sensitive, older type l cells, or that 

the type 2 cells may transform to younger, more resistant cells of the same 

type (Mustafa and Tierney, 1978). A number of workers have reported that the 

younger type 1 cells are relatively more resistant to the subsequent toxicity 

of 0 3 (Evans et al., 1976a; Dungworth et al., 1975a; Schwartz et ai., 1976). 

9-149 
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 0 3 exposure, there is cellular accumulation within the airways resulting in mounds of cells in the terminal bronchioles that may cause considerable narrowing of the airways (Berliner et al., 

1978). As the airways become more obstructed, the 0 3 molecules are le'ss likely 

to penetrate to lumen. This may result in a"filtering" system that removes 

the 03 before it reaches the sensitive tissue. 

Tolerance to 0 3 has also been studied by using a variety of biochemical 

indicators to measure the extent to which a pre-exposure to 0 3 protects or 

reduces the host response to a subsequent exposure. For example, Jackson and 

Frank (1984) found that preexposure to 0 3 produced cross tolerance to hypoxia. 

In these tolerant animals (0.8 ppm 0 3 x 7 days) there was a significant increase 

in total lung superoxide dismutase, glutathione peroxidase, glucose-6=phosphate 

dehydrogenase, and catalase. Such an increase in these antioxidant enzymes 

occurs with 03 exposure. Chow et al. (1976a,b) compared a variety of inetabolic 

activities of the lung immediately after an initial 3-day continuous exposure 

to 1600 pg/m3 (0.80 ppm) of 03 with the response after subsequent re-exposure. 

At 6, 13, and 27 days after the pre-exposure ended, the animals were once 

again treated to the same exposure routine. If tolerant, the animals should 

have shown a diminution of response. However, the re-exposed rats responded 

similarly to those animals tested after the initial exposure. The lungs of 

the naive animals had equivalently higher activities of glutathione peroxidase, 

glutathione reductase, glucose-6-phosphate dehydrogenase and higher levels of 

nonprotein sulfhydryl than controls and were comparable to the animals that 

were exposed and tested immediately after the initial exposure. The authors 

state that this indicated that by the time recovery from the pre-exposure is 

complete, the lung is as susceptible to the re-exposure injury as a lung that 

has never been exposed. 

In a follow-up study, Chow (1984) pre-exposed rats for 3 days (apparently 

continuously) to air or 1490 pg/m3 (0.76 ppm) 0 3 and challenged them at various 

times with an 8-hr exposure to a higher level of 03 . As expected, the pre-exposure protected the rats from the lethal effects of 6860 to 7840 pg/m 3 (3.5 to 

4.0 ppm) 031 whether the challenge was 8, 11, or 19 days later. Generally, 

9-150 
all 03 exposures decreased the GSH peroxidase system. When rats were challenged 

with 7644 pg/m3 (3.9 ppm) 03 11 days after the 3-day pre-exposure, there was. 

no tolerance immediately after the challenge exposure; 18 hr after the challenge 

exposure, a dampening of the decrease in the GSH peroxidase system was observed. 

When the pre-exposure and challenge (6860 pg/m 3 , 3.5 ppm) were separated by 

19 days, the decrease in the GSH peroxidase.system measured 16 hr post-challenge 

was less in the animals receiving pre-exposure (except for GSH reductase, for 

which values were equivalent); 114 hr post-challenge, no tolerance was observed, 

but some increases in enzyme activity were observed. 

Gertner et ai. (1983b) presented data showing that the development of 

adaptation and tolerance to pulmonary function changes is rapid and mediated 

through the vagus nerve. These investigators used a bronchoscope to expose an 

isolated segmental airway of the lung to 0 3 and study changes in collateral 

resistance (Rcoll). During a 30-min exposure to 0.1 ppm, the Rcoll increased 

31.5 percent within 2.min and then gradually decreased to control level in 

spite of continual exposure to 0 3 . Fifteen .roinutes after the 0 3 exposure 

ceased, the Rcoll returned to normal. Subsequent exposure to 0.1 ppm of 0 3 

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 3 and the term tolerance to describe 

resistance to subsequent 0 3 exposure. 

Thus, the available'evidence from animal studies suggests that tolerance 

does not develop to.all forms of lung injury. The protection described against 

edemagenic effects of 03 does not appear to offer complete protection, as 

illustrated by the following examples. 

1. There is no tolerance (i.e., no protection occurs) on the part of 

the specific pulmonar.y defense mechanisms against bacterial infection 

below the edemagenic concentration; whereas above the edema-inducing 

concentration the effect of tolerance (i.e., inhibition of pulmonary 

edema), can lower the expected mortality rate because the animals do 

not have to cope with the additional burden of the edema fluid. 

2. Specific cellular functions of the alveolar macrophage (i.e., enzyme 

activity) are incapable of being protected by tolerance. 

9-151 
3. Various biochemical responses were found in both naive and pre-exposed 

animals. 

4. Tolerance fails to inhibit the influx of polymorphonuclear leukocytes 

into the airway. 

This last finding is interesting considering the effective tolerance for 

edema production. This suggests that the chemotactic effect of 0 3 may be 

separable from the edemagenic effect. This may also explain why chronic 

morphological changes in the lung may occur after long-term exposure, even 

though there may not be any edema. 

The possible explanations for this tolerance phenomenon have been proposed 

by Mustafa and Tierney (1978). The primary mechanism of tolerance may not be 

due to hormonal or neurogenic pathways, because unilateral lung exposure does 

not result in tolerance of the nonpre-exposed lung (Gardner et 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 3 exposure but did after 

challenging with histamine, indicating that the parasympathetic system may 

pl ay a rol e i n response to 0 3 i n the peri phery of the 1 ung. Ther,e i s some 

evidence that 0 3 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, 0 3 could possibly cause anatomic 

changes, such as an increase in mucus thickness, that may, in effect, reduce 

the dose of 03 reaching the gas-exchange areas of the lung. 

It shouid be mentioned that the term tolerance carries with it the connotation that some form of an insult and/or damage has occurred and there has 

been an overt response at the structural and/or functional level. The response 

may be attenuated or undetectable, but the basis for the establishment of the 

tolerance still persists. It is possible that the cost for tolerance may be 

minor, such as a slight increase in mucus secretion; however, one must also be 

aware that changes in response to diverse kinds of insults to a host's system, 

such as the immune system, are adaptations that might even suggest an undesirable 

effect of ambient oxidant air pollution. 

9-152 
9.4 EXTRAPULMONARY EFFECTS OF OZONE 

9.4.1 Central Nervous System and Behavioral Effects 

Despite reports of headache, dizziness, and irritation of the nose, 

throat, and chest in humans exposed to 0 3 (see Chapter 10), and the possible 

implications of these and other symptoms as indications of low-level 0 3 effects, 

few recent reports were found on behavioral and other central nervous system 

(CNS) effects of 0 3 exposure in animals. Table 9-17 summarizes studies on 

avoidance and conditioned behavior, motor activity, and CNS effects. 

Early investigations have reported effects of 0 3 on behavior patterns in 

animais. Peterson and Andrews (1963) attempted to characterize the avoidance 

behavior of mice to 0 3 by measuring'their reaction to a 30-min exposure on one 

side of an annular plastic mouse chamber. A concentration-related avoidance 

of the 0 3 side was reported at 1176 to 16,660 pg/m 3 (0.60 to 8.50 ppm) of 0 3 . 

However, the study had serious shortcomings, including a lack of positionreversal 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 3 . Mice were individually exposed to 0 3 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 

photobeam. The delivery of 980 pg/m3 (0.50 ppm) of 0 3 was reliably turned off 

for a greater proportion of experimental trials, compared to control trials 

with filtered air. At 19,600 pg/m 3 (10 ppm) of 0 3 , all animals turned off 

100 percent "of the trials with an average latency of approximately 10 sec. 

Studies by Murphy et al. (1964) demonstrated that wheel-running activity 

decreased by approximately 50 percent when mice were exposed to 392 to 980 pg/m 3 

(0.20 to 0.50 ppm) of 0 3 for 6 hr and decreased to 60 percent of pre-exposure 

values during the first 2 days of continuous exposure to 588 pg/m3 (0.30 ppm) 

of 03 . Running activity gradually returned to pre-exposure values during the 

next 5 days of continuous exposure to 588 pg/m3 (0.30 ppm) of 03. If the same 

mice were subsequently exposed to 1372 pg/m 3 (0.70 ppm) of 0 3 for an additional 

7 days, running activity was depressed to 20 percent of pre-exposure values. 

Partial recovery was described during the final days of exposure to 1372 pg/m 3 

(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 3 (0.20 ppm) of 0 3 for 7 days. Konigsberg 

and Bachman (1970) used a capacitance-sensing device to record the motor 

9-153 
t: 

71 

Q1 

~J] 

F 

~ ^ 

G C ^ G ao 

rn 

ro 1~ 

rn~ b ro ro o o ~ ro~ 

tf ~ d •• aJ d d d w ^ 

c .n c d 

.0 

'Od 

V- •^ d C U 

V1 

ri 

• 00 

d 

C1N 

O.CO 

C.~ 

L lD 

d 

O,M 

O.oD 

O 4- 

C 4- 

T ro 

d 

C 

Cl 

W 

O ro 

Ym 

6fCl 

311 

dQf 

1-14 

01 

ri 

d01 

F-ri 

dL 

C 

U N Vl L E 

c. ro ~ rt ro o 0 o i. 

v~ a 

~ 1 v- o 

d U•

;; 

d 

d O N d 1 01 O N 1 ~ 

C vl ~- f.. tn O, C bf 

10 

^ c 

O 3 

C d 

~ ~( t N 

3 A. 

•N 

•e~ T 61 7 U 

r ro> Vl 

7 d U 

d 7 rtf u Of o 10 • t- r 0! N•O 01 d 4J M1f 

O V 31 O 

L r G• 

L 111 C 

U C d L 

N 

d d 

c r 

'CI GO•- 

7 O d L ~ i 

•O ~ i V- •Q 3 7 7 

uf C 

d d 

d 

L. 

U ff'! X 

I~ r d 

o 

d O L o 

'Q •~ V- i•~ 

i

3 

o 

V7 L 

10 7 

dtn 

d C C+f 

vl C f0 

roOf ~ 

d +~ O d O y Ul 

uf aQ G Yl r O O 

f0 i~ owrorc.c. 

Y U 

O c o.o d u 

u ••N L W >> t0 c E L o d c i ~ d t0 u>> y o x x u 

~ 

YI 

Vl R! ~1 O 

d C 

r L•.- 

d•N Y 

t1 G 

d x 

L•~ d 

U ul W a•7 Vf 

L f0 L 4- d d 

t.f 'O O'O tl 

d 

N M 

•O 

L 

v 

N 

GJ'O C C 

G d Cf O 

> G d 7 

d w O 

~ d d 10 O U C 

V d d O 

d d i•1 d ul M'O 

V uf tf9 •O 10 O d 

O C 

C 

l,l 

d.1 

u 

d 

fO }J 

t-r- O i~ 

U a i

j 

 ro 

•VE Ci 10 .0 

1/! c d+J 

d O L v- 

!J O) 

c 

• c •~ 

L. Vl W. •- 

>> u 4•f V- +f 

•1•1 c G ro 10 

ro 07 3 O 

>> d c•i.> >> E c 

bf i••- x+f d O.•r 

N >> ftf 

.G 

•O 

c 

ro 

V•. 

4- 

• c 

r t Of•rf. G U 3 

C) C 

O L. 

41 7 

• •~ d L 

> c^+f 

U C d f- O. 4 f 

•> N i C> 7 G •O U d 

Gl d C C ~ 

L•'- •rO M•~ 111 

L O 

O .O 

C 

L O C 

•1•f +f O.r 0! 

•O 7 tn 1, O 

~1 i•t d ~ O U 

d C 

•1•1 d O. 

Z7 O C d G i•f 1O O L U•'- E c0 tl Cf >I d L U O.O 10 7 E 

d 

> 

>f L

4

1 

•> +./ 7 

Cf L> 

>> c Cl i 

E o d 3 0•'- c 

f..) +~ O 

d i•f L+~ ro X L 

•> 

d a+f 

c Q' 

•E 

d•O •w 

L 1O •~ .f.~ L i•~ •'- •'- ~f L •> Of >f C U Of ^ 07 Q! .0 •~ f- d 

d 

v! 

.0 > d d 

d — L i•~ 

trf r d-~ 

•^ 10 

O 10 

\ L 

C r y C In C C E 3+1 

a• 

L 4- u7 

d 10 

~ 

O 

~ ,G ~•1 V- • d N

~ 

~ d •C 

L M 

IE C d O •G 

d•f O. >> C 

U 1O L •C O.r C d +1 OI d 

C U d 

41 R1 d 

d•f L 

U V . C 

C.p 

10 

C 

7•i- •r- V- L 

d~ 7 C d O i 

7 'O 7 n'f •FJ •r- R1 

C L. 

01 •r- U 

C 

O 

i L 1A 10 C E•r- i•~ L C+~ •~ Ol L O)N L . Ip +1 O. L Vl C 

d d 7>> 

> vf u1 ro 

'- O. E 

L • f1 1 

L C 

10 d 

O f0 •CJ •- 

r.F3 i L 

C v1 O••- ro a 

.- +f r. •O .FJ d 

7 ro•'- 

ul d 

+J 

ro 

O ro O'Q 

L C. 

O L M 

Y+1 10 d L 

c d 

d O d•~ d 

d C G 

 L. 

• 

d • c Vl d d c d O. 

d c L> L d E 10 uf 

O S. C 

d ti O 

> d 

d E 

O d X N Z+f dr•1 O•e- 4- z 30 0 • O. 

~ 0 L O d d V! 3€ c~ 3> 

L 7 n- d 7 L E'O ro 

3 ~ wr vl S•'-¢ 3 

X C•'- 

w•.-++ w+f 

0 

U Ul 

O 41 7 

O 

N 

7 

O 

S. 

7 

C 

O 

7 

G d 

•r- ii C 

L '6 

7 C 

C 

O 

+J 

C 

N 10 

O 

(J O 

ti 

O. C 

11 O Vl 

C 

^ 

N C 

N y ~E L L i ~ ul •B L L L L O 

I •O T t0 40 40 t- . tD M 

.O I 

~ 

CE}1•Q 

d N N W ~ Y t~/f W 

c=.~ v z ~ c=~ °z 

ro 

2 

0 0 0 o n o 

~-i ri o 0 

ID 1 N N1 O r1 

C E O O rl ri N M Iff tf'/ 

O fL 

•r- C. O O O O O O O O 

I+fO 

81 X. 

O 

O O O N O•. 

O C O~

1 ON1 T 0~0 M 0

to

1 

N d 

~ E 

ri M ~ Of r-1 r~ 

C 

O 0] 

id 1 

O 

r•1 

r-1 

CO 

Ol 

tO 

C1 

r-1 

tPf 

.t'M 

N 

N ~1p 

Of OO 

M tn 

O 

00 

01 

O 

Oo Q1 

9-154 
M a to 

C Q1 ro rt 

C •• 

+d•+ a 

cd¢ 

d 

~ 

E 

r 41 

•; 

a • 

ro ~ 

L ro 

c .i r c • d i U 

O C 

> U 

r M 

~ O 

d,C 

U •r c Vl 

a d 

i 

• O U 

> C Q .r 

n 

a 

d 

7 

4-) 

C 

O 

u 

v 

W 

Z O 

N 

O 

UO 

N 

H 

U W 

~ 

W 

Q 

O : 

O_ 

7 Q 2 W 

m : 

Cf 

Z 

Q 

f 

wI.- , 

, N ' } , 

N ' 

N , 

~ 0 

~ 

W ' 

Z 

J 

~ 

F~- 

Z 

V 

N: 

rt 

i 

01 

W 

m 

H 

N¢Y 

d S 2 2 Z 

d U 

C 

d 

L 

w d 

6.' 

N 

d 

• U 

d 

N 

n 

U_ 

N 

tn 41 

u 

4- 

4~ - 

d 

a 

> di 

y y 

O ' 

O 

U 

r••) 0 

a d 

L a 

~ C w ro O 

X O 

W •r 

+~ 

i 

7 

a 

I 

Jc 

ro 41 

w o 

d +~ 

~ E H 

ro 

~ 

C E 

O Q 

~ 

ro 

d i 

O C 

O U E c O Of 

U 7. 

ro 

~ d 

(0 

i 

f0 

c c1Oo rn 

X r-I 

+~ ro ~ 

t 4-) ~I 

C Vl r 3 

• d ra o ro U 

V- L 

~ O . 

L L L 

Of d 7 

~ 7 O a . O. 

..°~ a aX) 

N roN 

a~ ro 

H L. 

i U ad 

N 

d cQ d Y 7 L 

O uf U > .r d 

W 9 a 

C 

L •E 

r-1 M 

Y 

m 

Z 

O 

LCf 

rt 

t1Y 

O 

0 

~ 

Q1 

rf 

O 

rn 

a M 

C 1~ 

~ a rn 

rt i 

C r 

U d 

a

4-)i a 

~ H 

4.) 

ro o: 

O 

C 

~ 

> O 

Z ~ u' u 

10 C 

rn~ 

C U 

C VC d 

L a 

W 

d C d .r 

3 ~ O 

~ > ro 

C 4- C 

O O E 

+J +J d U !•1 - 

7 d O. 

d 4- 7 OC (U N 

N 

0 ~ 

e 

Y 

C 

0 U 

N 

~ 

a 

~ 

O 

r-1 

O ~ 0 

01 

9 

ro 

~ 

d 

ul 

H rt 

rn 0 

c 

a~ a etav 

a O L Nro L N N O L E ++ 00 

t

4J0 ¢ t0 00 ~ N . LO 

a rl a E +1 

V1 }) t» L d ro 

ro ro ro 4- m a d d E d 

U d U d s ro •om vroei d d •r U 

~ ~ ~ u >a 

•~ •r ~ •r •r a 

UN U ii~ ro 

ro > ym.-. ~ n roro 

U Rf U N F L 

H -# 4 

O O O 

~ 2to cl 

r N rd 

155 

ro 

+J 

d 

C ~ O a 

t n 

7 ~ 

rn 

c i 

W 

O 

i N 

v~- c 

ro o E 

i r0•I 

4J L 

ro~ 

O_4- 

ro 

W d3 w0 

C 

r 1-1 

7 

C ~ 

i C Ul 

Q Ol 6X1 

M 

O O 

rnm ,4 O 

~ 0 

C 

II 

c 

z 

d 

a 

a 

O 

E 

7 

.N 

ro 

a~ 

0 

G 

a 

d 

i 

O1 

~ 

4- 

~ 

i~ O 

~ 

d to C L 

II U 

1 C Y d 

Z i

0

. 

~ 

U 

d d 

U 

cu 

C 61 

U 11 

N C7 

C W 

W 

SE 

d 

N 

e ro 

d 'O 

L 

U • X 

O 

d 

V1 d 

10 C 

t 

C E 

m 

~ n c 

OI E 

II II 

z 

= v 

• o E 

U a 

O d ~ r N 

L 0 

~ 7 N 

E vlC 

4Jul ~ L 

ro o 

v 0. a r 

U d 41 •r 

i L d Ld E u 

N V- O O 

o a ~ ai 

o >, 41 

'O 

j 4-) iJ ~ 7 r-i 7 R7 L N ]C d U i)O 

11 C 11 3 ~ 

1- 1- a 

m U iS? Z d a 

E O 

N 

O O d •r t t L V1 L +1 

~ +1d•~ 7 4• ~ • 

d d d 3 

E E c O C 

C O 0 O 0 

E m 4.3 ro G 0 

d ~ rti •r O .L L •r L > L- ~ . 

Uf U 

:JR 

 L. U iJ 

roaim ro ao... 

~E a U ¢N Y 

~o .G U a d 

O 

1 tD 

O 

O 

t0 

1 l0 

ro 

a 

~ L 

a 

.s 

n vi 

N ~ 

C a 

4-0

C 

+~ 

C a 

EL Et0 EL 

~ O ~ O ~ co 
activity of rats during a 45-min exposure to 98, 196, 392, 980, and 1960 pg/m 3 

(0.05, 0.10, 0.20, 0.50, and 1.0 ppm) of 03 . Compared with control rats, 

motor activity following 03 exposure progressively decreased with increasing 

03 concentrations up to 980 pg/m 3 (0.50 ppm). No greater reduction was obtained 

at 1960 pg/m3 (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 3 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/m 3 (0.12, 0.25, 0.50 and 

1.0 ppm) of 03 . The 3 days preceding an exposure were used for control observations to measure running activity for each rat in a wheel attached to the 

home cage. Decreases in wheel running activity occurred at 235 pg/m 3 (0.12 ppm) 

and progressively greater decreases in wheel running activity occurred with 

increasing 0 3 concentration. An analysis of the running behavior showed that 

the components of running were differentially affected by 0 3 . An increase in 

the time interval between running bursts primarily accounted for the decreased 

motor activity at the low (235 pg/m 3 , 0.12 ppm) 03 concentration. Postexposure 

increases in wheel running were seen following this low 03 concentration. At 

higher 03 concentrations (>490 pg/m 3 , 0.25 ppm), an increase in the time per 

wheel revolution, a decrease in the burst length as well as the extended time 

interval between bursts contributed to the reduced motor activity. These 

higher concentrations also caused a decrease in performance compared to control 

for several hours after exposure was terminated. 

Effects of 0 3 on behavior were further investigated by Weiss et al. (1981) 

in their studies on the operant behavior of rats during 0 3 exposure. The term 

operant refers to learned behaviors that are controlled by subsequent events 

such as food or shock delivery. In this case, rats were trained to perform a 

bar-pressing response maintained by a reward with food pellets delivered 

according to a 5-min fixed-interval reinforcement schedule. The rats were 

exposed for 6 hr to 03 concentrations from 196 to 3920 pg/m 3 (0.10 to 2.0 ppm), 

with at least 6 days separating successive exposures. Two groups of rats were 

tested, one beginning in the morning and the other in the mid afternoon. Ozoneinduced decreases were linear from 196 to 2744 pg/m 3 (0.10 to 1.40 ppm) for the 

first group; for the second, the decreases were generally monotonic from 196 to 

3920 pg/m3 (0.10 to 2.0 ppm). Analysis of the distribution of responses during 

9-156 
the various 0 3 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 3 concentrations. In contrast to other types of toxicants, 0 3 did not disrupt the temporal pattern 

that characterized response during each fixed-interval presentation. Based on 

the sedentary nature of the task, the authors suggested that the inclination 

to respond rather than the physiological capacity to respond was impaired. 

Kulle and Cooper (1975) studied the effects of 0 3 on the eiectrical 

activity of the nasopalatine nerve in rats. Ozone exposure to 9800 pg/m 3 

(5 ppm) for 1 hr produced an increase in nasopalatine nerve response (action 

potential frequency) to amyl aicohol, suggesting that the nerve receptors were 

made more sensitive by prior exposure to 0 3 . One-hour air perfusion following 

the 0 3 exposure reduced the neural response to amyl alcohol, but not to preexposure levels. The nasopalatine nerve is a branch.of the trigeminal nerve 

which responds to airborne chemical irritants. Because most irritants, including 0 3 , 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 3 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 pg/m 3 (1, 2, or 3 ppm) of 03 . In 8 hr/day exposures, 

reported decreases (35 percent) in the catecholamines norepinephrine and 

epinephrine were not statistically significant, although 0 3 exposure at 3920 

pg/m3 (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 pg/m 3 (1 ppm) of 0 3 , but not at 3920 or 5880 

pg/m3 (2 or 3 ppm) of 03 . Increasing daily exposures to 1960 pg/m 3 (1 ppm) 

from 8 to 24 hr/day caused a significant decrease in COMT activity, but MAO 

activity increased at 8 and 16 hr/day but decreased at 24 hr/day. Concurrently, 

Johnson et al. (1976) measured electroencephalographic (EEG) patterns in the 

same dogs and noted alterations in EEG patterns after 9 months of exposure to 

1960 to 5800 pg/m 3 (1 to 3 ppm) of 03 , but not after 18 months of exposure. 

The authors noted that it was difficult to correlate the observed EEG changes 

with the alterations of inetabolic balance described. Furthermore, it was even 

more difficult to assess the metabolic and physiologic significance of the 

changes without more information abcut chronic 0 3 exposure. 

9-157 
9.4.2 Cardiovascular Effects 

Very few reports on the cardiovascular effects of 03 and other photochemical 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 3 . After a 3-week exposure to 392 

Ng/rn3 (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 0 3 on pulmonary arterial 

pressure i n dogs. They exposed 31 dogs to 1.0 ppm of 0 3 dai 1y for vari ous 

hours for 17 months. Ten percent (3 dogs) of the animals developed pulmonary 

arterial hypertension, and approximately 30 percent (9 dogs) had excessive 

systolic pressure, but there was no proportional relationship between pulmonary 

arterial hypertension and 0 3 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 03 and cadmium, singly and 

combined, in rats. The rats were exposed to 1176 pg/m 3 (0.60 ppm) 0 3 , 

5 hr/day for 3 consecutive days or to 3 mg/m3 cadmium for 1 hr or to both 

pollutants. A11 exposure treatments resulted in increases in systolic pressure 

and heart rate. Neither diastolic pressure or mean pressure was affected. No 

additive or antagonistic effects were seen with the pollutant combinations. 

Costa et al. (1983) measured heart rate and standard intervals of cardiac 

electrical activity from the electrocardiographic (EKG) tracings of rats 

exposed to 392, 1568, or 3920 pg/m 3 (0.2, 0.8, or 2 ppm) of 0 3 6 hr/day, 5 

days/week for 62 exposure days as part of a more extensive evaluation of lung 

function (Section 9.3.2). Heart rate was not altered by 0 3 exposure. The 

predominant effects occurred at the highest 0 3 concentration (3920 pg/m 3 , 

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 pg/m3 (0.3 and 1.0 ppm) of 0 3 on the pulmonary gas-exchange region of 

dogs ventilated through an endotracheal tube. Pulmonary capillary blood flow 

and arterial 02 pressure (Pa02 ) were decreased 30 min following exposure to 

both 03 concentrations, and arterial pH (pH a ) was decreased following exposure 

9-158 
to 1960 ~ag/m (1.0 ppm) of 0 3 3 . Decreases in pulmonary capillary blood flow 

persisted 24 hr following exposure to 588 and 1960 pg/m 3 (0.3 and 1.0 ppm) of 

0 3and as 1 ong as 48 hr fol 1 owi ng exposure to 1960 pg/m 3(1.0 ppm) of 03 . 

Persistent decreases in pH a and Pa0 2 were observed 24 hr following exposure to 

1960 pg/m3 (1.0 ppm) of 03 . Pulmonary edema, determined histologically and by 

increased lung water content and tissue volume, was observed 24 hr following 

exposure to 1960 pg/m3 (1.0 ppm) of 0 3 . The data indicate that 03 exposure 

can cause both acute and delayed changes in cardiopulmonary function. 

9.4.3 Hematological and Serum Chemistry Effects 

Hematological effects reported in laboratory animals and man after inhalation of near-ambient 0 3 concentrations (< 1960 pg/m3 ; < 1.0 ppm) indicate that 

03 or some reaction product of 0 3 can cross the blood-gas barrier. In addition 

to reports of morphological and biochemical effects of 0 3 on erythrocytes, 

chemical changes have aiso been detected in serum after in vitro and in vivo 

0 3 exposure. Hematological parameters are frequently used to evaluate 0 3 

toxicity, because red blood cells (RBCs) are structurally and metabolically 

simple and we11 understood, and because the relatively noninvasive methods 

involved in obtaining blood samples from animals and man make blood samples 

available for study. 

9.4.3.1 Animal Studies - In Vivo Exposures. The effects of in vivo 0 3 

exposure in animals, including studies reviewed in the previous 0 3 criteria 

document (U.S. Environmental Protection Agency, 1978), are summarized ~in Table 

9-18. 

Effects of 0 3 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 pg/m 3 (1.0 ppm) for 30 min. Goldstein et al. (1968) 

reported a significant decrease in RBC acetyicholinesterase (AChE) activity 

after exposure of mice to 15,680 pg/m 3 (8 ppm) of 0 3 for 4 hr. Menzel et al. 

(1975a) observed the presence of Heinz bodies in approximately 50 percent of 

RBCs i n the bl ood of mi ce exposed to 1666 'pg/m3(0.85 ppm) of 03for 4 hr. 

About 25 percent of RBCs contained Heinz bodies after continuous exposure of 

mice to 0.85 ppm of 0 3 for 3 days. Heinz bodies are polymers of inethemoglobin 

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. 

9-159 
m 41 

o~ O 

u 

s%. 41 a ro 3F c 

4- ro 03 r l~0 'C 

0

O ' Of 

W ro Ol U ri 

'L 01 

O] ri Gl 

> 

n~i +~ +) 

u .n a 

ai 

a 

a~ ++ a a ~ 

a ro 

N 

ro ro o 

L - d 

V! VL ro 1 U N 4D oi 

N ~o 

ro 

t0 +L- c 

u•~ ro 

•~ L io aUv G oc C! ro :30+1 L u ro 

a = ~ ° co +•Ti aro u 

cr ° m °u c u u o 

~«+ u ~ c 

U ~ S. 

v o O) c o rnvi 

ro6 4:~~ ~O ro a 

kV 

4~•. 

O O 

C 

Vl 

A 

1-

U Y_ C. 

¢r 

r0- 

a° 

N E L•.C

- 

u G~. 6~ O7 

N i ~ V- 

+.~ o 

~O G) € c rn 

1000 

L L C 10 

GO ~ L 47 ~ 10A 

rt1 O c 

V~1 r 

O V1 d N L ~n ~ n u 

~ n 7 U > O O rt1 

° IOiI L L •%- fO N tOnU tA ^ Z 

fU L N W .t G/ L 6ro1 ~ 

U•

u 

 O. U rl O u.G u f0 ~ 

O~ C! C.' O 41 rC-~ V

d

i C-i ~ b 

'O 

ro rar roaY

i 

~ O ~ V1 

T 

L 

L 

\ ul \ N L uf Y 

cu 

W~ a M tl .c .00 v 3 

~ O1 N a• CO u7 M 

ro.1 

G 

~ O 

O N 

ro~y E U O Z O 

S 

to 

C P O t0 rlNat N N O G 

O, 

41 

O 0000 O O 

C L 

O ~1 

O ~ v 

u 3. r~-1 r~i N ~P Q

~

1 CN~f M .. 

W oc 

CD

~ 

W 

>I 

~ 

ZI 

~r 

~ Jg S_ 

~ 

~ 

9 S 

O 

~ 

~ 

Ol 

~ 

~ 

00 

~ 

~ 

~ 

o ro Ol r 

ro 

t0 

ro 

O 

ro 

~ d ~ d v 

a rn C c rl to 

L rl L O 

' C • c CO 000 O N 

> 

Gl Ol 

>~ 

O Of 

f14 2: (11 

rtl 

m 

Of 

n 

4' aroi 

a 

a 

m n a. 

ar ai 

a) n 3 c 

C F 

m 

N Z C..1' 

L G. 

•G1 

~ to 

T7 N 

ro U C 

+) 

+~ C C O •>Ol "O Cl E N 4- L 

° c G7 4~- U — ° 

a T Gl eo in 

 i.0.1 7 L 

". . 

i•~ o~cq~ a 

2 ~ ro •EO E U 

ac—o L 

O Of Ol 0 U N m r•~ o c L c t v c i c a c E= u m r E•.- ai •"- i-) 

O 

O. N O Gl 

en E .• Of 

O) 

ul • '0 O 

r V1 c'O 

'O E t+~ Ol +J 

~ L•.- L L C L N ~ G. p1 r O 

a c'~ i o v vi •~ oj, cii '"~ m a ~ 

 i,_ , d'f;, C 

a E u .c 

ro++ 

c.t a ai o 0 or d aiv mv u

~ m~v u0i Cd X X a'Oo 

a 

~s- 3 cii L O "U O C E O - . 

L d t0 t~i7 t~n = vr > E ~~ y O ro U•^ L Ol U O 

U L. O O ~0 •U 'O N N +~ -O ..U-. r- 0~p L7 G) O a> u To.c.E m > v•r .. U n aiO ar ~ 

c 

° m ~ v ~ v°1- o u o ro T r 

ro 'c 

E>•,- •'- r..- 

N•r E E G.+> 

ui 

>> 

ai ~ n t ~ n 

L ro ro v- U v v E v- a~ 

Gl u1 O ~~ L f0 G7 •r 

tn i~ 

IV i.~ u 

O. /0 r-1 M•rC• .% 

U r 

C O 

u 

~C-i G. ~ O. 

~ p O U+~ 'O > 

Z J.O v0 ' '> U 

~ 

Taroi~ 

n t C N t 

 tD r-1 N M 7 v CM 

I 

> > > > > I 

I 

I 

I 

N N O N ~ n en M 

O O 000 O O 

1O N 00 

N 

c07 

N tD O O 1- CO 

~ rl d• Q N 

0

1 •-M-1 Lf7 

9-160 
io 

41 

ar 

aa 

L. r-1 

o ao 0 0+ 

E ~ 

G 

N 

t 

N 

S N 

t.7 

G1 iJ 

I u 

I O 

I L L 

I 4-1 

I i 

N 

a 

a 

O 

ro 

d 

L 

u 

G! 

O 

t 

~!f 

n 

N

L 

(~ 

0 

O 

~ 

o+ 

Y 

u 

> w 

•~ Cl 

• Of C 

ro t ... 

w- w 

.0 

N Q 

N 2 d 

U C;I t0 

~c. a cI 

d 

vl G7 O i J 

C a O 

~ i0 

ta 

d d 

~ 

a d 7 

C 

~ 

C 

0 

v 

W 

C 

~ 

N 0 

X 

w 

>I 

ZIN 

~ 

~ 

H 

Z 

Q 

} 

J I 

O 'I 

E ui 2 

r0-I 

01 

W 

J 

m 

Q 

H 

L 

G7 

.~ 

ai 

~ 

a 

c 

~o 

a 

u 

c m 

L 

O1 wrL 

ai 

C 

G/ 

.0 

a 

t 

N 

•1•~1 v w w a) 

a 

N 

L 

d 

0 

a 

aror L O 7 C V 

O ~ 4j 

X ~ Rl L 

W i C. 

a 

c0 

41 

C N 

E a m o 

3 41 

ro E N 

S 

c E 

O G. 

T

1 CL 

ar ro C L 

O r~ N C 

° u E 

c 

o rn 

u Z 

O 

O 

Ol 

ri 

m 

4J ai 

m 

L 0 

~ 

N 

7 

Z 

a 

i0 

°a : 

I U) w 

CD I GI C 

N a S 

U O > 

fO..v ro 

i67r~~ 

aaa a~ ai a~ m •'- 

N N 111 a 

aroi aroi aroi t 

L L L 41 U V U O 

caica 

H a H V 

L w 

i~ +E 

L r~ tp 

L ~O+1 

lD +I > 

> 

~ 

N 

M 

O 

n 

i 

G7 

.~ 

ar 

~ 

a 

c 

ro 

C -1 

ron _ o 

d rl 

i~ 

a 

~ 

ro 

C 

L 

O 

~ 

a 

.L 

C 

C 

y 

a ~ 

E 

7 

i• 

O1 U) 

C ~ 

ro •~ 

u ~ 

CL 

Z U 

Y 

C1 

aU)++ 

L •ro0 E 

4O u7 t0 

G 

Z 

II ~ 

I O 

~ 

M 

n 

o+ 

~ 

i 

ai 

rn 

ar 

"7 

i•~ 

a a 

~ 

CI 

N 

L 

Cl 

4J 

ai 

C 

N 

4J 

L 

7 

a

tn 

cn 

L 

Gl 

C 

c~ 

ro 

u 

u 

C 

H 

Y 

ar 

a\ c 

L L.ro0E 

W ttf r°•1 

G 

Z 

a 

O 

~ 

n 

L 

N 

O) d ~ 

a 

c ro 

C N 

ron _ o 

1 rl 

N 

n n 

z 

N 

LOL+~ 

Gl O .0 ~ 

v •~ -u c 

O m E ^ 

U 41 41 Ol 

C C O C 

az ro 

7 C U 

~ o • V N O 

ro a ~ z 

7 N 7 • Y1 

L ro C C C 

N Ol O •r •~ 

~ V O) 7 4.1 

ac~ .no NH.o o a 

L C ro 01 7 

c 

C1 ~ 23 •r H 

Y 

ro~~ 

a Y1 C 

L V ~ 

tOLnr°1 

Z ' 2 

v v 

O O 

n n 

9-161 

N 

n 

~ 

~ 

ro 

4J M 

ar 

N 

c 

ai 

Z 

ro 

C 

N 

G! 

~ w ~01 

O C •r 

L •r d 

-.5 ~ aui 

i ~ L 

GI > 4J 

O O i fn 

wacc 

uf 4J t E 

A ai 3 a ~ 

r 

°E ani rn a 

L am w 

a r u w 

ouErc i 

a`

o

icue L 'OUf f0 

cw- o ~ 

H O Z > 

U1 

7 

C >> 

C ~ 

f~ N 

~ N 

~ 

~ 

O 

co 

o+ 

Ln 

n 

r-4 

co 

+~ 

C! 

3 

0 

t 

(..) 

d 

O 1~Q I S L 

> ~ 

rov2i>, 

L Oy 

O G! C 

1!f •r~ . 

>~ O ai •,- n 

2 L O 

C7 d-O 

L 

~ S O 

Ol V- UOI c o ro 

t w c

+)i vi 

V 61 7 G/ 

O+' aal T Z •.^ L .0 

1~0 

t 'rod 

ao n 

Y 

m 

2 

Lf! 

O 

0 

rn 

M 

W 

Cl 

rl 

ro 

a-) d 

C 

Y 

L 

~ o 

J 

N Y 

c 0 

Z 

a u; u 

roai a ar v c 

L t7l O O 

uai aroi e

410 4J 

a L E U 

C ~ Y O

O

- ~ H W 

T d 

00~ ul U1O 

t TT 

L O O 0

0' U N C7 C E N•2; O 

OVO aY 

~ 

 O ~ 

C V- V- L 111 

O O QI O 

aar L L a ~ L GI N ul O 

a c c (aU- 

~ 

a 

L `!f 

00 r-1 

~ 

~ tD 

O 

~ 

un 

~ 

G t0 

mn 

0. r-1 

iJ 

d 

a 

~ 

C7 

N 

L 

d~ 

a~i 

C 

.~ 

i•~ 

O 

L 

a 

E 

L N 

co 

C 

d 

N' 

V 

C 

H 

O 

L 

C 

E 

I~i r°•1 

O 

n 

Ol 

~ -i 

~o 

~ d 

Y 

i 

ro 

C-) 

d 

~L 

c 0 

Z 

G Z 

L!9 

n 

O 

0 

n 

~ 
Y 

m 

Z 

C~ 

O 

~ 

~q 

Y 

m Z 

~ 

O 

t

coD 

un rt 

Y 

a 

c ro 

Y 

~ 

C ro 

on .0 Q1 

C7 

4-) ro d: 

~ 

a 

• i~~ 

-0

rC 

N G.G -a u.0 

r• a•r 6• N ~0 O 

f0 •r .0 C R1 r• 

f0..'O V-C4- a ~ EL~ ac oto aNC;JN =a 

dr0 9r•+~ Iv ~4-a 4jt 

s c_ acw 00 .G 

(N,'J tn ro a ~ C Of N•p ro ~ 

ror• u — 

a~ rxo 0 

O Y ~~ d ~na-•r~-• Lt O7 

T c av• 'o a a+a o 

4. i0•~ Ol U W a 'O+> >V- N N > ro o•a L a u N o.c+-+ 

•r- > L a C iJ i•~ ~II Y T d..1 7•D L•r O G 9 N a U 

f.l L>> ~ a C C r• € o 

roC a•'

G

•• ~ C N•r 

Val a aN •> •N C C VOi ~ C~ 

ro N 4-1 ro V•• r' = ro ro c ro a 

L~~ L O ~ N !.1 10 •r V L 

u•r u us•r ~ .N o cov 

N o ~ v CM.7 ¢ ro c u A L ro 

N 

'O b 

n 

n 

n 

~ 

~ ro }1 

a 

3 

G V 

I d .X c 

s° 

C 

O a 

~ Y 

~ ro d 

v a 

id ~ N roc a 

1O N U i r• r O 

L 

4J Q 41 

49 +.T+ a 

C •> L 

r O 

ac

4Jiro 

c ro ~ 

r a ~ 

u C d 

b V to 

.LC •O 

G) n 

ro ~ ..A 

I ro 

~ d 

a 

c a S 

i 

I a N 

~ 

~ 

I 

a i 

~ N N 

O 

0~7 X 

z a 

4 m ~ 

i aNi ~ ~•~1 

0 0 .a u 

2 3 a 

N a 

~ aroi 

L cii 

cv tr v 

O 

7 N c T 

C ~ 

L I 

L 

~ 

d• 

I ~ 

I~ 

d 

3 O 

L 

C.i 

ro m 

I 

d 

47 .; 

a=+ U ro 

a 

T N 

O 

-

>1• 

v d 

N 

aroi ai 

rGr V 

v G ~ o 

en 

G! 

~ 

i M 

+~+~ a^

i 

Y ~ d= 

rr- 61 

u i N 00 

4J a 

d 

L 

O 

' O 

ro 

a N 

~ 

~ 

1 

L 'O d•a 

0 Z 

47 

LU 

~ C 

L ~ a 

~ 

•> C 

I ol C •ro0 

eo o •r 

~ 4J o 

W 

C 

• E 

IO N .; 

L 

t 

-It 

~ 

ro ro 

'a

J

i aci d~ N Ll7 

u 

CI•^Ul (U oM•r N N I,- L a O1 L•r ErIC.)C7 

a N 

~ 

4-) 

u 

O i 

4J 

L a 

O y 

a C7 

c 

~ O +~ N 

vi a Of c 

ro 

r U 

'z 

b 

'O 

n I 

L 

O 

:L 

rE~ 

O 

M 

ro 

41 a 

L 

'O 

aci r~ 

rorn 

V ~ 

tm 

• a 

ro a 

~ •~ 

9= rn 

a L 

d ~ N N 

N ~ 

U r= 

c a r, t 

u G 

a L 

W 

a 

S 

N a 

e 

a d N 

.O 

N 

v 

~ 

d ~.. 

d 

v a 

> 

L 

a 

a 

O 

~ 

G i 

L ro o 7 cu 

N O O 

~ ro s°. L d 

V 

% 

c a ey v a o L .G 

7 a.1 

N a 10 ~ 

a 

S 

C ~ 

~ d 

~ ro G L O ij 

N G 

O ~ ~ 

c 

°u 3• 

a Y/ 

~ 

N 

L a 4J 

Et m u 

C 3 u L 

o c ++ c r+ T 

o ° aii 

qJ U •r 4-

roa ~ o 

v~i c •c a 

Va ra C ^ i C rtJ 

0 4

4.,

- 7 N a ro •r N 

Lo 

Grol N E L L •r N O 

C fr+~ a ro 

Y 

a 

L 'ro0 E 

CO Lf9 t0 

5 

G > O = 

CO 

O r-i rl rl N 

1 

LO 

1-1 r~-1 r~-I ~ M 

~ 

7 

c 

~ e O 

u 

W ¢ 

~ N 

a 

W 

o) S 

7 

M xl

1 

J 

~ 

~ 

~ 

~ 

O 

S 

t"1 1 

Qf 

W 

J 

.r 

■ • 

9-162 
ro° 

C E 

.G L O 

pf N 

L 1J 

X S. 

O C 

C N o u 

NO 

+•1 U 

U 

40-• d c 

w•o• - m t a M C 

Z N a 

L 

~ 

N 

v 

U 

G1 

V- W 

N 

a 

N 

> 

i 

d 

a 

O 

co 

r, 

.. a 

N 

~ G 

c 0 

v 

W 

O.' 

7 

N 

O 

d 

X 

w 

~ 

Z~M 

I 

J 

~ 

¢ 

r 

c~ 

O 

0 

F 

2 

co 

01 

W 

6 

F 

rn 

~ 

r ^ e0 

N 10 0

4J

1 

c ++ c 

tn 

N 61 f0 

4- N ' 7 00 

rn . Q 

N at +•~ ,o 

a 

•i~ n c 

N G' C.7 

~ ,C 

o aNi 

, 

}1 C c aCl 

Nai ro•dc~io 

O c O w 44 

L •- L 9- U 

U Ol G. ro Ol 

E o C .Gtv- ~ 

i O.r O O 

N C~ ~Z 

d r•,n+~ N` G c V1 Uf 

,0 +2 G7 Gl GI N 

Ol••- •O Lr•a 

cii c a G E •iC N O 0. C/ 0! M a r 1 O ~ F i.7 

•O 

a ror 

L O 

7 C U 

O r +J 

W L G. 

•~O 

C E O O 

~ G ri M ri 

C L ~ 

N C 

° u @ o 0 0 

O 01 01CO 

tD O1 

u S r•1 tn rl 

~ 

cn 

,6 

ro ro 

y C 

y ~e t. 

O ~ f 

~ d 

o ~ a ab . 

~ G S. L aL 

10

+i1 i0 lO..i0~1L tYA ~, 7a ~ ~ tLO, 

c+t e at o at a, r a> etl d+- 

a 01 0 ~ r- +•t i •~ r Cf 4- N 

c U+,r c • NaL o•~ c ro t0 u 

ro C O•.~ C! G1 O L i •.- Gl v L Y1 . OC L Gl N. O.a u Y i 1•,,C 

N CJ •r-• Ol +~ t0 O•~ C + U h-1 -> 

ad N•O.. i u r ~ N t Y C-~ rr 3tF . 

°• E ro ~ o aVi ~a~ovi.. 61 x otS d O. 

r G Gl O O. a ro•~ O 1 N G. • +t CI 

C. L L•~ +J N C ro. C7 ~ •t ro> 

E u U r ..• O c O•~ G! /'~ N t••, r•.- Ol 

+1 CN O L tD . ^ Gl +101 

i C7 ~ O a 11 4-1t u O C G) N L_ Rl RI 

C1 a•f • 41 E O Cl O> G1 L U F~ 

N t0 t. L. •- Cl ~ . = L C. L a O N Gl +~ O•~ ~o 

IE 4- N.}f N ~ L. Df, C. r>> u a a 

c N 41 1 C N C! GI •'- O •^ II c U c 

O G G C N N a N L r 4~ O r O N•r ia r•~ o•.- 10 O. G G! ro 

ij GI e- •Gl t O i a ~ L r O v +J t0 •i- +t ^•r 

U ~ i•,L L GI O! r•1 Gl O U v L>> N r 

d O a O Of U U N +~ ~ N 61 u u 07 d 

4- i 61 L. •~ >, 10 O N i•~ O GI Y- N C! O C L 

V- O_}1 O.L r r 41 0l •~ a 4,• O C 3 ~ 

a o ro o ro ot i 11 N Il • d•~ • ~ o 

araa.~•~ . u oc ~ a 3 +~ r a 

O•~ Gl •. C O L C O..0 C) G. Gl O t0 U U C C 

Z r L - 10 14 +- r v U Z7 v U Z L. S G) •- G7 

a et o 

v ro 41 

ISS 

O C E 

N G! 

Of L 

O ~ ~ II 

a 

W 7 L 

u a+1 

 U 

N N a 

C. 

a II 41 ~a d 2 u o 

i tn ro r 

rn 4- O 

V- ^ 11 E 

o a+ at a N xt ro c 

ro iai J u 

L. iJ • a 

G1 C U 

c - ro e 0 

11 t ~ •N 

rr U + ~ N 

m >, v% 4J 

Z ~ II a 

L ro ~ II 

~ 11 Y 

O L tn 61 

L aU a 

 to 

nG1 a X 

C 1-  y 

t Of C n 

> O O d 

O L t0 L 

c v ~ ~ 

u 4) O) 7 

c 

% 11 tT 

a a u 

' z ~ a E a U 0 

L, y y a) c o • 

u N L ro 

N U t/1 7 N L ocm Nw 

rLU 'ol6l a •~ N •r II . ~n a 

CI •N G 'ro0 '0 IGO a, o. •r ..- r-• 

' 110 ~o 0 o ro c a 1 L i.F ~ Z ~ • w O CA OJ N N a 

S •- O_ G O • 

U 7 L L II r d vN C. u 

C f6 4) H v O 

+

L

•1 °o lJJ >f 

•LL O R1 N GS L 

U GI O p 1 • 

N f/ 

a f0 N C ~ a 

++ a+ N II L tL •.- 

° i n °N'i,~ ' 

u.. rora a 

U a O1 c O CD u m•i o ro L 

z L ~ d 

. C~

! . 6L

1 O ~ E 

a E a GL Ul U 

O O G! O. O • .Cr NS•~ f-. L 

m o o ~ v a ~ 

E u ~ •^ M11 Eo 

1•a / O N N 2{ 

E Y t0 ~ il CU3 G 

i II •>a+a ~ s 

N1-- LbJ ~ iJ U 

f0 NQ L 7 M•r- N 

~ S L6 O1N U tN 

L 

L 

d' 

~ 

3 

C 

C 3 

U N 

G ~ 

c o 

+~ • L„ .. 

f~0 •N ~ 

a >, >, 

°r-4 ~ 

ro 

a 

, 

\ b ~ O 

LaL•,- 

,n r-

`n+ 3 n 

aaa 

M -* -t 

10 

G! ~ 

ro w d 

E 

Ln 

n 

.-1 ,••1 M 

O O O~p 

r~•1 ~ ,If 

~ 

. w x 

c~ 

O lD 

~ 

~ 

u"f 919 

ri ,O C70 

O O O 

t ^ 1 ~ 

N

O

~ ~ 

9-163 
In more recent studies, Clark et al. (1978) investigated the biochemical 

changes in RBCs of squirrel monkeys exposed to 1410 pg/m 3 (0.75 ppm) of 0 3 

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 3 bn the oxygen-delivery 

capacity of erythrocytes. After exposure of rabbits to 1960 or 7880 pg/m 3 (1 

or 3 ppm) of 03 for 4 hr, no changes were detected in RBC 2,3-diphosphoglycerate concentration, oxyhemoglobin dissociation curve, or heme-oxygen binding 

of RBCs. Analysis of blood parameters 24 hr after exposure revealed no delayed 

effects of 0 3 . 

Alterations in RBC morphology have been previously observed in 0 3-exposed 

laboratory animals and man (Brinkman et al., 1964; Larkin et al., 1978). 

Similar observations have recently been made in monkeys exposed to 1254 pg/m 3 

(0.64 ppm) of 0 3 for 8 hr/day over a 1-yr period (Larkin et al., 1983). 

Ultrastructural SEM studies of RBC's following exposure to 0 3 demonstrated 

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 0 3-exposed animals. 

Moore et al. (1981a) reported reduced RBC survival in sheep exposed to 490 pg/m 3 

(0.25 ppm) of 0 3 for 2.75 hr. Similar reductions in RBC survival were reported 

following 2.75-hr exposures to 0 3 concentrations as low as 118 and 235 pg/m 3 

(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, pyruvate kinase, and LDH activities, and a decrease in RBC GSH after exposure of 

vitamin E-deficient rats to 1568 ug/m 3 (0.8 ppm) of 03 continuously for 7 days. 

These effects were not observed in vitamin E-supplemented rats (45 ppm of 

vitamin E for 4 months). The activities of G-6-PD, catalase, superoxide 

9-164 
dismutase, and levels of TBA reactants, methemoglobin and reticulocytes were 

not altered by 0 3 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 pg/m3 (0.32 ppm) of 0 3 for 

6 hr. Animals were maintained on vitamin E-deficient, or supplemented (3.9 mg 

tocopheroi/100 lb., twice the minimal daily requirement) diets for 6 weeks 

before 0 3 exposure. Mice on the vitamin E-deficient diet showed a 24-percent 

increase in G-6-PD activity over controls after 03 exposure, and mice fed a 

supplemented diet exhibited a 19-percent increase. Decreases in AChE activity 

were observed in both vitamin E-deficient (19-percent decrease) and vitamin 

E-supplemented (12-percent decrease) groups. 

Dorsey et al. (1983) evaluated the effects of 0 3 on RBC deformability 

after exposure of vitamin E-deficient and supplemented (105 mg of tocopherol 

per kg of chow) male CD-1 mice to 588 pg/m 3 (0.3 ppm), 1372 pg/m3 (0.7 ppm), 

or 1960 pg/m3 (1.0 ppm) of 0 3 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 

of mice to 1960 pg/m 3 (1.0 ppm) or 1372 pg/m 3 (0.7 ppm) of 0 3 and incubation 

of RBCs for 6 hr resulted in a significant increase in filtration time of RBCs 

from 0 3-exposed mice, and a lack of protection by dietary vitamin E. The 

hematocrit of vitamin E-deficient mice exposed to 1960 pg/m3 (1.0 ppm) of 0 3 

was significantly greater than that of nonexposed vitamin E-supplemented mice. 

The increased hematocrit was attributed to a ioss of RBC deformability, and 

sphering resulting in decreased packing of cells during centrifugation for 

hematocrit determination. No TBA reactants were detected in the blood of 

exposed animals, with or without vitamin E. 

The influence of vitamin C deficiency on erythrocytes of guinea pigs 

exposed to 588 pg/m 3 (0.3 ppm) for 3 hr and examined 0.5 or 3 hr post-exposure 

was studied by Ballew et al. (1983). Ozone caused no effect on reduced glutathione levels in erythrocytes. There was a' slight increase (p = 0.08) in 

percent methemoglobin in 0 3-exposed animals. Vitamin C levels did not signifi - 

cantly influence these results. Plasma L-ascorbic acid levels were not affected by 0 3 exposure. Hematocrits were decreased in animals on the low vitamin C 

diet that were exposed to 0 3 . Hemoglobin and red blood ceil counts were 

unaffected by 03 . 

Moore et al. (1984) infected mice with Piasmodiumber hei (a blood-borne 

malariai parasite) 1 day prior to exposure to 588 pg/m3 (0.3 ppm) 03 . The 

9-165 
exposure lasted for 3 hr/day until death, or approximately 2 to 3 wk. Mice 

exposed to 03 did not live as long as the controls. Ozone-exposed mice also 

had an increase in the number of parasitized red blood cells. The authors 

hypothesize that there are 2 potential mechanisms responsible: 0 3 may have 

altered the erythrocyte membrane, making it more permeable to P. berghei, or 

03 increased the reticulocyte count, reticulocytes possibly being more sensitive 

to P. ber hei infestation. These results are consistent with those of Schlipkoter 

and Bruch (1973), who reported, without statistical analysis, an increase in 

infestation with P. berghei and higher mortality in mice exposed for 6 mo 

(8 hr/day, 5 days/wk) to 1686 pg/m 3(0.86 ppm) 0 3 . 

9.4.3.2 In Vitro Studies. The effects of in vitro 03 exposure of animal 

blood, have been studied by a number of investigators, and these reports are 

summarized in Table 9-19. 

The effects of in vitro 0 3 exposure on human RBCs have been evaluated by 

using a number of different end points, such as increases in complement-mediated 

cell damage (Goldstein et al., 1974a), formation of Heinz bodies (Menzel 

et al., 1975b), decreases in RBC native protein fluorescence (Goldstein and 

McDonagh, 1975), and decreases in concanavalin A agglutinability (Hamburger 

et al., 1979). Exposure of RBCs or their membranes to 0 3 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 3 caused spherocytosis and 

increased fragility of RBCs after 03 exposure. (See Tab•le 9-20 for a summary 

of the human in vitro studies.) 

Kesner et al. (1979) demonstrated that 0 3-treated phospholipids inhibited 

RBC membrane ATPase. Addition of semicarbazide to 0 3 -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 03-containing 0 2 through the solution at 4 ml/min (2.5 pM/min of 03 ) 

for 1 or 9 min. Semicarbazide (40 pM) or PABA (40 pM) inhibited the crosslinking of 03-exposed spectrin. The inhibition of AChE and hexokinase activities of RBC ghosts exposed to 0 3 was also partially prevented by these two 

agents, as was K+influx into whole RBCs. The authors attributed the inhibition 

9-166 
=I I O 1., 

V r-i 1O 

O N 

~ ~ ~ 

m n 

rn 14 

.~ 

+~ 

N 

a 

0 c~ 

LLJ

~ 

N O 

X 

W 

~I 

f•' 

> 

M , 

I ~ 

J 

~ 

¢ 

} _ cM 0 

0 

. ¢ 

~ 

W 

x 

01 - 

~ 

rn 

W 

J 

m 

F•` 

ai N 

rtl O 

0: E 

~ 

N F~1 

~ ro a v 

N N r- 01 

da 

ro ~ 

ro a +J 3 

U N 

~ E 

T•~- 

o ro ~ 

L O 

ba. 3 L 

Q G C 

+~n ° E 

N 

L n I.c v++ 

0~3 

C 

•E ~ 

CD 

Q1 d• 

M 

Y ~. 

Z 

N u 

c 

d L 

N w ai o: 

N 

a 

u Gl 

C. 

N 

w 

v 

41 

V 

4- 

u- G1 

~ 

d 

> L 

N 

a 0 

~ 

C 

v ro ~ 

L O 7 C U 

N O O 

O •'- +J 

W L C 

7 

~ 

to a-) c 

a E •O 

G! O 

L L 

. 

7_~ N ,0) i0 E d Z 

C 

O 

4J 

C L 

O d) 

O C! 

u 

c 

O 

V 

o+ ~ 

c ,•-I ' 

ro 

L C - 

rnv L +J 7 (n av 

~ o 

x co 

4.) 

~ 

0 L .L 

~ 

i 

N 

I w O 

o • . a 

C •rC7 > 

.- f0 

b U 

C 

wa ~o 

d N 

U 4J 

da 

o u 

i 

L 

N 

Z W 

x . t~ 

cn 

r- 0

, 

m 

4J 

G! 

G1 U) 

~ 

ai 

N 

7 

O E 

~ 

a a> rnt 

C 7 

L ro 

u N N 

E V 

Z N iJ 

10 N 

Ni~ 

c_ ro ro aJ 

O ~ N 

~ ~ O aroz 

E L 

c 4- fJ 

+~

u a 

U C N 

a m V- cnz 

V- L O. 

N O W 

z•~ a 

L 

L 

~D 

~ 

~ 

~ 

r1 N 

l0 CO 

Ln o 

N -0' 

ai 

~ 

v 

0 

~ 

•N 0 

ro +j 

O a 

'v 

ar L 

d W i 4- 

~I 7 .II 

ro L 4J 

~ 

41 

C 

II 

Y 

m 

Z 

L 4J 

E 0 

44 

L 

a 

> ~ 

u 

> 

~ 

N u 

e 

a~ 

u 

N 

N 

~ 

.~ 

G1 

L 

V 

C/ 

N 

ro r 

a 

t/N 

f6 

01 

f 

W 

2 

U 

9 

O 

L 1, 

Gi 

E 

i! 

C 

d 

N L 

7 

N ro ar 

S 

ro 

9-167 
W 

C 

~ 

~ 

O 

x W 

OI 

h•• 

> 

2H I

1 

S 

~ 

s 

~ 

C:J 0 

S W 

S 

O , 

~ Q1 

W 

J 

m 

9 

m 

a 

C 

Y 

~ 

O L 

~ 

~ V 

E co 

S v 

~ .; 

~ 

U m 

N 

~ 

¢ 

a d 

N 

ro 

i 

uai 

C7 

d 

N 

C 

~ 

I C 

m 

E 

S 

a 

N {,t 

I C I ar > 

I N 

L 

I1 

C W 

O 

+o CE ~ 

o 4-~ v- > 

o ro 

a +~ 

ar 

N •rC a 

• d T 2 d 

~ 

~ 0

, 

~ 

~ 

ro 

L c~ 

a 

~ 

a 

L7 

r, 

m 

~ 

m 

+1 

Cf 

b 

4J m 

c 

~ 

O Q^

i C.7 r-1 

C 

m 

E 

~x 

i 

O1 u 

a 

a~ ~ m 

a 

N 

E 

~ 

C 

E 

a 

a 

E 

co~ 

a w 

N • 

m v 

L ro 

c ~ 

N a 

L 

L 

N 

f-1 Y 

m 

Z I 

^ 

E 

a 

a 

O 

v 

~ 

r= 

Of 

1 

p 

p 

V 

W 

n 

~ 

~+ 

~ 

i0 

4-) 61 

L G1 c 

G/ 

Y 

~ 

O L tm 

to V 

~

to 

2 v 

~ .; 

4J 

O 

ro 

1 

~ ¢ 

a 

ar 

~ 

ro 

N 

L 

d 

O 

a 

ro 

LI; 

~-i 

•• C 

~ •E 

..p 

tlJ N 

ai x 

a •,- cD 

00 

Nri •L • E 

O X \ 

C 

\ 

L p C 

+~ 1 

01 O 

O 

1f7 

t0 

O 

C 

E 

p 

M 

d 

U 

N 

i 

w 

d 

C 

N 

d 

. ~ 

d 

O. 

~ 

N 

v 

Y u 

w 

w 

N 

a 

c~ 

i m 

.G 0 

a 

c 

L O 

7 C U 

w O O 

O •.-10 

W L C 

7 

a 

' y 

c 

m 

~a 

L L 

m E 

SI 

G 0 

rmm b 

O +1 

N C 

O V 

C 

O 

V 

~ 

o~ 

~ 

~n 

.~ 

d 

i 

d Q1 

7 .n 

b 

S 

C 

ro 

~ 

S 

T 

.~ 

N 

U 

m C 

4- 

O 

c O 

ro 

C 

~ y ~ o 

~¢ 

ro ~ 

aro 

~ b 

ro c 

L t%i 

v c 

O7 O 

O U 

i 

L 

N 

t 

LO 

O 

~ 

G 

V Z 

^ 

.~ 

a 

~ 

E ^ 

a E ' o n 0 

L 

~ O. . d 

O N 

O • O 

s 

~.+ c 

cq •o 

ci p\ 41 

~, 07 ro 

S d 

pO ~ 

0.7 tD 1 Ol Ql c0 

P'i p 

t .~ m d 

x 

a 

ro 

N 

on 

Y r~-I 

C 

m 

~ 

S 

d N 

ro 0 ai4JH 

V1 L Q 

cm °n a 

¢ mm 

O 4J L 

41 C Gl u oa , 

v°

)

~ - ~ i V ~ 

a 

oa+) . 

za~ a>, w o +~ 

i ro 

L 

s •.- 

~ pr> 

U U U+J 

m N m U 

zc ~ m 

(0 

O 

~ 

~ 

S. C 

o n °1 ro M M> a o •~ 

7 

O O CI 

~ O ~ 

7E N rl C U 

I 

~i 

Y ` 

(D 

m 

~ 

C 

O 

c 

O 

'7 

C 

ro 

~ 

x 

a 

N 

4- 

i 

7 

0. O 

O •'

4J- 

~ L •> •ICi-) (l 

u b 

a ~ 

W 4-) 

ro o 

vI 

C t~ 

M ~ € 

00 

N u7 

..a 

O ~ 

I ro 

4-+ 

ar 

C m 

cn 

E 

i 6^1 

' LL ri 

I ^ 

w 

I 

4J 

O L 

01 

bu 

Ez I 2 v 

I 

I i 

I 

c G. ~ 

M 

d 

' T 

d 

a 

L y 

GJ 1A 

~'r u 

rna 4J 

aa n v a •~ 

n n 41 

m m m 

L L N 

u u 

O 0 Z 

G 

Z 

1•-1 Y 

Z 

c 

\ E 

C 

p 

p M 

'a a 

~ 

~a 

c I ro 

I ~ 

d00 

~~ 

^ 

w 

~ 

N O 

~ L 

C V! Of 

ro~ L. zm S v Z 

C 

0 

41 

u 

VO- Y 

v- m 

01a 

O 7 

Z 

G7 

0 

x U 

LO Cy r 

rn 

a 

ar c uf O 

ro 

i O 

aui a 

C 2 

L 

N 

O 

h-1 Y 

Z 

.G 

S 

4- 0 c 

~ •E 

E. g 

p 2 

Qi ~ 

1 0 0 

b 

4J 

v 

c 

I •ai a^ ~ 

I ^ w 

+1 

O L Of 

(Ctl u 

E m 

~ C 

x v 

, 

N 

7 

4-- 

C 

C1 N 

241 

C. W 

> ro 

~ a m cZ 

a 

a~ Y1 . 

m a~ 

L C U 4) 

a u G N 

c 

E 

p 

~ 

a 

m 

p 

N 

H-1 Y 

Z 

^ 

E 

o. 

a 

O 

~ 

.. 

M 

~ 

m 

1 

p 

~O 

Ql 

r'I 

E 

r. 

H 

C 

E 

E 

1 

1-t 

. p 

u'! et' 

! -1 Y 

C m 

Z Z

m 

Z 

9-168 

~ 
G1 

u 

G/ 

CL N 

~ 

~ 

N 

C 

~ 

C 

O 

U 

v 

w 

C 

~ 

N 

0 

X 

W 

d') k ~ -• 

> 

ZI.. 

Q 

f 

~ 

2 

>- 

c9 

O 

J 

O 

H 

Q 

~ W 

S 

0 

N 

Q1 

w J 

m 

Q 

F 

~ c dmr L O 

7 C tJ 

N O O 

O •r +J 

G+~ O 

x ro L 

W L d 

7 

V 

.. ~ N I 

~ 

41 

U , 

m w 

4- 

N , 

V ' 

d } I 

G1 '' 

N 

.G 0 

a 4J c 

a 

E 'O 

G! O i t 7~ 

UI N 

m B d 

~ 

0 

ro 4-) v m C L 

O N 

N C 

O G1 

U 

C 

O 

U 

N 

~ 

~ 

O 

E 

7 

N 

N 

m 

++ 

O 

O. 

V 

N 

i 

ai 

w 

~ 

a 

~ i. 

4J 

C7 

C 

Y 

m 

Z 

ei 

U 

G7 

u 

d 

C 

.~ 

7 •E ai 

t 

U 

Gl 

N 

•ro 

N L 

~ °• 

r N 

m m } pf 

v 11 O1 

4J f 

L W 

O x 

O.0 

N 

L ••• 

~ 

T d 

a a 

'O •L 

ai u .1.1 N 

N O1 

•r• ~ 

r ~ 

•-+`- Q 

C C 

~ 11 ~ 

mc 

i Z 4J 

c 

ar .. 

U •O 

C O 

O .0 

U +- 

N 

TE 

a~ 

a c 

a~ a~ 

Y E 

C C1 

L 7 

N 4J m 

O G/ 

Z : 

roa 

N 41 

Vf 

O 

~ 

C 

m U 

E a~ 

3 O: S ~ 

V 1 

Uf t0 ro dv 

d r C 

u i mim 

dY N G/ 

m Oio C 

G1 N G1 

c roa a'i 

•L L ro O 1-) U U 

u c•-v 

. GN 

~ 

E •rua

l N ns • 

O ~ . UU N O G/ 

Of }UN4- 

C •r- G] CV- 

~ i~ C GI CI 

Y U a 

c ro E c9 

r Gl L C~ vNi b 4- •E vOi oxdma 

LU Of 

 c.

1 C 

c~ Qro++ 

C 

m 

E 

7 

S 

N 

c~ 

•~ M 

4J 4) 

L ~ 

at 

O V 

cr 

mmai 

i L N 

adm 

E U c 

E orn o 

4F• L. O O T OI .0 

cca~ 

~ O 'O 

C 4J Gl 

= i ro 1 • .0 

v1 i~ d 

w tU ~n 

S- C L O 

U •- Q 

ai 

u 

c 

ar 

L 

G1 

4- d C' 

~ 

~ 

~ 

~ 

m 

~ ai 

C 

ro 

L 

U 

0 

Go cn

r•1 

C Y 

m U 

c 

•n•~ 

•~ c 

ar a~ 

~ 

a 

} 

~ > tN 

r-I 

c oo 

mrn 

> ri 

C Y 

ro u 

c .~.t 

•.- c 

ai ai ~ } 

a) .•~~ > tN 

V 

L 

~ 

ar 

E 0 

~ 0 

'-+ 

~ 

N 4~ 

N 

O 

L 

Of 

C 

m U 

E C1 

7 C 

2 v 

~ 

~ 1 C 

L r 

4•1 41 Y N ac v GJ 

r O •N 

•m ^ a N a .~T.1 J L 10 } u u o 

c E c 

+~ N •~ Gl •~ rua+•01~ N c 

d C L +J r Cm. w O 3O 

v N O1 

G! O1 O 

y 

V 

m i

Z =6/ 

N c•r- ) m m 41 , ~ d = 

L ~ V GVl +•> > u E •r +3 OJ O 

C E O•00 S. G 

v 

m 

C C 

~ •E 

O C 

N t0 

y rti o-•~ 

c c C 

\ 

•e • E 

£ 

~ 

1 

'E 

1 

C 

O 

a N N 

O 

t0 

v 

m 

O 

C ~ 

•E ~ C 

O •'• 

V• N E 

N 

• ~ 
of ATPase to oxidation of phospholipids with subsequent cross-linking of 

membrane protein by lipid peroxidation products. Because the reaction of 

ozonolysis products with semicarbazide and PABA during 0 3 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 in vitro 

reaction of 03 with sulfhydryl groups of human RBC membrane, proteins, and 

cytoplasmic contents. After exposure of RBCs to 03 in 02 at 20 ml/min (0.84 

pMol/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 

980 Ng/m3 (0.5 ppm) of 0 3 continuously for 23 days. 

Although invitrostudies using animal and human RBCs have provided 

information on the possible mechanism by which 0 3 may react with cell membranes 

and RBCs, extrapolation of these data to in vivo03 toxicity in man is difficult. In most invitrostudies, RBCs were exposed by bubbling high 0 3 concentrations (> 1 ppm) through cell suspensions. Not only were the 0 3 concentrations unrealistic and the method of exposure nonphysiological, but the toxic 

species causing RBC injury may be different during in vitroand invivo03 

exposures. Because of its reactivity, it is uncertain that 0 3 per se reaches 

the RBCs after inhalation but may instead appear in blood in the form of less 

reactive products (e.g., lipid, peroxides). However, during in vitroexposure 

of RBC suspensions, 0 3 or highly reactive free-radical products (e.g., hydroxyl 

radical, superoxide anion, singlet oxygen) may be the cause of injury. 

9.4.3.3Changes in Serum . In addition to 03 's effects on RBCs, changes have 

been detected in the serum of animals exposed to 0 3 . P'an and Jegier (1971) 

investigated the effects of 784 pg/m 3 (0.4 ppm) of 0 3 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 valuesremained within normal jimits. However, after exposure for 10 months, the TIC 

had progressively increased to about three times the normal level (P'an and 

Jegier, 1972). Microscopic evaluation suggested that the rise in TIC may have 

been due to the thickening of small pulmonary arteries. The results from this 

study`are questionable, however, because the rabbits may have had intercurrent 

infectious disease, which was more severe in the exposed animals (Section 9.3.1). 

9-170 
P'an and Jegier (1976) also reported changes in serum proteins after 

exposure of rabbits to 784 pg/m 3 (0.4 ppm) and 1960 pg/m3 (1.0 ppm) of 0 3 . 

Following exposure to 784 pg/m 3 (0.4 ppm) of 0 3 for 105 days, the albumin 

concentrations began to decrease, and a- and S-globulin concentrations began 

to increase. At the end of 210 days of exposure, the mean albumin level fell 

16 percent, the «-globulin level rose 78 percent, and the S-globulin levels 

fell 46 percent. No significant changes were observed in total protein concentration. 

Chow et al. (1974) observed that the serum lysozyme activity of rats. 

increased significantly during continuous (24 hr/day) but not during intermittent (8 hr/day) exposure to 1568 pg/m 3 (0.8 ppm) of 03 for 7 days. The increased release of lysozyme into the plasma was suggested to be a result of 0 3 

damage to alveolar macrophages. 

Veninga et al. (1981) reported that short-term exposures of mice to low 

0 3 concentrations induced changes in serum creatine phosphokinase (CPK) 

activity. Ozone doses were expressed as the product of concentration and 

time; the maximum 0 3 concentration was 1600 pg/m 3 (0.8 ppm), and the maximum 

exposure time was 4 hr. Alterations in CPK were detected immediately and 

15 min after termination 6f 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 3 doses employed. The authors concluded that 

these responses may represent adaptation of the animals to 0 3 toxicity by 

enhanced metabolic processes. 

Serum lipids and lipoproteins of rats exposed continuously for 2 wk to 

1960 pg/m3 (1 ppm) were determined (Vaughan et al., 1984). Serum from each 

guinea pig was sampled before and' immediately after the 2-wk exposure and 

30 days.after exposure ceased. Thus, each animal served as its own control 

and there was no air-exposure group. Immediately after exposure, cholesterol, 

low density--lipoproteins ; and ve-ry low density lipoproteins were elevated in 

males. Generally, females had similar effects, but no changes ,in very low 

density lipoproteins. Triglycerides were not affected in either sex immediately 

after exposure. Although statistical comparisons of sex susceptibility were 

not performed, it appears that males were more affected than females. Statistical tests of the post-exposure group were unclear, but it appears that 

levels of cholesterol, low density lipoproteins, and very low density lipoproteins had returned to pre-exposure values. 

9-171 
Serum lipids and lipoproteins have also been evaluated in male rats after 

repeated 03 exposure (Mole et al., 1985). A concentration response study 

involved exposure to air; 1960, 3430, and 5880 pg/m 3 (1, 1.75, and 3 ppm) 0 3 

for 5 hr/day for 10 exposure days within a 14-day period. For a given rat, 

serum samples taken 2 days prior to the first exposure were compared to samples 

taken 20 hr after the last exposure;.each animal served as its own control. 

Another group of animals was exposed to air and sampled pre- ancl postexposure. 

Shifts in the 0 3-exposed rats (pre- vs. postexposure) were compared statistically to shifts in the air-exposed animals. Ozone caused a concentration-related 

linear increase in total lipoprotein-free cholesterol and high density lipoprotein total cholesterol (both the free and esterified components) and a 

decrease in total lipoprotein triglycerides. There was no effect on high 

density lipoprotein-tryglycerides or on total lipoprotein-free fatty acids. 

At the 1960 pg/m3 (1 ppm) 03 level, none of the values was elevated significantly over controls. In a sampling time study (Mole et al., 1985), rats were 

exposed to 1960 pg/m 3 (1 ppm) 03 for 5 hr/day for 15 days in 5-day segments 

within a 19-day period. Serum samples were taken from each rat 4 days prior 

to the first exposure and at 7 times (0 to 44 hr) after the last exposure. 

Ozone increased serum total chlolesterol (p = 0.1), high density lipoproteincholesterol (p = 0.08), and high density lipoprotein-free cholesterol (p = 

0.006), and decreased triglycerides (p = 0.06). The changes appeared to be 

maintained over the 44-hr post-exposure period and were greater than those 

observed at the 1960 pg/m 3 (l'ppm) 03 level of the concentration-response 

study described above that used fewer days of exposure. Thus, 0 3 caused a 

mild hypercholesterolemia and hypotriglyceridemia. 

Both the Vaughan et al. (1984) and Mole et al. (1985) studies report 

increases in serum cholesterol. There is some disparity between results for 

other lipoproteins. The Vaughan et al. study did not account for changes that 

can be produced by exposure stress alone, as, i.ndicated by,.Mo.le._e.t__a1. _(1985).. 

The Mole et al. (1985) studies showed only marginally significant effects. 

Thus, a possible conclusion from the rat and guinea pig studies is that shortterm exposure to 1960 pg/m3 (1 ppm) has the potential of elevating cholesterol 

in animals. Elevation in human serum cholesterol is a risk factor in human 

coronary heart disease (Gotto, 1979; Dawber, 1980). 

9.4.3.4 Interspecies Variations. The use of animal models to investigate 

the effects of 03 on the blood is complicated, because few species respond 

9-172 
1 i ke humans. The rodent model has been most commonly used to predi ct the 

effects of 03 on human RBCs (Calabrese et al., 1979). However, the reliability 

of this model was challenged by Calabrese and Moore (1980),on the following 

grounds: (1) ascorbic acid synthesis was significantly increased in mice 

following 0 3 exposure (Veninga and Lemstra, 1975), (2) ascorbic acid protected 

human G-6-PD-deficient RBCs in vitrofrom the oxidant stress of acetylphenylhydrazine (Winterbourn, 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 3 exposure (627 pg/m3 , 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 

animai model for RBC G-6-PD deficiency in humans (National Research Council, 

1977). However, Dorset sheep were found.to be no more sensitive than normal 

humans with respect to 0 3-induced changes in GSH and also differed from humans 

in the formation of inethemoglobin. Further studies (Calabrese et al., 1982, 

1983b,d; Williams et al., 1983a,b,c) demonstrated that the responses of sheep 

and normal human erythrocytes were very similar when separately incubated with 

potentially toxic 0 3 intermediates, but G-6-.PD-deficient human erythrocytes 

were considerably more susceptible. Consequentiy, the authors also questioned 

the value of the sheep erythrocyte as a quantitatively accurate predictive 

model. 

9.4.4Reproductive and._Teratogenic Effects 

Pregnant animals and developing fetuses may be at greater risk to effects 

from photochemical oxidants, because the volume of air inspired by females. 

generally increases from 15 to 50 percent during pregnancy (Altman and Dittmer, 

1971). Before 1978, experiments designed to investigate the reproductive 

effects of photochemical oxidants often used complex mixtures of gases, such 

,as irradiated auto exhaust (see Section 9.5), or they used oxidant concentrations greater than those typically found in ambient air. Brinkman et al. 

9-173 
(1964) exposed pregnant mice to lower concentrations of 0 3 , but the results of 

their experiments are difficult to interpret, because the time of 0 3 exposure 

during gestation and postparturition was not specified. They reported that 

mice exposed to 196 or 392 pg/m3 (0.1 or 0.2 ppm) of 0 3 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 

03-exposed mice, even at the exposure level of 196 pg/m3 (0.1 ppm) of 0 3 

(Table 9-21). Unfortunately, without more details on the period of exposure, 

it is impossible to ascertain whether the decreased infant survival rate was 

due to development interference in 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, 

Veninga (1967) found that mice exposed to 392 pg/m 3 (0.2 ppm) of 0 3 for 7 hr/day, 

5 days/week during embryological 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 03 for precise periods during organogenesis. No significant teratogenic 

effects were found in rats exposed 8 hr/day to concentrations of 0 3 varying 

from 863 to 3861 pg/m.3 (0.44 to 1.97 ppm) duri ng early (days 6 to 9) , mi d 

(days 9 to 12), or late (days 17 to 20) gestation, or the entire period of 

organogenesis (Days 6 to 15). Continuous exposure of pregnant rats to 2920 pg/ 

m3 (1.49 ppm) of 03 in midgestation resulted in increased resorption of embryos. 

A single dose of 150 mg/kg sodium salicylate followed by 1960 pg/m 3 (1.0 ppm) 

of 03 during midterm produced a significant synergistic increase in the resorption rate, a decrease in maternal weight change, and a decrease in average fetal 

weight. Exposure of pregnant rats 8 hr/day to 862 pg/m 3 (0.44 ppm) of 0 3 

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 in utero 

exposure to 03 can affect postnatal growth or behavioral development. In contrast to the results of Brinkman et al. (1964), neonatal mortality of rats was 

not increased by exposure to 2940 pg/m 3 (1.5 ppm) of 03 for periods of 4 days 

during gestation. Pups from litters of females exposed to 1960 pg/m 3 (1.0 

ppm) of 03 during mid- (days 9 to 12) or late (days 17 to 20) gestation exhibited significant dose-related reductions in weight 6 days after birth. Pups 

9-174 
0) 

~ 

~ ~o 

y N 

Y 

U 

O 

> 

ro 

Y 

4J 

ro 

z 

~ 

~ 

N 

C > 

ro ~ 

Of O 

~ 

L v 

Of cu 

•3 s% 

ro o_ 

~ ~ 

m o 

E U) 

cm 

L 

G/ i-> > QI N 

ro 4- r 0 

II O L 

G/ Cl .J 

N N C 

E tC O 

Cl C/ U 

L L 

U U i 4•- 

N 

1 

~N II 

L c E 

a-~ rn ar 

C O ~ 

°J ro ~ •L 

L Cf LL7 7 

0) 

O OO N 

F +1 7 C 

NT O tG 7 r 

-a a ~ +•~ 

\ O N • t0 

L L fi C n ~

00 0.v C..) GI 

~r LD 

~ 

~ 

a~ 

61 

C 

ro 

E 

Y 

C 

L 

m 

Q1 

N 

3 0 £ 

U~ 

t0 

O 

iJ 

C) 

c!• • 

~ 

~ 

+~1 r ~ o 

ro41 41 c 

L O 

E v L 

row 

ro 

O • 

Gl r~l 

c O 

II 41 

a 

N tD ro . N r-1 

L 

U . 

d 

G! 

3 

\ 

T 

ro 'Q 

t1f ~ 

V 3 

i M 

L O 

n w 

n 

tD 

rn 

r-I 

ro 

OI 

C . ~ 

N 

> 

d 

N 

£ 

G1 

U 

C 

N 

II 

U C 

~ 

tn 

L 0 

u m^ 

•^ N4- r 

O 0 

L +~ 

G U 

L 

rnc 

II •r 

a ae N 6~ 

E Ci 

C N 

> > 

' Y 

~ ar I ai 

3 

\ 

T 

ro 

II 

t2] 0 

II 3 

i M 

L L 

n W 

Ln 

Q1 

ri 

IL I y 

a i~ 

II 

c 

f0 

N 

E 

.1~ 

ro 

~ 

U 

4J 

N 

E 

O 

II 

O 

II 

a 

L U 

N 

G/ 

V 

~ 

O 

C 

u c O Z a~ 

ro 

7 

-o m o > L ai 

+~ v r 

E ro U 

~ • 

C +~ 

N E • ~

L f+J0 

N N 

roO1 O 

E Z m a 

W 

Z ' 

O 

N 

O 

O 

N 

H U 

LLI 

tL 

W 

LLI 

U_ 

Z 

CJV 

~ 

G 

W 

H 

0 

a 

¢ 

w 

~ 

H 

U 

~ 

O 

O 

a 

K 

rl 

N 

Q1 

J 

Q 

v u 

c 

a 

i 

d 

4- 

a 

C 

N I 

N 

U 

o. N 

~I 

~ i 

U 

w , 

4•- 

G1 . 

a 

d 

L 

N 

a 0 

O u O 

~-+ 

L 

a 

L II 

3 C 

O ro 

0. C 

lL ~Z . 

ro L 

7 

II 

m id 

C 

a~ II E O 

L 41 

7 G1 

N E 

ro 

~ 

O E 

~ 0. 

~ 0. 

C L 

N C 

O GJ 

u E 

C 

o rn Ul 1 

ri N 

O O O r-1 

to N N O 01 ri 01 M t0 N 

co rn 

N 

o 

~ 

cn ~ 

ro 

4~ 

0l 

Y 

U 

O 

> 

f0 

Y 

4J 

ro 

z 

O7 

C 

~ C C 

p/ •r 

4J 

i II 

O O a 41 

O C 

N 

(

4-

^ 0 T 

c ro N c 

L II . o II ~ ro 

c v a 

v- ai (1) 

O E L 

d O 

4A > •- 

U 

7 

'O 

QI 

i. 

Gl 

' aci E ro 

s0- 3 

O 'O a L M 

cn 

> O C 

ai N •rN 

3 

rl 

~ 

II•r i 

L O 61 ~ 

co 

G7 

O II II G/ E 

tn t0 1d Q M 

c .. 

O N 

ro II+O.+'~ -.'ro+°J 

E ~ 0 N 

O1 

r G! 

O) 01 

r 1~ 

Qf rl 

G C L C 

L 

O ~ L O N L 

7 7/~ N 7 O 

II IIrNi O 

Q v 

N U) +J N 

O r7 C 3 ~ ' O r + 3 

O 

^ Ql 

• N 

'- +J 

R1 

C N C f~0 f~0 O N O II O ~ C N O Cl V C7 c.7 vv U Of 

O O 19 

r-1 r•i r-i 

O O O 

cD l0 Q Q1 

r•1 

O~ r•1 T 

N 

9-175 
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 

to 2940 pg/m3 (1.5 ppm) of 03 during late gestation were also significantly 

slower in the development of early movement reflexes and an the onset of 

grooming and rearing behaviors. The authors pointed out that it is impossible 

to distinguish between prenatal and postnatal contributions to the behavioral 

effects, because foster parent procedures were not used to raise the pups. 

9.4.5 Chromosomal and Mutational Effects 

9.4.5.1 Chromosomal Effects of Ozone. A large portion of the data available 

on the chromosomal and mutational effects bf 0 3 was derived from investigations 

conducted above 1,960 pg/m 3 (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 3 may produce genotoxicity, this discussion will not be limited to data derived from research conducted 

at or below 1,960 pg/m3 (1 ppm) of 03 . Data derived predominantly from in 

yitro experiments conducted at 0 3 concentrations in excess of 1,960 pg/m 3 (1 

ppm) of 03 will be discussed,first (Table 9-22), followed by a discussion of 

the genotoxicity data from both in vitro and in vivo research conducted at or 

below 1 ppm of 03 (Table 9-23). 

The potential for genotoxic effects relating to 03 exposure was predicted 

from the radiomimetic properties of 03 : The decomposition of 0 3 in water 

produces OH and H0 2 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 pg/m3 (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 03 was slightly more efficient in the production 

of chromatid breaks than 50 rad of x-rays: Significant mitotic delay was 

measured in neuroblasts from grasshoppers (Chortophaga viridifaciata) exposed 

to 3500 to 4500 pg/L of 0 3 in a closed system (Fetner, 1963). 

Scott and Lesher (1963) measured a sharp loss of viability with Escherichia 

coli as the 03 concentration was increased. Viability was reduced to zero 

when cells were exposed to 1 pg/ml of 0 3 . Damage'to cell membranes was evident 

by the leakage of nucleic acids and other cellular components from cells 

exposed to 0.18 pg/ml of 0 3 . 

9-176 
C I 

L 

Gl +~ p. N 

L+J ~r I 

C E N •O +%1 I ~

r ~ V- r• C . 

+1 10.1 L d ; Sa o 3r- a a 

U - NL}j}j 

G1 NaN r• L•~ 

N X a +°

•i 34- 

cv- c a) ro L 

+-ro1 ° L •r9 ~ O. 

7 i 

E. •rN •r M•r 

xIGQ C Op• 

pi G 1p11 O L 

N J 

31i1 mQ L V flSL EZ 

cEwo ]Gc 

M- 4JM Cr rodd rro 

L L 

=CU 

F~ 

N 3 •~v- x ro•r N c 

O Lw cr4++tM cro mrn o a ai ro 

+1 1~ 410LL N 

7 ri T +1 +) C 

G/ •r0 

cn 

~ OO L 3r7 c 

crt'+J ¢ 44 N ° 

e0 ~ OG GI O. U ~ 

Ea~oaicaxi 61 UU°1i 

Tn > ro ML ro rl•r LO 3 L 

mgN O ~O CJ a0 T 

Z]G N4- LY~- OI~OO 

Cv N O C1N1-1G• 

N 

4J 

G1 

w 

w 

N 

V 

N 

> L 

a 

N 

a 0 

N 

C ~ 

O 

~ 

d 

d 

V 

'O 

~ 

E 

i 

L 

U 

N 

z 

0 

., 

~ 

~ 

~ 

z 

w 

U 

Z 

U 

w 

Z 

O 

N 

O 

x 

c~ 

x 

FO- ~ 

W G..' 

~ 

N 

0 

X j 

W 

Q:I 

H 

~ 

Z) , H 

S 

O ' 

Z 

lL 

N 

1-- U 

W tL 

LL 

W 

J 

Q 

Z 

No 

g 

O 

D: 

S U 

N N 

I 

01 

w 

-1 

m . 

¢ 

H 

v U 

C 

a 

L 

G/ 

W 

a 

z 

N ~ G1 

u CI rL t 

N ~ O\ 

1-~ 

i 

a~ 

C 

41 W 

LL 

Y 

N 

7 N 

x u 

~a C 1~ mrn rt 

C 

~ rn 

E O 

S U 

rol 

u 

L~°vI 

~o 

Y 

CI 

C 

ro dE n 

ro ^ 

S rml 

a~ U 

W 

U_ 

O U 

O 

41 

L 

C 

O1 

L Q C 

7 C vl ro •E C C 

a C ` u'f •E - •E 

1X O N O O +) 1Y) C7 M 

ro i 

•O 

a 

4J 

C O N O E t a +1 O 

r 

V fL 41 

vOi E 

tN 

t0 w 

~ 

^ 

ro C 

c 

~ E Q 0 0 

a o 

++ ao u~ 

ro L v v 

+1 M M 

O GI ~~~ -\ \ 

C Z Z 

O 

tJ O~p O 

cz 

t0 O 

rt ~ 

•- a 

E n 

S r0•1 

O 

u 

W 

41 

C 

Ol GJ C ~i 

.a NN O •O O! ' O r- L C}j C 

r0•1 ~ E N ro x•'0 

~ C U 01 O CI ~ v•rr- 3 O+~ _ 

Uf ro 7 N ~ G! 01 

C Gl 4- d G1i tA 

L E>O7N 

•L ~ Im C N+~ ~0~ .Fi T•r C •r v 

N a> 01 N O 

4-3'O .L L y ~ +' 

~ ~ a aaCM °I 

=rtiTais. 

Ea M LE N L 

O OOQO+~ 

C~ L C 

7 O Ln 

 ~ ~- O N L 

E r•~ 3 0 +~ •C +~ 

C 

•E 

O 

M 

E 

o. 

a 

0 0 

~ 

\. 

O 

1 

O 

O 

O 

O 

4m 

M 

w 

.>'z 41 •r

> O u C Q m Ol i d •r •.Z ~•-~ L 

+-+ M o ci•ro ¢ c.cr a 

N C V O 41 C GJ 

4- C Y- •r C t0 ••- •r O L 

vu°i ° •o ~ 0 vc m 3 aai . 

L i+> >~ N N 07 +~ Cf o ur• N ara T3 ~ ro 

c. ~ w > uN 01 O N 41 C ~ G1 •O W c t0 O u N N •r •O 

L y y•r L 07 ro C•O i 

¢ a u N E ~ L •• •i0 > Z ,. Z G/ •r ~6 T 

O o L 

ii ... N L 

- 

 G o++x 3-aia1>a+' o II O ro N>•O 

+~ ai +a r, 12-4j C 3 c ai 

O/ 01) d+1 TGlyJ + 

m•r 

aa- N u L a ¢_ 3 0. i - c'. 

4- c .00 E ~ nU•rL-•o 

m 7ro 

4 ~o++_T10 rtf C++O L t1'O •o p. f0 L 

tn +~ O O Gl C C y ro 4- 

rl N dJ O. S. i0 ro_L Q°_ O 

N 

L 

.0 

M 

O 

~ 

O. 

7 

6 

a 

a 

0 

~ 

v 

\ 

~7 

1 

O 

O 

O 

m 01 

~ 

~ N 

C 

~ 

_ ~ r ~ 

~ o 

C 

+0 

C 

E 

c. 

o. 

0 

~ 

v . 

M 

\ 

al 

S 

O 

O 

O 

O 

Ol 

E ~ 

S rm-I 

9-177 
C O 

y 

N 

L 

a~ 

O tU u c 

u o

d 

U 

C 

L 

W 

d 

C 

N 

U 

W 

V^ 

d 

~ 

d 

i 

d 

.No O 

co 

. 

t~ ~n 

.~ 

r N 

J • d to 41 d u~'f b d C ro 

Q1 m.-1 01 

rl 

+~ 

d U7 Cl i 

C ri i-1 Y ro •FI 

d 

.. 

d . 

^ 

L 

C 

r0 

~ 

+J ^ +J 

•CM ~ C 00 

M 

O tD 

+1 

ro 

L ro 

u aJ 

JM 

u f , 

N N 

•L d 

d 

N L Ol U Q1 L IE }f d 10 G1 .0 •r ro V) t-/ N rl O. tn d l.- 7'£ r-1 u C7 C 

O. 

ro 

a 

+-o~ I I 

+NJ ro ro L !- 

N 

ro N 

ro u 

t

4- 

d d 

c7 

•O •O 

O.G d O•r 

N 

T 4J Y N L L tdif •r~~- •~ i~ O L 

ra 

u u u 

d 

;i T• W W 

t E ~ 

t7 d 4- 

O 0Gl 

E ro u 

r ~ 

c,> > ~ 

Cf ' 

. Z 

N 

E 0 

+~ 0 

c 

> 

~ 

+J u N 

10 r~ > d •• 

yf 

cn 

^ 

d N 4- O +1 C 7 

4-ro N d 'Q O • u rtl O 

O Y tO d'O u 7 O ro N•r ~1 0 • d L p. • N 'O C~ C•r~ 

C d 

•

N 'O 

•^ 

N d 

C N 

• C N 4- .. O 

Uf E 

U O dJ C O N 

U O 

i 

d ~ O r d t0 'p •O /~ 

•U 

N'O € • • r- O GJ 7 N L > O 0/ O L 

•U 

'O •.- C d O N ~ N E C d O• C ~ 1.1 r ro 

ro T•Y 

C O) ro d ro 

U d 

,E ro c 

d O+J i. d 

c.•r- c.c ol ~ a i t~p 4- a Z U •E 

•L N.}~ d U'O O d L O N ~ ~ L. V•~ O] N U 

•d 

UN 

0. •d Y 

C ro E 

C i 

T•U 4~- ~ C d~ ~. •N O) T d ro ftf ~ Idi7 L ro f0 ••• O O d N 

~ Q1 ~ 

U c ~ 

•N +r

.f 7 C N d +%` LIO G E ~ F (%) ~ r~o ' m c ic z o °d ro ~ 

c•~ 

•3 

 cn lo o ro o o u o c^ I: 

Of N 

~ L O G7 

L~ 4- ro E d L t0 b N O N ST- •rU- d C L N GN1 2 E 'O 

C 4.1 L r 

U O.•r 

7 N O. 

O L 

4 1 N O C 

4 1 L•r . 

iJ E E E 

ro O C O 

N O C 

O+J d d 

'O CI G1 •O O• 

d N•r 

~ X 

O 

d 

L d 0_.O c O ~ O 

O L 

 L. d d U V- 'O ~ d N d'O i ~ ro O) L. U M1S 'O ro 4.1 i i.l 1 ro 

Vd- ro rL0 1~1! LO 

NrCro! r 

ro r r 

•.- L d•E 

'p OY d L 

E C O. u i•r 7 

'O •~ L N +~ i •O LCOi 

 L. 

G 4~- 

~i 

a~ Tu ~ O~€ • c.o o O O •'o ~ w ro = d Q OI i Z 7 t7 C N 1 O O d d N L ti1 e! 10 i.~ L i i 7 ~ 

G1 L L r- C O. N E O• R1 d 7 X d.l C 

.0 

p.•r- U V10 U 7 ~ u.G 

O. ~ N C LL'f €€ C u O.G v O 6•1 u N u N 6.' i4-1 d m Q C Id C.G C _ 

ro 

ro 4J 

N = •Op 

r. c "' W c .a 

c ro II _ L 

E € 

C 

L 

7 m Z O N 

O N o O CGl 

v 

M 

v •€ 

C 

•E O C t~p X ~ c VOi. 

c c o i~ o d' o E ^ d ~ II N 

•~ •E Z G cq O Z M ~ y- E Z 

O O ^ L .N d 

lG M LC) C]. r0 d UQ 

L 

T u 

ro + ~ 

s 

G 

.Sro c 

• 

r +d 1 

~ +> > N 

~~/l O C i •E 

t+ 

y 

Y 

1-q 

Y 

H 

Y -1 I- 7 

41 

t 

7 

N 

L 

rtl 

> > Y m m m Y G. o G7 3 ro 3 =D d C7 

O -i ti'~O ~ E- i 

.- ]G O C 41 d 

•11 - O +) 

t% II E \ C O 

C N •~ E 1d ~ 

E td J ~ N v 3 C 

r~ 

E 

o. 

O_ 

c 

O 

ro 

++ 

c 

•r M 

lo 

a. 

CL E 1 U O O L 

d O ~ ~ p Qi S. O ~ N 4- .0 

C. 

IZ 

O. O. 

O. G 

1 rl 

4] lD 1 O 

1\ N 

ri N • ro 

t'O 

+~ d 

> O N 

0. 

N9 N C 

tff O 

N M 

C O ri rl 

r'~ 

E 

u rl 

E 

r-i 

E T 

d i 

E d 

•Of N 

c~ 

Li 

N u LL~'1 u u~ d C• O. L 4- d o 1 O. E O. O. w +J • L 

Of b N 

r E.- \ E o =. o 

3 +J 

~ ~ 1~ 7. Q1 10 r0 L r 0 C ro 

S 71 3. tD 1 ~ c!• N Op L N ~ I 4- u t0 

e^1 ln 

N 

1••1 O 

1 

t!') 

O 

ri 

u I 

L17 

u u i~ 

O 

10 H 

d Y_ M 

C 

O M 

O O O 1-1 C rl ~ 

~Q ~Q 

M tV C6 10 d u •O d 

9—l7g 

v ; d 

5 

C 

~ 

C u o

v 

d 

r 

cr d 

O. N 

N 

N 

~ 

~ 

R 

u 

0 

.°a 

O 

d n 

~ C 

N Ip 

W •~ 

.1~ 

L 

17 

.0

a 

c~ 

H .c o

d a+ 

7 ~ 
EP a, 

a, 

~ I 

v 

M 

E 

\ 

rn 

1 

0 

l0 

~ 

rl 

3 

C

J 

W 

m cc 

O 

Q 

N 

Z 

O 

~ 

Z W 

U 

Z 

0 

U 

LU 

z 

O 

N 

O 

E 

O 

0: 

lL. 

N 

h~- 

C7 

W 

lf.. 

LL. 

W 

J 

Q 

g 

O 

2 

LLI 

O 

2 

U 

th 

N 

1 

Q1 

J i m 

N 

d io o ro 

c +.+ +-> +i 

m a d d 

i 

'E U t0 rtr~i , - 6rroi d O 1, K 001 G/(n N01 C,7 rl N rl N r•i 

N N N 

4J 4) N 

L U U U L L 

Gl G1 +1 C L C L C t i.~ 1~ N N m d ro a ro a N N •U 7 E E E E E 

n. x tn 

N C 

Cl C1 •r N L •r N I N.N N i.) +J i t O EcO ctl O¢ C O 6l +)• C•r N L a2 c E C 1• • c ' G! N r N 41 X ~ L L X G. •r O O 07 E d ai 3 ar s N u ai (1) i•r i ro ro ro • o- >T 3 c a 5-61 L+1 4- E L L L'p . L N ~ ~ ro ro O•r V T N N L•r 1p N N O) C.7 ro O Cl G7 N N .0 C•r C 'O CI N L ~ Gl U O Ol C/ (l i•) C i d L}3 'O G/ .0 i d U O L•r N L O U ro+j O d +j +j O'O ~ 4- C0 L.N .0 L 4- c u 7 41 • G1 ro~O "O i•) E L. /0 +j X T 4- c•r T R1 .0 O C +~ U rt1 <0 7 N a•^ 3~"~ I C G! r 10 N u Rl i•~ u U Gl 'O C ro U r G! N N M i•r .0 7 E O 'O rtJ O L N U C L L M O G/ O Ol Gl T•O ' v o L G. E L Of L i al E t.0 O E c . r O L o L N E L N G) t+) u c I 

~ u N•r •r y C dL 3 O a C O O M aL a Vl'O d..) T C t0 U T ~ C i+7 a•r E o 4. E~D N u O Gl C1 ~D O ~ N E ro~ u 07 

4- O•r a O r-1 in U G1 E T 4- ro o 'C C t0 r• L N•r .0 O' C/ N N N 3 ~ N L L ~ M'O q N E M +~ — Y T 41 C O 7 t0 

4- 'O d.~ N C — ^ 3 o c o N N L N ro o N ~ U c d G1 aJ s . Gl G1 T ro Ol 07 O r• 'O G/ Q L 'O G! O L ~p L O O • L i L'O C O~ E L fO 7 U U L Vl +J O O L. S O O f•. t.L E u E C 7 ~~ O r• N T4- N 'Q 7 C i q T+i w O 7 G O 7 N u d~ d C O N O N M U O +~ U r +~ O GJ V N Gl O U u r• +~ G) r+~ ro a r L r-1 •r o m G/ G1 r• .1~ ~0 •r Y c a. 

> C O.D E O 61 Y d.- L E .0 r• ^ 4•- • L N d N 4- E d C L L'O ro Gl X L r Q(0 L V- ; 7+~ O 7 C O N M d••~ U N/C X I w O ~0 L}3 O N L O 

y C1 L a~F r U•r N r U O C N c C r• G11~ N N r- - U N C O L rtJ N .F) i•O O E G7 m ro 3 O ~O • T O • Gl ~F- O 7 N O ro r.p N r• L u V- ••- >> i c E i C+~ u•r f~ L o•r E 'O N Cl E L. 4- ro U d a c 

o ai +~ L'- o ar a•r c o d•r ro c+~ ro +~ •r ar .. > o d v ro•r V- G! rp C! C U L O C- .0 N ro+~ a T ro+~ C • i.0 •C N'O r Q! 3 4- .0 E+~ •O d M•r L a M O 7 L d a U L N • C+~ L d Of E t0 N r• O v+~ o N o E o++ u o E a'L ai ro c s a o•r u•.- r• o rni ror•r• L O Q L ro •L • Gl GJ U G) Ol Q E•r 'O i •N tn C N Gl •r r 

Z O U~ a 1•rC• d~ 7 ~ Gq. Y u1 4- Mi0 N Z O' 1~0 1 U N N O G •2 E•L C U t~if 90-• . 

O U O 4J O 

U a OI OI OI C OI 

L'O L i i L+~ 1 +~ +~ E a~ i 4 7 C L L L L•r L> L> 

a C t.t) tn N tfl C Z•C Z•C Q1 C 47 ^I m r' 

X O •rI •r' •r~ 4'f •r, v v 

i-~ W •r v v v ~ 

ro i 7 

~ 

.0 

'O iJ . C 

E O Y Y Y Y ]~G t~¢/f i--~ N r-+ 

f0. ~ Z 2 . Z 2 Z 

o i E ro N S 

'. c E Ln ri rnM i ~n d• i O a rl N 01 N M t.[) tD N O N 

I +~ a O o O O r-4 I- r-I a' N O O O 

L r-I 

i~ 

O U7 1 O 1CN+f u E o 00i~ 4m o ~ C d• N d• r•1 ,h ch •• N N O m O 07 01 rf Q1 LL9 u7 eh N 1~ 01 01 I, CO G> 1 N d 1•1 d• N ri M N C) M :t 1n , 

Y~ • ~ 
~ 

d 

~ 

Q 

~ 

C 

O 

u 

v 

6 

O. a 

rl 

v 

~ 

t 

Qf 

J 

~ 

u 

c 

+-+ m io ao 

rn at rn eo r I 

O L Q ^

f 

i ~ 

N iJ 

Cl O S L •• 

+1 

y O 41 00 

N 

C 

Gt . C! c0 U I- (O 

C7 Rf , tn r0•1 F- r0-1 U 

N 

4-3 tn 

N ro 

U C O c L Ul 41 

C7 ro f.. ro O r• N N N 

y S 4- 2 Lt U 

N 

E 

O 

4J 

O 

1 a 

N 

E C OV 

••• C T O C O ^ 

> 

S 

~ 

1 N +1 •O 

N N N r• •r tp O 

i C •L L. t . 

N ro 

i.- 

N a) •• 

IIINO Gl O E c ~0 u.G N 

ZJiJ N•r O ro L a 

x t 7 Ol d > 4- ,p N•r Q1 G tA T N L U i O r• U ~ 

W L •p ~~ u~l O ~~ r• u 4•• .G CL C Oro 

r 

•C 

t15 .•• N O 07 •r a•r d 

'C 

~ 

N•r 

W 

w C r r• 

C1•r a 

N 1~ U N L T ar' •O r d Ofr• N a u 

E C E N 

o tn 

V 01 

•O 

N c c c o T ro G) 41 +1 L. ro+1 •r c 1/1 6l i•~ ~ ~F 'O 

v 

y1 

e y 

• 

O G! f0 L 

.- i L.II 

~..1 i N RI f0 O N E T L Gl Cl .G •r d d•t 

~ i N ro E r O 

u 

fY 

CI O N r-• C1 ~ •r 

E 3r 4- 

1 N O E N O U 0J 7 ~ 

~ .0 O. N O O ~ N O O. O• a 

P. C L 

060 

 O L G! N O C O. 

C a a~ O a 

r 

E V4- 

ro a 

Cl •r C! o N i N d•p 

~ •r pl O.0 X C E a C L O 

t0 i t• U G7 O€•r 4- L 

r x C•r 

E 01 ro iJ Lt7 

7 ~ 

Ql LV L. 4- L L•r E Ol Mi•) ro Ol L T a G7 O. U U •N • U 7 O Cl O o C.O N L L.0 r- T W E N L ~ M•r 7+1 N M 

I •O 

. d C CD •N 

r C7 

N E Gl r• 

r• 3'O a 

i G Cl G7 U u U T L C 4-• Gl •r .1..1 3 3 c N r• ro 4- O M- 'G tO yC ro N }1 f0 

> 

. L 

G1 

1 7 'O r U 

d 3 

r• ~ 

N L'O u Gl Cf 

U•r O .O 4- O O d G1 ro N C L 3. C'O 

C N N C N C T. Q N r G 

5- Q 4- •C •r N 

U Z O O) Y 

01 O r}7 ro 

. O N O• O 

a N 

.n 

iJ C 

ro C•r 

C i.~ L w•r tp O •r C y•r ro N O ••• ~6 C) Gl O 

O 4•3 01 T•r +~ E L+) G1 r •O N •N C GJ W. r- tQ L •O 

y V 

° N •r •O ° Cif L ~ 

T N i ro N C ~ T Cf.c 

tOif+t u N uII N N N u c 

N O r• Cf u a 

d c•r ~ L al ai 

a i O1 

d L i~ Of +1 ro ro O ~O C r• i c w O ro r• a•F~ u•r 4- 'O q- 01 c 

~I 

t u N 

N N a 

.t.> > C 

3 rO O 

O~ C 

a) i.c a7 •r ~ a) o c.) L .c ro L i a L. ri C/ V- ~ VE C. U i 

U 61 U ~ aV- E Gl 

ro t E•r c 

• D7 dl O C ro 

L•r ~..i U O L 

v- 4- m 

7 4- .0 

.0 7 u 

. 0 7 x L c o c.G ~ c C i~ T•r c c T o.0 ro Gl ro Ol •r i~ .O x 

. C1 C• N (;~ f0 +J f0 1••• ro r• 1-1 t0 ro+J 'O •r •r r• z a > 3 •C M N N <tl ro ~ 

+i,+ s% ° 

~ 4J ~ 

C N E 

t0.1 er IC U 

Y m Y L 

tn 

a Z Z N 

L'O 

O N 7 O 

TtO 

O O 

L•.>-I L ~I 

O 

O C 

i~I 

L•r ~+~I •O C•r t> L> 

L ~I 

> 

i Gl •N 

QI •O 

O ro rl > CO i> > ttf ~I t0 ^I NrI N'O II 

W ~ vl U~I 

v v v ~ y 

.0 

~ ro ~ i z c O 

•O 

.. in 

N • C 

U M11 

•L 41 

+~ O 7 .0 a ~ 

cn 

O 

a 

Cl 'O 

7 Gl N 

~ 

C 

U 9-. O 

1 7 41 

@ o w]C W Y Y C _ L 

2! i•t > > > Z C~ Z II a N E r II II 

V 'Q •O •O 

.c° t a u0i 4-) ++ • o 

f 

N N L 

C E E E U 

O C3. 

t 

N 

9 

 O 

1 M OD M 

et O 

1 O O 

1n C 

iJ C C1 O 

r a 

ro O r~ O O O, N r~ tn 

C O O 

~ ~ ~ ~ 

L 

4J 

N ro O • 

L i c > 

MC O 92 y 

° c

w

i ~ 

c 

~ o 

o~c 

t a0 ao ta 0 

M N 

0 o 

4c o 

e`na 

en ro o.o 

O O 

t7 Z 

01 Ot 

~ r) 

GD li] 

t17 r-1 

~ 

M r0-I 00

1 ro S .O U'OQ 

9-180 
The molecular mechanism for the clastogenic and lethal effects resulting 

from 0 3 exposure are not precisely known. Bubbling 8 percent 0 3 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 pyrimidine bases, nucleosides, and nucleotides. In a more recent report, Shiniki 

et al. (1983) examined the degradation of a mixture of 5' nucleotides, yeast 

t-RNA or tobacco mosaic virus RNA with 03 (0.1 to 0.5 mg/L). The guanine 

moiety was found to be the most 0 3-labile among the four nucleotides, whether 

the guanine was present as free guanosine monophosphate or incorporated into 

RNA. The sensitivity to degradation by 03 among the four nucleotides was 

found to be, in decreasing order, GMP > UMP > CMP > AMP (GMP = guanosine 

monophosphate, U= uridine, C= cytidine, A= adenosine). Even after extensive ozonolysis of yeast.t-RNA (0.5 pg/m1,.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 3-iabile, 

the sugar-phosphate backbone was intact and extremely stable against 0 3 . Prat 

et al. (1968) investigated the reactivity of the pyrimidines in E. coliDNA 

with 03 (0.5 to 6 mg/L, 0 to 5 min) and radiation. Ozone preferentially 

reacted with thymidine, then with cytosine and uracil, in decreasing order of 

reactivity. The results are slightly different from those reported by Shiniki 

et al.'(1983) in that the reactivity with uracil and cytidine are in reversed 

order. 

There is evidence that single-strand breaks in DNA may contribute to the 

genotoxic effects of 0 3 . Radiosensitive lex mutants of E. coli , which were 

known to be defective in the repair of x-ray-induced single-strand breaks in 

DNA, were•found to be significantly more sensitive to the cytotoxic effects of 

0 3 than the repair-proficient parental strain (Hamelin and Chung, 1974). 

In an effort to investigate the nature of the 0 3-induced'les3on in DNA, 

Hamelin, and co-workers investigated the survival of bacterial strains with 

known defects in DNA repair. Closely related strains of E.coii K-12with 

mutations in DNA polymerase 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 

N : 
degradation of DNA in response to 0 3 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 03 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 lon mutant strain of E. coli K-12 (Hamelin et al., 1977b). The 

lon mutant appears to have a full complement of DNA repair enzymes. With 

these mutants, there may be an enhanced DNA repair activity (evidenced by the 

extensive degradation of DNA), and the enhanced activity of the lon gene 

products was thought to be responsible for the increased cell killing observed 

with these strains when they were exposed to 0 3 . 

Although DNA polymerase I was shown to be involved in the repair of 

0 3-induced DNA damage (Hamelin et al., 1977a), E. coli cell strains with 

mutations in DNA polymerase II or III were not found to be more sensitive to 

0 3 than the wild-type, suggesting that these enzymes are not involved in the 

repair of DNA damaged by 03 (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 03 . Therefore, the DNA gene products are probably not involved 

in the repair of 0 3 damage. Recombinational repair mutants, rec A and rec B, 

only showed a slightly increased sensitivity to 0 3 than the wild-type, suggesting that the rec gene products are only partially involved in the repair of 

03-induced DNA lesions (Hamelin and Chung, 1978). 

Other effects have been observed than those described above on bacteria. 

In the commercial American oyster exposed to 0 3-treated sea water (MacLean et 

al., 1973), fertilization occurred less readily and abnormal nuclei (degeneration, fragmentation) were observed approximately twice as frequently. Sachsenmaier et al. (1965) observed a rapid loss of glycolytic and respiratory capacity 

and subsequent loss of tumorigenicity in mouse ascites cells treated with 0 3 . 

These authors also reported that chicken embryo fibroblasts exposed to 0 3 (1 

to 10 pl/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 predominantly examined the in vitro effects of extremely high 0 3 concentrations on 

biological systems or biologically important cellular components. Although 

9-182 
these investigations may be important for the eiucidation of the types of 

damage or responses that might be expected to occur at lower 0 3 concentrations, 

the most relevant data on the genotoxicity of 0 3 should be obtained from 

investigations where the 0 3 concentration did not exceed 1960 pg/m 3 (1 ppm). 

Research,conducted at or below 1 ppm of 0 3 will be ,presented below (See Table 

9-24). 

Several investigators have examined the in vivocytogenetic effects of 0 3 

in rodents and human subjects. Until the reports of Zelac et al. (1971a,b), 

the toxic effects of 0 3 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 3 , }ittle systemic absorption was predicted. 

Zelac, however, reported a significant increase in chromosome aberrations in 

peripheral blood lymphocytes from Chinese hamsters exposed to 392 pg/m3 (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 3 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 3-exposed animals 

when compared with nonexposed control animals. Zelac et al. (1971b) reported 

that 0 3 was additive with radiation in the production of chromosome breaks. 

Both 03 and radiation produced chromosome breaks independently of each other: 

Simultaneous exposure to 0.2 ppm of 0 3 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 ,addi ti ve. 

Chaney (1981) investigated the effects of 0 3 exposure on mouse peritoneal 

exudate cells (peritoneal macrophages) stimulated by an i.p. injection of 

glycogen. Mice were subsequently exposed by inhalation to 1960 or 9800 pg/m 3 

(1 or 5 ppm) of 0 3 for 24 hr. A significant reduction in the average molecular 

weight of the DNA was observed i;n the peritoneal exudate cells from mice 

exposed to 9800 pg/m3 (5 ppm) of .0 3 but not in animals exposed to 1960 pg/m 3 

(1 ppm) of 0 3 for 24 hr. The reduction in the average molecular weight of DNA 

in 0 3-exposed animals indicated the induction of singie-strand breaks in the 

DNA. It should be noted, however, that the alkaline sucrose gradient method 

of determining the average moiecular weight of the DNA does not discriminate 

between the frank strand-breaks in DNA, produced as a direct effect of the 0 3 

treatment, and the induction of alkaline-labile lesions in DNA by 0 3 , which 

9-183 
N 

I •r 1 

rtl N C 

N 0) C7 

7 C N 

EOf N 

rL 

r• O N 

ro ++ 

G7 L rU 

i-)V7 ~ d 

C N 01 

rtro v- a ro c O 4-) 

O N 

O

tn 

61 ~ L 

Z7 m

.

0 d 

Rl+roi 7 r 

N O i 

V w (d O rn a 

C L N 

O L 0) f0 

++ •O 3ai 

7 C L r 

O O O1 Z 

c •r d•r 

1--1 4i N N 

~ 

C~ 

N f0 N C 

C L tA o 

• ~ O w •O ~ Cl •ri- E •L > 

 L M17 G/L N O+ C. ~ •r 

'~N r v•~ ~ G1 Gl N+J 

Of }J L f-. i- +~ •N 

iJ C•O •r GJ C C 

C•r C N L+J ••• Gl G1 

f0 N f0 C}1 L/~ •r N 

+1 i N•r OfM U 

O Y- i•t N O•r • >f 

E O

c~~ a~i G7 GJ L Gl L t0:+L•O i•~ 

>

maE•N v s -L 

a~ d~ ai c•r cr 

r C/ O N O)t •r ro•O 

•N L'O L C+1 IE d O 

C GI •r L Gl E 

N C'II;r i+J L 

N•r C r O N N 

C N ro Y.X d4J ro 3 

O>— ro d C C 

a-iu~i s%. ~~..Ti ~ ~t~o 

ro d•O a U •O ~~ro'OO 

'0 V- (O Q r• 4- C ro 

t0 G) L Z M•r G! •r L O 

C•O v C1 0 3•O .G —•1J 

C 

L 

.0 

M 

C E •r \ 

E E 

Mr1 

N 

~ 

r 

t N 

N •r 

L vLC > 

ai 

U 

L N 

I f0'r 

LC7 G>1 u L 

ro d 

tn u 

t 

u 

i U! 

O1 N 

L 

V vi ~W 

N 

> 

i- N •r 

I C C N G) 

 1 ~7 •r N •0 

O +- G/ •r 

~ E+J X ro 

e0 N CI L 

ft 

L O r- O U oa)mcro 

N L +- f0 N 

t0 > 07 +J ~ 

V- O Z. L O 

tl td O) d 

.G d d•r 

N 

L L L= 7 

CM 

3 

 4- 

 O d 

 tn 

'0 4J ro 

V Cf U 

4-07 Gl ~ Vro+JV-•r O 

. v > 

7.~ F~

ro 

 •r

- 

 •r C 

E N'O+J O 

N U N N }1 

7•r C C U O L •r- C7 

i L O 7 N O

Ip 1- 41 C L 

>+1 N -- d 

4) 

C 

d 

L 

w 

d 

K 

N 

U 

ei 

rL tN 

N 

4-3 

aui 

V-• 

v- 

(U 

~ 

a~ 

i 

a~ 

N 

a 

O 

a 

U 

O 

+3 

GJ L 

L. d 

m 

N •O 

°c. m 

W C 

O 

4.) 

L ' 

7 I 

V 

~ 

C 

E o y L 

N E 

ro 

d 

~ 

C E 

O d 

•r d 

a~ 

i0 

L 

N 

MC 

O (V 

U E 

c 

o rn 

U 3. 

LL7 

Z 

O N 

O 

W 

0 

~ 

W 

ti 

W 

-J 

~ 

C 

M 

P• 

1•~ 

~ 

~ 

N 

Ot 

W 

..d 

~ 

L 

4J 

41 

E 

O 

N 

O 

t 

d 

y ~ 

il 

~ 

N 

•O 

b 

O 

E 

7 

. N 

41 

O 

d 

a 

L 

4- 

4- 

n 

L a••i Z, 

N 

C 

I 

H 

]C 

m 

Z 

~ 

L 

N 

4-) e 

+J N 

N 

f ro ~ r• 

u • ro r > 

• L ro 

~ 

G1 GJ 

E i 

o ar r• L 

~ 3 0 

U 

1 ~ 

i-+ C 

Y •r 

E 

II \ 

F- E ¢ v 

~ C 

. c 

-a ar 

o > 

L •r 

•F•) 01 

d 

E N 

N 

4-1 }i 

c ro 

C1 L 

E ai 3 

L O 

7 r 

N 4- ro 07 M 

~ o 

ro .n 

N 

C 01 

~O rl 

7 •• 

I N C 

7 L 

Cf C~ 

I N ro 

U l 

i N 

(C 

L > 

fJ i 

T O/ 

N tJ 

C 

N •r rt1 X I 

tC C C O O U 

roN O•L 10 d•~ N N E 

 N\.+•r d-) i. f0 O 

' C N N 4..1 C N U S.. 

O N L v C•r O 

+•1 •N > •r •r E ro •O rovaiuw o3+•~ N 

7~ L ~ O G~/ M •7 N 

•i7 vai O t7 o 

u 0 

O X 

•V •r g C ro•r O m u cr,ro- 

~ a•r c 7 

CJ 

G!G L+3 

+•~ V O1 •b~ t 

O N C.7 O) ^ L C C. 

4- C 7 N• U•r 

rTJlO • +> 0•rr• ~ w C C ^ 

O GJ •r +J +J CJ ~ 

C ro • ro ~ -o.N.ld= 

O /~ C rl C 41 O 

V- U o ro N 7 Y 

C7 O1-1+~ N u C EN 

7•r 1 R1 r'1 •r rp N d 

~ U t0 +1 C}1 N L 

H ~ V EX Of E u0i 

c 

• E 

O 

[+7 

~ 

~ 

O u) 

O 

O 

0 

m Ol 

ro 

•o u'f 

C 1- 

ro(n 

C 

Of d C 

S C.) 

•OI u 

ro 

N 

co C1 

rl 

CN 

ro g 

C C m ro 

I V L 

W S 

01 

/0 r-~•1 

ro ~ 

C! C 

7 L 

CI U 

~ 

C 

c E •r \ 

E ~ 

UD r-1 

N 

v 

I--• N 

N Y 

~ m ? 

Z ~ 

r-I 

O O O 

m Ln 

t0 O O 

Ol 

•1 

O 

ao 

O 

0 

00 

tI7 

00 

01 

• • ~ 
would be converted to strand breaks under the alkaline condition of the assay. 

Although these experiments do not prove that 0 3 exposure can cause strand 

breaks in DNA, they do indicate an 0 3 effect on DNA at 9800 pg/m3 (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 3 exposure by 

inhalation, Tice et al. (1978) tried to repeat the experiments of Zelac as 

closely as possible. Chinese hamsters were exposed to 843 pg/m3 (0.43 ppm) of, .

03 by inhalation for 5 hr. The authors investigated chromatid and chromosome 

aberrations in peripheral blood lymphocytes and bone marrow of control and 

0 3-exposed animals immediately after exposure and at 7 and 14 days after . 0 3 

exposure. They also investigated the sister chromatid exchange (SCE) frequency 

in peripherai blood lymphocytes of Chinese hamsters. - In separate experiments, 

SCE frequencies in C57/B1 mice exposed to 2 ppm of 0 3 for 6 hr were examined 

in peripheral blood cells collected from the animals immediately after 0 3 

exposure and at 7 and 14 days after 0 3 exposure. 

The authors reported no significant increase in the SCE frequency of the 

0 3-exposed hamsters or mi ce at any sampl i ng ti me, nor di d they observe a 

significant increase in the number of chromosome aberrations of phytohemagglutinin (PHA)-stimulated peripheral blood or bone marrow cells. The only reported 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 3-exposed animals, frequently in the range of 50 to 

100 percent increases over the control values. Statistically significant differences were assigned at the 1 percent level of significance. It is not 

clear how a slightly more rigorous evaluation of significance (e.g., p< 0.05) 

would have influenced the interpretation of the data. 

Although both Zelac et al. (1971a,b) and Tice et al. (1978) reported significant increases in chromosome aberrations in peripheral blood lymphocytes 

foliowing 5-hr exposures to 392 to 843 Ng/m3 (0.2 to 0.4 ppm) of 0 3 , the types 

of lesions observed in the two studies were clearly different. Tice et ai. 

observed chromatid-type lesions and no increase in the chromosome aberrations, 

whereas Zelac et al. reported a significant increase in the number of chromosome 

9-185 
aberrations. There were a number of differences in the experimental protocols 

that may have produced the seemingly different results: 

1. The animals were exposed to different concentrations of 0 3 . 

Zelac et al. (1971a) administered 470 to 590 pg/m3 (0.24 to 0.3 

ppm) of 0 3 to Chinese hamsters for 5 hr, whereas Tice et al. 

(1978) exposed animals to an atmosphere of 840 pg/m 3 (0.43 ppm) 

of 03 for 5 hr. 

2. Zelac stimulated peripheral blood lymphocytes into DNA synthesis 

with pokeweed mitogen, which is mainly a B-lymphocyte mitogen. 

In the experiments of Tice et al. (1978), lymphocytes were 

stimulated with PHA, which is a T-lymphocyte mitogen (Ling and 

Kay, 1975). 

3. Zelac cultured lymphocytes with the mitogen in vitrofor 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 aberrations, 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 3 

exposure were examined in different populations of lymphocytes. If one of the 

populations of lymphocytes was more sensitive to 0 3 than the other, different 

cytogenetic responses could be expected when PHA was used as a mitogen, compared with the results with the use of polkweed mitogen. 

There is evidence that the B-lymphocyte may be more sensitive to 0 3 than 

the T-lymphocyte. Savino et al. (1978) measured the effects of 0 3 on human 

cellular and humoral immunity by measuring rosette formation with human lymphocytes (See Chapter 10). 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 formation with B-lymphocytes was significantly depressed in eight human subjects 

exposed to 784 pg/m3 (0.4 ppm) of 0 3 by inhalation for 4 hr. A similar inhibition of rosette formation was not observed with T-lymphocytes from the same 

• :. 
subjects. The depressed B-cell responses persisted for 2 weeks after 03 

exposure, although partial recovery to the pre-exposure level was evident. 

It cannot be stated with any certainty how the differences in the 0 3 

exposure, the choice of mitogen, and the length of the mitogen exposure may 

have contributed to the differences in the results reported by Zelac et al. 

(1971a,b) and by Tice et al. (1978). There are sufficient differences in the 

experimental protocols'of the two reports so that the results need not be considered directly contradictory. 

An assumption that is made in all of the reports in which lymphocytes are 

stimulated with mitogens is that the lymphocytes from the 03-exposed animals 

and the control animals ar2 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 significantly suppressed in blood samp1es taken immediately after the subjects were 

exposed to 784 pg/m3 (0.4 ppm) of 0 3 for 4 hr (See 10.7). Other reports have 

suggested that 0 3 might inhibit or inactivate the PHA receptor on lymphocytes 

(see Gooch et al., 1976). Because the ability to measure chromosome aberrations in mitotically arrested cells is absolutely dependent on the induction 

of GO or G1 cells into the cycling state, cells exposed to sufficient concentrations of 03 would not be stimulated to divide, and hence no 0 3-induced cytogenic effects would be observed in activated cells. In their report, Tice 

et al. (1978) stated that the lymphocytes of the 0 3-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 3-damaged lymphocytes were selected 

against in the cytogenetic investigations. 

In other investigations with rodents, Gooch et al. (1976) analyzed bone 

marrow samples from Chinese hamsters exposed to "451 pg/m 3 (0.23 ppm) of 0 3 for 

5 hr. Marrow samples were taken at 2, 6, and 12 hr following 0 3 exposures. 

In separate experiments, male C3H mice were exposed to 294 or 412 pg/m 3 (0.15 

or 0.21 ppm) of 03 for 5 hr, or to 1940 pg/m 3 (0.99 ppm) of 03 for 2 hr. 

Blood samples were drawn from these animals at various times for up to 2 weeks 

following 0 3 exposure. The mice were killed 8 weeks following 0 3 exposure, and 

spermatocyte preparations were made and analyzed for reciprocal translocations. 

Data from the Chinese hamster bone marrow samples and the mouse leukocytes indicated that there was no effect induced by 0 3 treatment on the frequency of 

9-187 
chromatid or chromosome aberrations, nor were there any recognizable reciprocal 

translocations in the primary spermatocytes. 

Several investigators have examined the effects of 0 3 on human cells in 

yitro . Fetner (1962) observed the induction of chromatid deletions in human 

KB cells exposed to 15,680 pg/m3 (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 

tumors was inhibited by exposure to 588 to 1568 pg/m 3 (0.3 to 0.8 ppm) of 0 3 

for 8 days in culture. - 

Guerrero et al. (1979) performed SCE analysis on diploid human fetal lung 

cells (WI-38) exposed to 0, 490, 980, 1470, or 1960 pg/m3 (0, 0.25, 0.5, 0.75, 

or 1.0 ppm) 03 for 1 hour in vitro . A dose-related increase in the SCE frequency was observed in the WI-38 human fibroblasts exposed to 0 3 . In the same 

report, the authors stated that no significant increase in the SCE frequency 

over control values was observed in peripheral blood lymphocytes from subjects 

exposed to 03 by inhalation (Chapter 10). Unless the lymphocyte is intrinsically less sensitive to the induction of SCE by 0 3 than the WI-38 human fetal 

lung fibroblast, the results indicate that exposure of human subjects to 980 

pg/m3 (0.5 ppm) of 03 for 2 hr did not result in a sufficiently high concentration of 0 3 or 03 reaction products in the circulation to induce an increase in the SCE frequency in the lymphocytes. From the authors' data on the 

induction of SCE in WI-38 cells, the concentration of 0 3 required to induce 

SCE in human cells is approximately 490 pg/m3 (0.25 ppm) for 1 hour. 

Gooch et al. (1976) also investigated the effects of 0 3 exposure on human 

cells in yitro . In these experiments, lymphocytes were stimulated with PHA 

for 12 or 36 hr before the 0 3 exposure to obviate the potential problems of 

03 inactivation of the PHA receptor. Human leukocyte cultures were exposed to 

3920 ag/m3 (2 ppm) of 03 for various lengths of time to accumulate total 0 3 

exposure doses of 3234 to 27,832 Ng/m3 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 pg/m3 (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 di d not report how, or i ndeed i f, the data were stati sti cal ly 

evaluated for differences in chromatid aberrations. 

In summary, in vitro 03 exposure has been shown to produce toxic effects 

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 3 exposure has occurred.atsufficiently high levels and for sufficiently long periods. 

9.4.5.2 Mutational Effects of Ozone. The mutagenic effects of 03 have been 

investigated in surprisingly few instances (Table 9-24). No publication to 

date has investigated the mutagenic effects of 03 in mammalian cells. 

Sparrow and Schairer (1974) measured an increase in the frequency, over 

the background level, in the induction of pink or colorless mutant cells or 

groups of cells in petal and/or stamen hairs of mature flowers of various 

blue-flowered Tradescantia. No 03 concentration was reported in this publication. 

E. coli, strain MQ 259, were mutated to various growth factor auxotrophic 

states, including requirements for most common amino acids, vitamins, and 

purines and pyrimidines (Hamelin and Chung, 1975a). Ozonated air (196 pg/m 3 , 

0.1 ppm) was passed through the bacterial suspensions at a rate of 2.1 L/min 

for 30 min. Many of the 0 3-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 characterized (Hamelin and Chung, 1975b) as producing excessive amounts of capsular 

polysaccharide. 

Erdman and Hernandez (1982) investigated the induction of dominant lethal 

mutations in Drosophila virilis exposed to 58,800 pg/m 3 (30 ppm) of 0 3 for 3 

hr. 0 3 induced dominant lethal mutations at various stages of spermatogenesis. 

The sperm-sperm bundle stage was the most sensitive to 0 3 , and the meiotic 

cells were the least sensitive. 

Dubeau and Chung (1979, 1982) have investigated the mutagenic and cytotoxic effects of 0 3 on Saccharomyces cerevisae. Several different strains 

were utilized to investigate forward, reverse, and recombinational mutations. 

ozone (98,000 pg/m3 , 50 ppm; 30 to 90 min) induced a variety of forward and 

reverse mutations as well as gene conversion and mitotic crossing-over. Soth 

base-substitution and frame- shift mutations were induced by 0 3 . 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 

0, (98,000 pg/m 3 , 50 ppm; 30 to 90 min) was compared with other known mutagens. 

The positive controis were UV light (1.54 J/m 2 per second, 1-min exposure), 

N-methyl-N'-r,itrosoguanidine (MNNG) (50 pg/mL, 15 min), or x-rays (2 kR/min, 

. ;. 
40 min). By comparing the induced mutation frequency at similar cellular 

survival levels for 0 3 , MNNG, UV light, and x-rays, it was shown that 0 3 was a 

very weak mutagen. Induced mutation frequencies were generally 20 to 200 

times lower for 03 than for the other three mutagens.. 

In summary, the mutagenic properties of 0 3 have been demonstrated in 

procaryotic and eucaryotic cells. Only one study, however, (Hamelin and 

Chung, 1975a, with E. coli ) investigated the mutagenic effect of 0 3 at concentrations of less than 1 ppm. The results clearly indicate that if cells in 

culture are exposed to sufficiently hi-gh concentrations of 0 3 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 0 3 toward these species can be determined. 

9.4.6Other Extrapulmonary Effects 

9.4.6.1 Liyer . A series of studies reviewed by Graham et al. (1983a) have 

shown that 03 increases drug-induced sleeping time in animals (Table 9-25). 

The animal was injected with the drug (typically pentobarbital), and the time 

to the loss of the righting reflex and the sleeping time (time between loss 

and regaining of the righting reflex) were measured. Because the time to the 

loss of the righting reflex was very rarely altered in the experiments described 

below, it will not be discussed further. Animals awake from pentobarbitalinduced sleep, because liver xenobiotic metabolism transforms the drug into an 

inactive form. Therefore, this response is interpreted as an extrapulmonary 

effect. 

Gardner et al. (1974) were the first to observe that 03 increases pentobarbital-induced sleeping time. Female CD-1 mice were exposed for 3 hr/day for 

up to 7 days to 1960 pg/m3 (1.0 ppm) of 03 and the i ncrease 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 pg/m3 , 1.0 ppm; 

3 hr/day for 7 days) and then challenged with a 3-hr exposure to 9800 pg/m 3 

(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-1) and 

two inbred (C57BL/6N and DBA/2N) strains were compared (Graham et al., 1981). 

9-190 
~ ..1 

J ~ 

r _ Z 

O 

W 

Z 

O 

N 

O 

O 

N 

H 

U 

W lL 

W 

ui 

N 

C1 

W 

co 

J 

F- 

^ 

.. 

+) io ia ro a ro ~o 

d + ~ N +~ 

U G1 Uf OJ 

N 

G1 

y E i L Of E 

Oil t ri ~ th E ~ •°I C e-1 •CO d• .C rl ro

K- 1O Cp Op n • C OD i n 16 GO 

C c.i~~•~i c~r~i' lir

m

i ~ i-~+ G.07r~+ C'LJr~i 

4.+ 

.. 

ro a 

N 

.. 

N G1 i G1 y 

$. c 

S. rtf ~ 

O 7 ttJ - i 0•7 N 7 q7 d+) GJ r 

u o E N N O E N ~ n r• eO 

y ~ y +1 E 

SId 

7 

0 

f G! 7 E(0 E 

tN v C E ~ ELv4.0- 

N G7 

' >> • •• U 1 ~ N 

10 N U Gl 

c 

• 

a rn m 

G1 N ro m r- ui >, al v 

G! atG a a d L 

Ol 

L. 

L Cl 

O L 

a ~+ t+J U 

d " N o•r 

a E 14 L ro L.0 

N G! E Cl ^ a U i•1 

u x r+ ro> 

~ Gl 

O >, u u v, >, E ui 

7>~ cp V- >> 

U v- 41 +J a c 

7 V- N C1 M•r C 

'a L a ~ 

C Of o ai ~ ro 

N 

ro 

~ 

10 . 

c0 L E 

O•r 

'fl Rl a O O 10 

ca +~ a 

a Y- E 4-1 ro 

c o ^ro ua G1 L 

.. 

N 

rc oro 

1•r In In c a 

.. 

o 

aNx 

d o ro 

r na 

1 M rn ~.n 

r..tatci.c+Jvi 

1 3ri " ar a N u aE o c L 

^ro 

cm d«•r w m 

vVi 10 

4•1 

N o o GV 4•> > 

+1 u• 

u 

7 

c 

Gl 

~ x 

a Gl >> 

i+1 G) L L 

+1 o i- O 

•r C r• f0 N v1 +1 

+J ro ro U Ol N 

U • E•^ al OI a a al • ar- M 4- • L a E•F+ al Gi r• E 

N d p a N ri 4- 

L 4- a>> 4- N 

Ol 

L U>> 

L C 

R1 O 

d N 7 1tl 

L v L X E. M+

~ C Gl O i L Rl 

S. N 10 ~F C 7 N L 

 W ro ro m>, • +~ u~ (0 L u a ro G/ N la 

Itl o+J • oaoa 

i 

v 

dJ •+- 

ro> 

aj 

+>ua 

CJ uf E a 

o++>,ma 

.G M ^a N u o 3 G1 

o Jc•N•ra 

. a +J •r o r~ aZ 

L 

> 

. 

a•r 

07+1 

ro¢ d 

Cl 4J 

+J n-.ro al"1 

- c IOa o 

+J c m m ro E x a ro 

c•rn L N L E. 

> 

L 

G/ •" L. rl N 

a m N o +J r 

L u 

ro 

d L 

r o 

G/ +1 1ff o 

o. a~ L ~ 

d ~ 07 d 07 Gl 

a c) c.~ .- o r V- +J vCl E- C •^ 3~ 4- C C i•1 a G . E G1 G1 •• E 10 C q1 O N 

N 

~ 

C•r Cl t0 N 10 ro 

r+~ E A a 

O N O Cl 

w 

c c d Gl 

r 07 L> 

C•r .0 U M a 

r+~ +~ C E G) 

C•r E 10 E E C 

r+1ri 

. O 

' 

10 SG) O) •01 E a rl O 

+J C) 

U> 

i•) (0 

U r- 

+~ 

Gl C ~ •Of 

O ro\ N 1 a 6J C>>L 

0l CI Cl C U d ro OJ 

d G1 G1 >~ N O O 

y Cf 

L. 

 Of O 

N C i' 61 1 a N C U ro•r L a d N 

f0 •r L aM d 

G/ a E S. 

4- ~ 

4- 

9- X 

V- O 

10 !~ C N 

N 

l0 •r O r X 

01 a C O G O! 

f0 •r ~ E;\ ~ Ip 

0! a •• L t0 G) 

L y N Lf~ f.. a O 

U N L N O a4- 

N a 01 L. 

a 

i a N G! 

U•r C> 

L. N +j O 1 

U Gl ~ 00 G) 

S. y Cl N N c L E L 

U Cl U+~ •- Cl U 

N tfl a Z •ro Z L O 1U0 E ~ •--~ N~ Z v p• '-C

i N •E i i. d v•r ~ 

O 

U 

O 

N 

O 

L. a Ln 

Q ~ \\ L X 

X O L e1 L L L 10 L .G 

Wi-) M r-i L t ~ ~E/ M LL7 

IE M M 

L 

a 

a 

ro 

c a 

L Gl Sd O• m C Sd 

C.7 C7 

N 

ro 

d 

S 

rl•1 M n 

~ 

ONO IXO 

O C. r a O 10 O O M O O v rl rl 

4J rr N L 

C 

a c 

rr ai 

o 

o ~ 

O U E 1 O ~ G C tC O X G O 

~ V 7. r~i ~ ~ r~•i CWO ISf r~•1 r~i v r~•1 r

~

l 

9-191 
u ~a N y 

N € IE E 

L R tl ro R L Cf 

N 

L r-i 

fR..T 

.G N 

iOOi 

L N 

C C7 r-1 L7 r-I 

LO01 

61 N r 7 m N r 

7 R N r 7 R 

Uai ~ Eai f aEi 0 aEi a a- v- v 

tn 

t!1 co V 

1V c•~ L d i i~ N C 1 d 1 Gl •r- a+J V U•~ L 

N O 1 N Gl N 07 •~ •r C C+1 

a N Gl m C 1 ro G/ d; X•- OI 

~ ••4- L 

o ro L'p O ro G/ € r o 

205 

 N 

• X n. •~ r r L•~ N+1 N M R 

N Tfp 

C+J V 

U T O ~ •fl ~ O C ~ R L 7 R N C1 

i•1N ~. O N O CJ N N d N L'O C L•p O.0 E i~ L Gl G/ 

G! r 

E•~ O•~ 'O L i 

~•'~ 

~ 'O T d f. 

G R L L. 

~~ G/ C IT }I C.•~ d•1 7}1 1m N 

C C€ O N L~•r- ro T.0 L ro 

O 5-4 C N N 

f- T Q N tC) > 

• 1 

M•O 7 7 R m•r- •r i a.- a'O O! C L R G) L a C R v•r G! N 

O v 

N U 

'Q 010 U 

+~ i~ a R ro N 0! L O ••i•1 r- S1 d U C 4 / Gl N•F/ L. G/ •- U 

U O R G! 

O c O • L E r C N m C C a O E N C ~ 1• r R G7 Cl E; O C 41 •~ L N U a C i~ i- R•r~ X R•~ •r pp• L N N L a•.- C•~ 

iE a•~ 

e N a •• fNp ~+~ 

+1 •FJ N V- L L 1•r iJ .• u R•rc•1•1 rl CJ N 

o Y•. i N d N U .O m, 'O •' 2 ~- C ~ N C a IT E o ro (~ „ 

u 4-• • C a> ~+~ c•° 

v d 

ro i o d 

L r-1 u G.VL c ro GJ .G R p U•r- . c•~ U; 

R•r- i x s +1 7 N¢•~ +7 G) +~ O • ro R+1 

o c 4- L a ro•~ R •• c ~ d1 G.127 a .G L R•~ s}/ > L R •O 

C N 

a- i~ •r- L C7 

c m L 

O 07 a E L 61 C7 C O 7 N d O7 O x Ol o r a•~ O tfl Gl c O! i•/ a d-1 O U•.- 'O U L 

CJ R! ro 4•1 c 

W > d L 1 O Gl r~ L C•~ 1'O i LL r•- N•~ L L ~ U R 0+1 

~ i 

M•/ G7 

a N•1••~ ~' C 

E•- N 

L N N i~ Gl i}1 r C Of N N a R.0 

U•'- L i 3 O R ro v [V y•~ 

14 y y ro Z L 

C C N N /~ Q7 J N G•'- ; •• C ~ 7; a•r C+.~ N C! L t O G! ro ro d L 

o 

a 

'O 7 

r C1 •O v C • •• r U C 

U C+~ o+~ ~ 07 ~0 i+~ N c•~ 

U N L • •N 

~ i~ c j~ j~ m ~; ai rna~ ai o 

N C N L i 

ai ~•~ c Na ~ i v o ~ 

N'C 1 67 L L.p R•~ C.f.l 'O N • 4-• 

u o GLrM N 

d U+1 +J T ~+ T T 

Z 

ro•r- R 7 Of 

Ol a CE N 

R c N E N ro .O E•~ ••- G/ .G N o N•r C+1 U U •• o a C.1 i-~ .G 

4- N GJ x Id 'O 

4- o E E O E•O o C L 61 L O L 

U 61 U a U 

L 1• R 7 1 Gl C ri G/ •r 7 

+

.t d 

U r E G! •O m C Ul 1 € L V'p N 

y ln N N L O_ a 

cf 'O 'O 'O a N 

W c r GI x R c ro R s c lD o.0 o•r- c C1 Gl ~ F- o 0 1 1 1 T o ZO N•O O/ C/ 1--1 a..i r}i •~ ~--4 > aU + 1 •~ L. N O .O Z O. Z O L!A X+~ 

O 

IL 

O 

Fy- O U U 

u

W. .~ 

W L 

ef a 

tf! 7 C 

N N R 

R O 

'O N 

1 1 aC L M tT0 W IX O LN V L 

L L L LL 

m NR `n rl a' `C' ''C' " 

F 5- 

v 

a 

la 

a~ 

cv 

a o 

aEiat~ w1— wF 

L d SO. wfy € U C7 U.C7 U C7 

ro 

v 

c r= 

o n r-1 rl rl ln 

iJ 

Cf L 

CN 

OC 

N Cf 

O U@ O O O O 

O Cf Ol O1 p

Im

1 00 

U 7. rl r-1 ri O1 

9-192 
y 

a 

I •L 

u 

I ~ N 

I y 

O C 

0 z 

~ 

~ 

a~ 

E 

O 

4-) 

O L 

d 

~ 

1 

~ 

~ 

•r 

'O 

O 

7 

tn 

cn 

Rf 

Q 

d 

b 

L 

~ 

W 

7 a 

ib 

i. 

4J 

7 N 

C 

II 

m 

z 

N 

u 

C 

N 

i.l N 

N 

C 

E C 

7 O 

~ 

E IE 

4) L 

L ~1 

U •r 

44 

d N 4) 

L

ul (O d L N 0. 

ffl N 

rn ro Cl 

I~ I ~ 

~ 

W FS d V (a 

'C7 •O 

L L 

4J 4J 

C1 43 

E E 

+~ C 

C O 

GI 

~ ~ 

i L 

7 a 

N •r 

~ m 

ro a~ 

~ 

v 

7 

a~ 

c 

O 

V 

v 

04• W 

? 

..J 

LLJ 

S 

~ 

Z 

P 

W 

z 

P N 

P 

LL 

P 

y 

H 

V 

w 

W 

ui 

N 

~ 01 

W 

m 

Q 

H 

Ln " a; b 

r-,n 

P, 

a, cc ai rn.0ao cr+ 

u rl N 01 +1 . 

d E E ~ 4-1 ~ O 

a ttni No io vm 

4) L. 

 •i7 c.9 ~-+ 

4-3 

C07 cOe r

~

i 

~ 

01 

3 /0 

c z a- ro ro tn v n C 

N 

> 

N 7 N 

ro U ro 4- N ~ 

N U V uEi U O C V- 41 N+~ •r ~ y 

~ 0 NC R! N C O/ 4- O E +J r4.7 

rv ror-a c.o 

d L d - ar c 

o i rov N> 

s. a~ ar •r m 

N 

v 

. ai 4- a~ c.•r 

C r 81 C r> O E"! 

r tp r•r ro L • .F> N 

t 

u 

O 

L rna+ ar 

Of d C U N 

C+~ 10 (0 

.•N+ O! ~ U 01 •ry- U c " C+0~+ 7f > ~0 cLi 61 >, 

u Qt N.O W Na oiJ•r•r 

r0 L O t0 L G1 •r ~0 u E 61 

•L E'O A C 

4- d'0 N 16 V+~ C! p U. d 7 W N•O 

qGl i. a N. L a C ttf C D1.0 

u O 

0. 

 U O ro L•r C R,1 

C N O C+) L+•)t0 ro+J ~ 

•r C 

+J O 

~3 •r 

OI •r N C•r 

'Cy C ~ N Y 

N ~y 

'O 

G1 

•r C r•r C ro C L L N 

G1 N d a a.a u E 

d4•1 

w ro O N L c 

> L ~Q d 0. r U C 

i-1 C ~ U C C O r•r 

L L 

~.1 

i r U O~'O 

N-Mi+ ro Clf O•r 

G) 

N 

t~• W. •r O V- O•r C rtf ro 

O rl O V- U +J L 

C C 

O 01 

C S. > L. 

•r O C1 

P GI LL7 61 ~ C 4f Of a a E W- C E'6- tl! O+f C C. L 

41 C ~~ V O C W.J •C I e>- 

0/ U 

d•r d••^ 0. •r N o r 

dr O f1r O•1•> a C a 1 . 

~ i U 

S. 

 N O E 

tl) u VE C Ql r 

W•r 4- N C) 

4- o E'F- ~ L.0 riv- Il Lnv- n

u~ a ro ro G so. a a~• a+~ ro >~ 

+~ m c. ~ w id C v- 0 3 a) ar o i 7•r m a) c d L vQ L•r N+a +0 d N Z G. N> O N•r 

C 

V 

0 

~ 

O 

L 

d a Lv 

oi ro c 

O 

d C L E 

\ 

S. 

`X O .0 O L 

~ tC) G1 Cp 

L 

,7 Q 

a 

ro 

~ 

C O 

cu 

E .0 0 x 

L N S

LLI 6~. C > = E C7 c.7 

Vf 

ro 

s 

O C . rl tf~ rl N 

r d

dJ 

41 f0- 

C ~ 

O C 

N 01 

I O O E 

. V 7. 

01 00 ri C1 Cf ri Qf 

m 

9-193 
Ozone (1960 pg/m3 , 1.0 ppm for 5 hr) increased pentobarbital-induced sleeping 

time in all strains. To determine whether this effect was sex- or speciesspecific, male and female CD-1 mice, rats, and hamsters were exposed for 5 hr 

to 1960 pg/m3 (1.0 ppm) of 03 (Graham et al. , 1981). The females of all 

species exhibited an increased pentobarbital-induced sleeping time. Male mice 

and rats were not affected. Male hamsters had an increase in sleeping time, 

but thi s i ncrease was 1 ess (p = 0.075) than the i ncrease observed i n the 

femal es. Thus, the effect i s not speci fi c to strai n of mouse or to three 

, 

species of animals, but it is sex-specific, with females being more susceptible. 

Female CD-1 mice were exposed to 196 to 9800 pg/m 3 (0.1 to 5.0 ppm) of 0 3 for 

3 hr/day for a varying number of days (Graham et al., 1981). At 1960 pg/m 3 

(1.0 ppm), effects were observed after 1, 2, or 3 days of exposure, with the 

largest change occurring on day 2. At 980 pg/m 3 (0.5 ppm), the greatest 

increase in pentobarbital-induced sleeping time was observed on day 3, but at 

490 pg/m 3

(0.25 ppm), 6 days of exposure were required to cause an increase. 

At the lowest concentration evaluated (196 pg/m3 , 0.1 ppm), the increase was 

only observed at days 15 and 16 of exposure. Thus, as the concentration of 0 3 

was decreased, increasing numbers of daily 3 - hr exposures were required to 

significantly increase pentobarbital-induced sleeping time.' Once the maximal 

effect occurred, increasing the number of exposures resulted in a diminution 

of the effect. Generally, effects were observed around an approximate C X T 

(concentration, ppm x time, hr) value of 5. Also, CD - 1 female mice were exposed to four different concentrations of 0 3 (1960 to 196 pg/m3 , 1.0 to 0.1 ppm) 

for 5 to 20 hr continuously in a fashion yielding a C x T value of 5(Graham 

et al., 1981). A11 regimens increased pentobarbital-induced sleeping time. 

The time to recovery was examined in mice exposed to 1960 pg/m 3 (1.0 ppm) of 

0 3 for 5 hr/day. Recovery was complete within 24 hr after exposure, whether 

exposure was for 1, 2, or 3 days. 

To determine whether the previous responses were specific to pentobarbital, other drugs with known and different mechanisms for termination of 

action were used in CD-1 female mice exposed to 1960 pg/m3 (1.0 ppm) of 03 for 

5 hr (Graham et al., 1982a). Ozone increased sleeping time induced by hexobarbital and thiopental and paralysis time induced by zoxazolamine. Within 

the liver, pentobarbital and hexobarbital metabolism is more related to cytochrome P-450 than to cytochrome P-448-dependent activities. Zoxazolamine 

metabolism is more related to cytochrome P-448 than to cytochrome P-450. 

9-194 
Other major differences in the nature of the metabolism (i.e., aiiphatic vs. 

aromatic) also exist between these three drugs. In contrast to the above, 

sedation from thiopental is terminated because of drug redistribution. Thus, 

it would appear that 0 3 might affect some aspects of both drug redistribution 

and metabolism. Although there are different mechanisms involved in hexobarbital, 

zoxazolamine, and pentobarbital metabolism, it is possible that some common 

component(s) of inetabolism may have been altered. 

CD-1 female mice were pretreated with mixed-function oxidase inducers and 

inhibitors with partially characterized mechanisms of action to relate any 

potential differences in the effect of 0 to differences in the actions of the 

agents. Mi ce were exposed to 1960 pg/~ (1.0 ppm) of 0 3or ai r for 5 hr 

before measurement of pentobarbital-induced sleeping time (Graham et al., 

1982a). Again, the effect of 0 3 was observed, but mechanisms were not elucidated. 

The effect of 03 on hepatic mixed-function oxidases in CD-1 female mice 

was evaluated in an attempt to relate to the sleeping time studies (Graham et 

al., 1982b). A 3-hr exposure to concentrations of 03 as high as 9800 pg/m 3 

(5.0 ppm) did not change the concentration of cytochrome P-450 or the activities of related enzymes (aminopyrine N-demethylase or p-nitroanisole 0-demethylase). However, this exposure regimen and another (1960 pg/m 3 , 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-mi n exposure to 1960 pg/m 3(1.0 ppm) of 0 3on 1 iver 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 pg/m3 (10 ppm) of 0 3 (Palmer et al., 1971). 

Pentobarbital pharmacokinetics in female CD-1 mice were also examined. A 

3-hr exposure to 9800 pg/m3 (5.0 ppm) of 0 3 did not affect brain concentrati ons of pentobarbi ta1- at -time of awakeni ng, eve -n though - sl-eepi ng ti me -was 

increased (Graham et al., 1985). Therefore, it appears that 0 3 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 roin postexposure (Graham et al., 

1985). Following this exposure, first-order clearance kinetics of pentobarbital 

were observed in both the air and 0 3 groups, and 0 3 increased the plasma 

half-life by 106 percent (Graham et al., 1985). Mice exposed to 1960 pg/m 3 

(1.0 ppm) of 0 3 for 5 hr had a 71 percent increase in the plasma half-life of 

9-195 
pentobarbital. This ozone exposure resulted in a decrease (p = 0.06) in the 

slope of the clearance curve. Clearance followed first-order kinetics with a 

one-compartment model in this experiment also. 

In summary, the mechanism(s) for the effect of 03 on pentobarbital-induced 

sleeping time are not known definitively. However, it is hypothesized (Graham 

et al., 1983) that some common aspect(s) of,drug metabolism is quantitatively 

reduced, whether it is direct (e.g., enzymatic) or indirect (e.g., liver blood 

flow) or a combination of both. In addition, drug redistribution is apparently 

slowed. It is unlikely that ozone itself caused these effects at target sites 

distant from the lung (see Section 9.2). Because of the free-radical nature 

of oxidation initiated by 03 , a myriad of oxygenated products, several of 

which have toxic potential, may be formed in the lung (Section 9.3.3). 

However, the stability of such products in the blood and their reaction with 

organs such as the liver are speculative at present. 

Reddy et al. (1983) studied the effects"of a 7-day (8 hr/day) exposure of 

rats to 3920 pg/m3 (2.0 ppm) of 03 on liver xenobiotic metabolism by performing in vitro enzyme assays. Although lower 0 3 levels were not tested, this 

study is presented because it indicates the potential of 0 3 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 3 exposure; no effect was observed in supplemented 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 3 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 

9-196 
of other compounds metabolized by the affected enzymes. For example, epoxide 

hydrolase, which was decreased in the vitamin E- and selenium-deficient rats, 

metabolizes reactive epoxides to dihydrodiols. The metabolism of a substance 

such as benzo(a)pyrene would be expected to be affected by such a change. 

However, because of the complexity of the metabolism of a given chemical, such 

as benzo(a)pyrene, a precise interpretation is not possible at this time. 

Veninga et al. (1981) exposed mice to 0 3 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 

determined. They stated that the maximal 0 3 level was 1600 pg/m 3 (0.82 ppm) 

and the maximal exposure time was 4 hr, which would have resulted in a C x T 

of about.3.2. Animals were studied at 0, 30, and 120 min postexposure. It 

appeared that immediately after exposure, a C x T value < 0.4 caused a decrease 

in the reduced ascorbic acid content of the liver. At a C x T value of 0.4 

and 0.8, there appeared to be an increase that was not observed at higher 

values. For the 30-min postexposure groups, the increase in reduced ascorbic 

acid shifted, with the greatest increase being at about a C x T value of 1.2 

and no change occurri ng at a C x T val ue of 2.0. The 120-mi n postexposure 

group was roughiy similar to the immediate post-exposure group. No effects 

occurred 24 hr postexposure. 

Hepatic reduced ascorbic acid levels were also studied by Calabrese 

et al. (1983c) in rats exposed for 3 hr to 588 pg/m 3 (0.3 ppm) of 0 3 and 

examined at 5 postexposure periods up to 24 hr. Rats had significantly increased 

ascorbic'acid levels in both the 0 3 and air.groups, with the greater change 

taking place in the air group. Thus, there were no changes due to 0 3 . Likewise, 

there was no 0 3 effect on reduced ascorbic acid content in the serum. 

9.4.6.2 The Endocrine System. A.summary of the effects of 0 3 on the endocrine system, gastrointestinal tract, and urine is given in Table 9-26. 

Fairchild and co-workers were the.first to observe the involvement of the 

endocrine system in 0 3 toxicology. Mice_exposed to 11,368 pg/m 3 (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 

corticai function, Fairchild and Graham (1963) hypothesized a possible interaction of 0 3 with the pituitary-thyroid-adr.enal axis. In exploring the hypothesis, they exposed mice and rats for 3 to 4 hr to unspecified lethal concentrations of 0 3 . Thyroid-blocking agents and thyroidectomy increased the. 

9-197 
W_ 

Z 

t•r 

t_Y 

~ 

C_1 

~ 

t 

~ 

h 

t0 

N 

C1 

W 

-1 

~ 

N 

~ -I M ch 

lD 

Q1 

Cl I , 

0

~

1 

'-1 

0

03

1

0, 07

r-1 14 

ri 

N 

I~ 

r1 Cl 

ri 

Ol 

r1 1, 

tU7 

Of rtl Of L 

C OI C 

• vt Q tn (U 

~ tco a 3 n c d c m 

~ 

F- io ro ro 

U 'C 

~ 

C 41 c L C T 

C 

0c. 

~ C 

`O u 

ro G7 (a tT M17 ro 

~ C m 

w ^ ~ i 3 3 3 E 3 t+-) 

~ W fY L roLL 6 Q C CO. Q S - 

07 }t 1 t 

•

u

¢ i J.1 i~ 4-3 a ~ Of t71 +J 

N ~ C 

ra 

C tY tY O C ~ 

4-• G7 

E •O • i 'O •r0• C 

a G) tp 

a 'O Vl .t. ~ 

u N - a •• 

1 O ql 

• •• ro L C 

'C ro'O 

•r G 

C L N 

fCl U'O 

C tE U 

lD W. ro C7 C1 

•L 

+J 

.• 

~•+ 

C tY 

t0 W 

07 L. 4.a 7 O r ro 

M 

1n O L V- V1 .y 4- C C •C 

In L N 7 ~ 

~ 7 r 6 TL •• r C 4- 

O D U Vtq •r d ty 

t0 N 

Cf.0 

c+7 

.y 

Of o T 'O L. 

C vl •r • 

ro O V- O 

.0 .N C 

~ O O 

'O •r 

'O 

O 

••+J o c Tnv 

O N L 47 

t- 

~ Yv+J . ar o 

r C V7 V7 }i ac +1 

Cf ro X•r C 7 

rov•r 

L C 1.1 

o c++ 

L 7 Ul 

m ro c 

C r- YJ o tA ln Cl O •o G/ G7 ro Cl u O, o ro tA d ro T O tll 

•

L i•r •r 

01 +1 U ro tU 

L o0 M tn 3 L 

•1•t . 

• 'O tn p! C 

L E•r N L 

y L L U 

'O O+.~ G/ •r a t7f 

•• N 

L 4- L 

1.t t7f d 

Z U U ~ 

N G1 L•r ~ Vl O ~ tJl rtl U GI •

O 4- uf +~ +t O 

7 L. Ql ro ~ V O T ro i~ N 07 S. ro to •r L V- L 

~t ~ ~l 

O C L. C1'C L. 

r n U 

L ro 6f 

Rf •r 

Cf +~ 

a..~ C r• C uf 

RI t0 — 

L L a U f.. 

O d N 

L OI t7f 

i~ RI Cf 

i C L 7 

ro O V1 

4- O O U 

O C X 

.D 

n Gf N.G c O 

X U iJ 4.1 ro PG L 4- 

•r 

4- 

U 

tU C7 

L i•r .{.t 

ro L. a c 

L•r tn Y 4- 7 

+J 3>f7 r'O 

L f0 

C L - 

n•r L O 

tn i~ n 

Vl •r d 

E 

~ 

NN 

G7 C V- r 

O¢ G3 V 

tn y 

/~ ro 

L•n 

C a ro ro • ro 

U m V 

ro ro 6l r L ~ •O r O G! r i+7 C 

O Cl O 

C 7•r X 

r 

3 d 

~ 61 Rf L 

• > O 

.~ 

N 

v- u ~ m 

O ar• +~ 

c3o -o•r c= r c 

r a1 'O 7 

roa L m 

a~1-~ f- n 

wEr•r 

Gl G! Gl I•t 

u .w 

•O C'O u7 

vm 

U 

67 

6f . a ro c 

'O C N C tv 

•E 

C 

>t 

L'O 

•O -~ 

L.+ 

Y G! Gl .G u1 T T ~ t7f.0 .N ro c•r •r O •

u r• y •• 

C CJ 

V- G•.• v lp a> v1 o a) 07 >,cn 

01 •r d-t U ro 

E! ~ f0 

G/ N L L T ro 

i t7f y a L E 

C ro L 

Rr rn•- •ht 

~ O n 

O C/ L 

ro C•r C 

r c•r 

V- 6f L -- u7 L. y M> w L.y ~ L r• 4- •• 7 C+.~ O rt7 L C nr• 4- Y T N N • • C Of 7 O O'Q L 

aS a! p~ o 

v O = N C N•r y.r iA O V- L r ~ A U•r 

- ul r- +t ro ~ d ro+~ ut 

v 

G! .0 i.t d 0J 

m CI u 

o L 

c Yl 

o o 

L tn L r• 

u 7 u o 

a f0 •r o 

x'O a•r 

L 7 C c 

ro r- o•r 

.{.t t ro V 

tq >t i 

(0 Of G) ro'O 

d..t c L d•r 

> ~L +~ U1 0.0 L 

Gl 

a C,7 OJ . c . ro uf 

~0 

U•r T~ (0 M tE ro u i u L 3 O i.1 y.t N 

•~•t 3 

VO>) > Yl 

i.~ L. 

f CU td 'O 

lLt = E C7 i.> • 

}t ro Vl u 0) . 

tn • E r- Vl 

7 c+~ G! 

U•r t0 i~ 

u O ar•1 

~ t 

L L\ u ro 

+~ U~\ 

tn .0 0f EU N 4- i-t Ol U L F- T a.t C ~ n o'O ~ T'O c tn L T 'O V- o • c o 

a 

O 

+~ N Y i o Rl 

~ ro 1 7 ~-1 E•r 

G.r ~ 

y 

y- ~• 

4- L ro GJ N 

•O O U r• C r• 

n N•r L c i t.o 

O1r• U f0 In •r 

ro ~ d u 

> IA Y- O 

G7 T o rl 

+~ L 

}t o U N c 

C ro O7•r 

r~ tC n R7 L ~ F tD C C O•r 3 T f. 7 C 01 }1 O TJ •r L RI O tn Of ~ L O E 

E S. r•1 O CI'O d•t Cl Gl CM t0 N 61 7 E C f0 u tll C OI U i C r- ~ n r•r-C y L U L RJ 

~I O U n> 

L C 4-• X Gf O 

LL7 L 3 

O O 

t 

O tD 

Vl L U O 10 . 

O R1 ro L L 

L r• N L O Of ro 

t0 C M17 ro v- = CJ i T 

r• • 

ro Gl Gl 

3.1~ •r +~ 

O•r i•r- N. 

{L. •r O 61 r Z rl E r• Z 01 ~ -i n7 a ~ W Gl > a+1 Vl L 

U~O .O E 

E O1 nr• U Z U L J 3 7 C rl 

'O 

C _ 61 N 00 

ro 

in 

3 

to++oo w> cb M~ at T3m 

o•.- +> >f c 

>~ 

ro 

oT 

a ro a'O rl O 

O 

X t0 L. 

17 •r 

y r.t 

C 'O ul 

\ T 

L 

L X'O L. ql 

X 

L G! 00 I a 7 

L O to 

~ VI 

\ T~ tL L. n C L •E L N L 41 O L+t rl L rl vt O L ro 7 7 

'O 

M O 

Of u 

L 

O 

L'O 00 

1 

vl N 

00 O t 

ul 1 

CO O N 

a n 

•• ro~ X 

L'O O 

L 

N M tn e}' ~ a• arl cl' arl ~ M'Q 07 tD <}' rO 

~ 

C 

d b 

~ O ' 

30- Y U 'O -t Z Z Y 

7 flf C1 C! 00 

w E 

to 

Z Z Z 

O 

~ I N 

G E 

O a 

O r•1 tn tn 

N IA t3Y 

tn 

O O O 1 , 

t2'f 

I , 

t0 

ItA 

i1 C Ctp LL7 ro 

41 a O C O O rl O r-I O O O O 

d L 

C 41 

O C 

~ 

G! 

O U O O O CS O O ~ O C 

c 

O t3! 

~ o rl rl o er 

01 C1 oto 

OD 01 

~ 

~ 

t, 

d' 

t~ 

d' 

r, 

ct 

to v ro 

t~ 01 i 

U 7. tl9 N ri st' N Ol r-i rl t--1 rl rl rl N v 

• • • 
E 

I O C. 

~ e c -c ~ ~~ ° 

G1. ~ U •r r C .-d-~ L S L a 

~E N ro ~ 

~ C Rt ••• N O ~O H N F- •• N O O N ~ 

•0• a(D ~ c L~~ ~ y 

N ~ i•~ +•~ ro 

F- O u L ~O O -E c L rN O. u 

1 L• ~ G) L L•r C O+ ~ 2 G! 'O ' 7 

~ o a727 ~ c N U L 1» ~~ c F•N- U N~ Cl 10A ~ 

•O L F- C•r C O i•1 ~- 7 U O f L r C E r 

i G) ~ 7•O •r C 3 - GI tq N • `-1 N Of OJ +r t0 - 

L C N d r C• ~ i U•.- 4- U t0 N II 3 ~ L 

++ m ~ ~ a•r N a•r i 01v°-~ •• ~r'o m o L 

~•• E o•r II O Gl -•• r O- Gl V~- w'O L a O ~ n 

oL arma>co T.t °r . o ~ i~ +•~ 0 61 •r w- tLO NC~ona 

4- o o ro r r C. i o d N+1 a> > 

d L L L OI o ~ ro~ c 4- N C74• G7 

> d a 7 C L N+~ Ol y d N N c O r 

O C 9 a•~ Of•r •o-~ C•r +~ N r• L Gl Gl .. 

r O• 'o N C+~ , O O O C'R7 1 L ~ i*- ~_ 

E - O c N.r U ~ U E++ a 3'~ ~o ~ cn 

E L L ~o c v ro•r L•r > .~.+ N•o E F- 

~ o T ~ cr+~ o aa a ic•r . o a•r 

L L L •• • O L L - L L L 7 r p••r X C ai ++ ~. c a L o E c. a~ •o-~ •— X w ro•r 

N Cf a•r• ~ O- U Of O L Cf•r G/ w•r• E 

C F- 'O - C L T F- C O. U - U Ol 

N+~ L G/ p~ •O •~ O •N ~~ O +1 L 2' ~ • O. ~ 0 Rf 

N~O R1 N G L. ~ Cl ~ . IO O N T7 N•r pl 

ro r+~ L ro T.d ~ /1 •e c u N I- G! c+~ L 

d 7•+- 4- L L N • O 91 r 7 •i L 7-C U i E o v++.ro d a E ~ d u a a a a a 

U•r +•1 'O Gl > ~ i•r > L a.~ L i~ L+1 'O 

y ~ •r C O V- L Gl 7 O O 6l •r C O r O O 

. C N a t0 c O u r r L L r u ro E ro 4- a R1 

~ 

C 

v ror 

L O 

7-C U 

N O O 

O •r d•~ 

X N i 

W L G. 

a 

ro ~ 

da N L L d~ 

7 N N E ro 

d 

E 

C E 

O ~ 

~ 

N L 

C 1~ 

O C 

O u E 

u 1 

i 

L ' L 

L- . ~ 

tP7 N 

G 

rr Z 

rf N ~ r-I 

000 0 

l0 N ~ 

r~IMI~ 

t0 

Ol 

r-1 

' a 

r M_ 

•L O . 

L r ` - 

+~ ro 

E o L}t : 

~ ~_~ 

~ , •r' 

.. +~ 

M U 

.•i G! .~ 

~ c 

O ~ 

E 

N ••~ C. 

N d•~ e1 

N 

OJ q 61 

s- a o 

~ 'L-a 

d L f0 

Gro1CO r' 

L M 6! 

U I > 

N 00 G! 

O ct r 

a_ 

N 

~ 

U 

d 

~ 

v d 

i a N 

a 

O 

~ 

~ 

. ro 

r C7 M 

ro r ~c 

+~ L MP~•1 l0 

OL G~1 O^1 

T7 •r ri 'O rl 

r Ip 

rw 

L E L 

U ••• M17 U 

i 01 L L 

Y 

7 

r tC) ro •r- N 

•f6 Ol i tff 

l.L e-i C:3 li 

7 

N 

ai 

N 

o ro E L 

ro 2' 

N 

C GI 

O L 

O

T >~ E ~ 

E i O O 

c~.7 +•O~ E L X 

aro umov

a~ 

O f0 U L O 

s. c ~ T T Ol '6 'L i Lica~a~ 

i~ 'O •r' +~ ro i 4- w- Mo m 

N Q O ' 

I +~ C C 

. C tp .I..~ O ~O 

D1 C ~ ~ 

~~., ro E •0 a ~ Ui i 

v o rn•r, ~ 

+~ roctn 

O U H i y y 'O 

i~ L U T ro 

i n ai +~ ni 

0 1 ~~r L 

.1• ~ O.'Or U: 

C T L f0 N 

Q L f].+•~ ~ 

C 61 

r .Of 

m ro i~ L 

~U 

+L.t c 

T 

L ~ 

~ . ~o 

L La 

~n c 

O N 

N 

/ N 

ro 

> L d u 

r y 

~ 

y .. N.. 

N 07 C 

L r Y 

u 

C C C 

rr •r r- 

~ 

0 

O 

~ 

01 

O 

u'i 

n 

O 0 

0 

01 

n 

~ 

N 

~ 

C 

i-~ 

C 

O 

U 

v 

w 

z 

1-i 

~ 

C 

z 

6 

H 

U 

Q 

C H 

Q 

Z 

H ` 

N 

!- 

z 

K 

N 

¢ C7 ', 

f 

w 

~ } 

W 

Z 

z U - 

o - 

Z - 

W 

S 

F••- 

Z 

O 

w 

z 0 

N 

O 

L3LL 0 

N 

U 

w 

~ 

W 

tD 

N „~ .. 

C1 

u1 

J 

¢ 

H 

~ 

~ 

~, 

~ 

a 

b 

~ 

+7 O 

N 'O N 

C 01 

G/ ~ ~Q ~ 1 u C N 

d L C 0 

L U q •r 

~ L N LU

. 

a ro r m 

1' lL U C7 

N v 

u ~ ro N O C 

a 

N 

~ 

~ 

a, 

~ 

.~ 

3 

~ 

C 

ro 

U7 

C 

O 

E 

01 

V 

a~ m 

Q: 

_ ~ C 

N 61 O1 

{- N ~ 

'O ~ E ai 

c o L L 

qU +•~ ~ 

V~ N O7 •r- 

~ L D7 •tl• 

M O N ~ 

4-L 3 ~ 

o~~v •3 

N C ~ 

y

rtcu ~ 

r +~ 

>0! ~OJ • 61 OC1 

r N N 6 

E E N ~ 

~ o L o.si +~ U G N 

G/ U Cl 7 N 

N~ a N 3 

a •r ai a 

G1 O L L 07 

N i i•~ d C 

a`0i t 3Y 

L 1.~ Vl N C 

U +•~ +~ • - 

61 c Rf ~ Q i 

G -+ L i O 

L 

L i 

L L L 

L L 

~ 

M 00 

M 

N n V 

C 

Z ~ W 

S 

U 

r-i 

0 

tD 

I Q1 

r•1 

E 

7 

7 

U U 

L ~ 

+~ OJ 

C1 SE 

o u a O E 

r N 

c0 

II 

a 

H O 

V 

C 

Q~ N 

~ 

L 

L ~ 

0 7 

O 

i 

E 

~ II 

Ul O_' 

N W 

~ ~ 

oo. a 

•a o 

G! U 

L N 

~w L «- u 

a E 

C 

N O 

L L 

a ~a 

7 U v a 

C 

C1 

iI 

~ O 

Y 

C] N 

Z E QI N 

U C 

c ro 

N L 

U +~ 

N 

d II c E s a w 

7 d H N 

r a c 

E s i, a 

N U G. 

L N O L 

u G7 U U 

'II N ~ 0 

G! O G1 O 

ro o u ~ v 

L c •r X > 

O. I IE L L 

N a-~ T o 

~ 0 C .0 .0 v 

rnz rn++ 

d 

II ^ r II o• 

w ~~n II F c ~ 

U ri ~ • u +~ a d ro 

c ++ c 

C O 

~ ro O C U N 

O E d O• O 

L N N_ L L 

CJ y- L d O 

E ~C N ~ E i 

N C O O 4~ 

C 9 ~ O O N 

aEi `tl ro ~ o ro 

L N > +~ L L 

7 L d i~ 

N Gl L IIU 3 

roaim a M a. o 

R vL ~ u ~ 'O 

9-199 
survival of mice and rats acutely exposed to 03 , and injections of the thyroid 

hormones, thyroxine (T4), or triiodothyronine (T 3 ) decreased their survival. 

This response in animals with altered thyroid function was not specific to a 

concomitant altered metabolic rate, because another drug (dinitrophenol), 

which increases metabolism, had no effect on the 0 3 response. 

Hypophysectomy and adrenalectomy also protected against 0 3-induced mortality, presumably in rodents (Fairchild, 1963). Hypophysectomy would prevent 

the release of thyroid-stimulating hormone, thereby causing a hypothyroid condition 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 3 toxicity and suggests that a decrease in adrenocorticosteroids reduces 0 3 

toxicity. Rats that have been adrenal ectomi zed and treated with adrenergic 

blocking agents are more resistant to 0 3 than rats that have only been adrenalectomized, indicating that decreases in catecholamines reduce 0 3 toxicity. 

Potential tolerance to the effect of 03 on thyroid activity was also 

investigated by Fairchild et al. (1964). •A variety of exposure regimens wpre 

used for the rats, and the release of 131 I was used as an index of thyroid 

function. A 5-hr exposure to 1960, 3920, or 7840 pg/m 3(1, 2, or 4 ppm) of 0 3 

inhibited the release of 131I at several time periods post injection of 131I 

(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 03 concentration showed an effect. Rats were also exposed for 5 

hr/day for 4 days to either 980 or 1960 pg/m 3(0.5 or 1.0 ppm) of 03 . At 96, 

192, and 384 hr post- injection of 131 I, no effects were observed. Thus, 

tolerance appeared to have occurred, a finding consistent with lethality 

studies with 0 3 (Matzen, 1957a). In another study, rats were exposed to 3920 

pg/m3(2.0 p3m) of 03 5 hr/day for 2 days and challenged with a 5-hr exposure 

to 7840 pg/m (4.0 ppm) on the third day. These animals exhibited a greater 

effect than rats that received only the 7840-pg/m 3(4.0 ppm) challenge. This 

difference persisted for 48, 96, and 192 hr post-injection. Thus, although it 

appears that tolerance qccurs, it results in a condition that leads to stimulation of thyroid activity after a subsequent exposure to an 0 3 challenge. 

Clemons and Garcia (1980a,b) extended this area of research by investigating the effects of 03 on the hypothalamo-pituitary-thyroid axis of rats. 

Generally, these three endocrine organs regulate the function of each other 

9-200 
through complex feedback mechanisms. Either stimulation or inhibition of the 

hypothalamus regulates the release of thyrotropin-releasing hormone (TRH). 

The thyroid hormones (T3 and T4 ) can stimulate TRH. Thyrotropin-releasing 

hormone and circulating thyroid hormones (T. 3 and T4) regulate secretion of 

thyroid-stimulating hormone (TSH) from the anterior pituitary. Stimulation of 

the thyroid by TSH releases T3 and T4 . A 24-hr exposure to 1960 pg/m 3 (1.0 

ppm) of 0 3 caused decreases in the serum concentrations of TSH, T3 , T4 , free 

T4 , and protein-bound iodine. There was no change in the uptake of T 3 , 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 pg/m 3 

(0.2 and 2.0 ppm) for unspecified lengths of time in a fashion to result in a 

concentration x time relationship between about 2 and 100. Plasma TSH was 

decreased after a C x T exposure of about 6. These data cannot be independently 

interpreted, because the specific exposure regimens were not given. The 

authors state, without any supporting data, that the decrease in TSH levels 

persisted "beyond two weeks" when exposure was continued. Therefore, it 

appears that tolerance may not have occurred. Thyroidectomized rats exposed 

to 1960, 3920, 5880 or 7840 pg/m3 (1.0, 2.0, 3.0, or 4.0 ppm) of 0 3 for 24 hr 

did not exhibit a decrease in the levels of TSH. Exposure to (presumably) 

1960 pg/m3 (1.0 ppm) for 24 hr di d not al ter the ci rcul ati ng hal f-1 i fe of 

125 I-labeled TSH injected into the rats, and therefore, there apparentiy is no 

effect on TSH once it is released from the pituitary. 

To evaluate pituitary function further, rats exposed 24 hr to 1960 pg/m 3 

(1.0 ppm) of ozone were immediately subjected to a 45-min exposure to the cold 

(5°C) (Clemons and Garcia, 1980a,b). The anterior pituitary released an 

i ncreased 1 evel .,of TSH_,_ i ndi cati nge. that the hypothal amus , was sti 11 abl e to 

respond (via increased TRH) after 03 exposure. The increase was greater in 

the 03 group, which might indicate increased production of TRH or increased 

sensitivity to TRH. In addition, the anterior pituitaries of the 0 3 group had 

fewer cells than the air group. The cells from the 03-exposed rats had more 

TSH and prolactin per 1000 cells, irrespective of whether the cells had received 

a TRH treatment. The cell.s from the 0 3 group also released a greater amount 

of TSH, but not prolactin, into the tissue culture medium. 

9-201 
The thyroid gland itself was altered by the 03 exposure (apparently 1960 

pg/m3 , 1.0 ppm, for 24 hr). Ozone increased thyroid weight without changing 

protein content (e.g., edema) and decreased the release of T4 per milligram of 

tissue. There was no change in T 4 release per gland. Clemons and Garcia 

(1980a,b) interpreted these findings as an 0 3 -induced lowering of the hypothalamic set point for the pituitary-thyroid axis and a simultaneous reduction 

of prolactin-inhibiting-factor activity in the hypothalamus. ' 

The effect of a 24-hr exposure to 1960 pg/m 3 (1 ppm) 03 on exogenous T4 

levels was examined in rats by Clemons and Wei (1984) to increase understanding 

of their previous studies. Normal, thyroidectomized, and hypophysectomized 

rats were exposed to 0 3 and serum T4 levels were measured. Ozone reduced 

serum T4 1 evel s i n normal rats , but not the other groups of rats whi ch had 

reduced levels of T4 prior to exposure. However, when the thyroidectomized 

and hypophysectomized rats received supplemental T 4 in their drinking water 

that increased their serum T4 level, 03 caused a decrease in serum T4 . 

Generally, the higher the pre-exposure T 4 levels, the greater the 0 3 -induced 

reduction in T4 levels. Similar observations were made for thyroidectomized 

rats when serum T3 levels were measured. Exposure to 0 3 also decreased plasma 

TSH levels in normal rats and in thyroidectomized rats supplemented with T 4 in 

the drinking water. These observations led the authors to hypothesize that 

the results are not due to a reduction in the hypothalamus-pituitary-thyroid 

axis as previously suggested (Clemons and Garcia, 1980a,b), but that 0 3 causes 

decreases in serum T3 , T4 , and TSH by peripheral changes, possibly changes in 

serum binding. 

The susceptibility of the parathyroid gland to 0 3 exposure was investigated by Atwal and co-workers. In the initial study (Atwal and Wilson, 

1974), rabbits were exposed to 1470 pg/m 3 (0.75 ppm) of 0 3 for 4 to 8 hr, and 

the parathyroid gland was examined with light and electron microscopy at 6, 

18, 22, and 66 hr after exposure. The parathyroid gland exhibited increased 

activity after 03 exposure. Changes included hyperplasia of chief cells; 

hypertrophy and proliferation of the rough endoplasmic reticulum, free ribosomes, mitochondria, Golgi complex, and lipid bodies; and an increase of 

secretion granules within the vascular endothelium and capillary lumen. Such 

changes suggested an increased synthesis and release of parathormone, but 

actual hormone levels were not measured. 

9-202 
Atwal et al. (1975) also investigated possible autoimmune invoivement in 

parathyroiditis of rabbits following a 48-hr exposure to 1470 pg/m 3 (0.75 ppm) 

of 0 3 . The authors stated that there was both a"continuous" and an "intermittent" 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 pro.liferation 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 alterations were observed by electron microscopy, including atrophy of the endoplasmic reticulum of the chief cells, atrophy of mitochondria, degeneration of 

nuclei, and proliferation of the venous limb of the capillary bed. The alterations to the parathyroid gland were progressive during postexposure periods. 

Parathyroid-specific autoantibodies were detected in the serum of 0 3 -exposed 

rabbits, suggesting that the parathyroiditis might be due to inflammatory 

injury with an autoimmune causation. Microvascular changes in the parathyroid 

were further studied by Atwal and Pemsingh (1981) in dogs exposed to 1470 pg/m 3 

(0.75 ppm) of 0 3 for 48 hr. They reported focal hemorrhages, vascular endothelial proliferation, intravascular platelet aggregation, and lymphocytic infiltration. A potential autoimmunity after 0 3 exposure was also observed by Scheel 

et al. (1959), who showed the presence of circulating antibodies against lung 

tissue. 

In another study, Pemsingh and Atwal (1983) studied cells (APUD-type) 

within ciliated cysts of the parathyroid gland of dogs exposed for 48 hr to 

1470 pg/m3 (0.75 ppm) 0 3 . Examination was made at several times post-exposure 

(3, 10-13, and 28 days). Ozone caused ciliated cysts in 5 out of 12 exposed 

dogs and 1 out of 4 control_ dogs. Cysts in the 03 -exposed dogs had different 

celiular content, namely the presence of APUD-type cells on the cyst wall. 

The interpretation of these findings is not- clear: The ultrastructure o-f 

chief cells, which are the hormone-producing cells, was also examined in dogs 

exposed identicaily to those described above (Atwal and Pemsingh, 1984). 

Mallory body-like inclusions (i.e., accumulations of filaments in the perinuclear 

area) were found within the chief cells of the parathyroid of'0 3 -exposed dogs. 

Additional alterations were observed. The highest incidence of this change 

was at 10 to 13 days post-exposure. 

9-203 
Also classed as a hormone is 5-hydroxytryptamine (5-HT), and it too 

interacts with 03 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 

pg/m3 , 5.0 ppm) of 0 3 for 3 hr, this study is discussed because extrapulmonary 

effects were observed. This exposure caused an increase in the 5-HT content 

of the lung and spleen, a decrease in 5-HT in the brain, and no change in the 

levels of 5-HT in the liver or kidney. 

Adrenal cortex function after a 93-day (continuous) exposure to 0 3 was 

investigated in rats (Egl.ite, 1968). Exposure to 110 pg/m 3 (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 

03 exposures were not sufficiently described. The authors described the 

effects as statistically significant but did not specify the statistical 

methods. No data were provided. Therefore, these results need to be confirmed before accurate interpretation is possible. 

9.4.6.3Other Effects . Rats were exposed for 6 hr/day, 4 days/week, for 

about 19 weeks, and analyses were performed on urine collected for the 16 hr 

following the exposure week (Hathaway and Terrill, 1962). Ozone exposures 

were uncontrolled, ranging from 1568 to 2940 (0.8 to 1.5 ppm). A11 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 

03-exposed animals. Titratable acidity was also lower on day 98. Ozone did 

not alter the levels of creatinine, creatine, uric acid/ creatinine, amino 

acid nitrogen/creatinine excretions, or excretion of 12 amino acids. The 

lungs and kidneys were examined histologically, and no consistent differences 

were observed. The authors interpreted the results as a reflection of respiratory alkalosis, assuming no kidney toxicaty. 

Gastric secreto-motor activities of the 'rat were investigated by Roth and. 

Tansy (1972). A 2-hr exposure to 490 pg/m 3 (0.25 ppm) caused no effects. 

Thirty minutes of exposure to 2940 pg/m3 (1.5 ppm)"inhibited gastric motility, 

but activity tended to return towards normal for the remaining 90 min of 

exposure. Recovery had occurred by 20 min postexposure. However, these 

results are questionable because ozone was monitored only by ozonator flow 

rates. 

9-204 
9.5 EFFECTS OF OTHER PHOTOCHEMICAL OXIDANTS 

9.5.1 Peroxyacetyl Nitrate 

Very little information on the toxicity of peroxyacetyl nitrate (PAN) has 

appeared in the literature since the previous criteria document on photochemical oxidants (U.S. Environmental Protection Agency, 1978). The document 

reviewed the results of inhalation experiments with mice that tested PAN's 

lethal concentration (LC 50 ) (Campbell et al.; 1967); its effects on lung 

structure (Dungworth et al., 1969); and its influence on susceptibility to 

pulmonary bacterial infections (Thomas et al., 1979). The concentration of 

PAN used in these studies ranged from 22.3 mg/m 3 (4.5 ppm) to 750 mg/m 3 (150 

ppm). They are considerably higher than the 0.232 mg/m 3 (0.047 ppm) daily 

maximum concentration of PAN reported in recent years for ambient air samples 

in areas having relatively high oxidant levels (Chapter 5) and are of questionable relevance to the assessment of effects on human health. 

Campbell et al. (1967) estimated that the LC 50 for mice ranged from 500 

to 750 mg/m 3 (100 to 150 ppm) for a 2-hr exposure to PAN at 80 1 F (25 1 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 50 (less susceptibility) was seen at 70 ° F 

(125 ppm) than at 90°F (85 ppm). In a follow-up study, Campbeli 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/m 3 (2.8, 3.7, 5.5, 6.4, and 8.6 ppm) of PAN for 6 hr depressed both the 6-hr and 24-hr activity when compared to similar pre-exposure 

periods. Depression was more complete and occurred more rapidly with higher 

exposure levels. However, the authors indicated that PAN was less toxic than 

0 3 when compared to similar behavioral data reported by Murphy et al. (1964) 

(Section 9.4.1). 

Dungworth et al. (1969) reported that daily exposures of mice to 75 mg/m 3 

(15 ppm) of PAN 6 hr/day for 130 days caused a 30-percent weight loss compared 

to sham controls, 18 percent mortality, and pulmonary lesions. The most 

prevalent lesions were chronic hyperplastic bronchitis and proliferative 

peribronchiolitis. 

Thomas et al. (1979) found that mice exposed to 22.3 mg/m 3 of PAN (4.5 

Wm) for 2 hr and subsequently challenged with a Streptococcus sp. aerosol for 

1 hr showed a significant increase in mortality and a reduction in mean survival 

9-205 
rate, compared to mice exposed to air. No effect on the incidence of fatal 

pulmonary infection or survival time was observed in mice challenged with 

Streptococcussp. 1 hr before the pollutant exposure (27.2 mg/m3 of PAN for'3 

hr). Thomas e.t al. (1981a) published additional data that extend observations 

of reduced resistance of mice to streptococcal pneumonia over a range of 

exposures to PAN. A single 2- or 3-hr exposure to PAN at 14.8 to 28.4 mg/m 3 

(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 3 at 0.98 mg/m3 (0.5 ppm) and challenged with 

theStreptoccus sp. aerosol resulted in a mean excess mortality (38 percent) that 

was almost equivalent to the excess mortality for the group*exposed to 28.4 

mg/m3 of PAN. The results agreed with earlier reports that PAN is less toxic 

than 0 3 to mice exposed under ambient condi,tions. Exposure to 7.4 mg/m 3 (1.5 

ppm) of PAN 3 hr/day, 5 days/week for 2 weeks had no appreciable effect, 

although no statistics were provided. Neither exposure routine altered the 

morphology, viability, or phagocytic activity of isolated macrophages, although 

there was a decrease in ATP levels. The other noticeable effect was that 

macrophages isolated from the animals that were repeatedly exposed failed to 

attach themselves to a glass substrate. These investigators also studied 

whether a chronic infection initiated with an exposure to Mycobacterium tubercu - 

losis(RIRv) was influenced by subsequent exposure to PAN. The exposure to 

this oxidant (25 mg/m 3 for 6 days) did not alter the pattern of bacterial 

growth in the lungs of mice. 

9.5.2Hydrogen Peroxide 

Hydrogen peroxide (H2O 2 ) has been reported to occur in-trace amounts in 

urban air samples (Chapter 5), but very little is known about the effects of 

H2O2 from inhalation exposure. Most of the early work on H 2 O 2 toxicity involved exposure to very high concentrations. Oberst et al. (1954) investigated 

the inhalation toxicity of 90 percent H 2O 2-vapor in rats, dogs, and rabbits at 

concentrations ranging from 10 mg/m3 (7 ppm) daily for six months to an 8-hr 

exposure to 338 mg/m 3 (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 H2O2 gas with a concentration of 0.71 mg/m 3 (0.5 ppm) 

in the presence of respirable ammonium sulfate particles. No significant 

effects were observed in body weight, lung lobe weights, and protein or DNA 

9-206 
content of lung homogenates. The authors suggested that because H 2O2 is highly 

soluble, it is not expected to penetrate to the deep lung, which may account 

for the - absence of observed - effects. 

The majority of studies on H 2O 2 explore possible mechanisms for the 

effects of H 2O 2 . These include direct cellular effects associated with in 

vitro exposure to H 2O 2 and biochemical reactions to H 2O 2 generated in vivo. 

Hydrogen peroxide may affect lung function by the alteration of pulmonary 

surfactant. Wilkins and Fettissoff (1981) found that 10 -2 to 10-1M of H2O 2 

increased the surface tension of saline extracts of dog lung homogenates. The 

authors estimated that a dog breathing 1:4 mg/m 3 (1 ppm) of H2 O 2 for 30 hr 

would build up a pollutant concentration of 10 -2M in the surfactant,' assuming 

that all the H 20 2 was retained by the lungs. HoweVer, as s -tated above, this 

estimate of tissue dose is not realistic, because most of the H2O 2 would be 

absorbed in the upper airway. 

Another mechanism by which H2O 2 may affect ventilation is by changing the 

tone of airway smooth muscle. Stewart et al. (1981) reported that 10 -4M of 

H2O 2 caused significant constriction of strips of subpleural canine lung 

parenchyma and of bovine trachealis muscle. In the distal airway preparation 

(canine), this contraction was reversed by catalase. Pretreatment of both 

proximal (bovine) and distal muscle strips with meclofenamate or iodomethacin 

markedly reduced the response to H2O2 . This suggested that the increase in 

airway smooth muscle tone produced by H 2O2 involves prostaglandin-like substances. 

The potential genotoxic effects from in vitro H 2O 2 exposure have been 

evaluated in isolated cell systems. Bradley et al. (1979) reported that H 2O 2 

produced both toxicity and single-strand DNA breaks but was not mutagenic at 

concentrations up to 530 pM. They observed a significant increase in the 

frequency of reciprocal sister chromatid exchanges in V-79 Chinese hamster 

cells at a concentration of-353 pM of H 2O 2 . 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 unabl e to detect any DNA-protei n or DNA-DNA crossl i nks wi th 353 pM of 

H 2O 2 for 3 hr at 37°C. Increases in the frequency of sister chromatid exchanges 

in Chinese hamster cells have also been found by MacRae and Stich (1979), 

Speit and Vogel (1982), and Speit et al. (1982). Wilmer and Natarajan (1981) 

9-207 
reported only a slight enhancement in the frequency of sister chromatid exchanges in Chinese hamster ovary cells followinq treatment with up to 10 -3M of 

H2O2 . In comparison, cells were killed with a concentration of 10- 1M of H2O 2 . 

Similarly, H2O 2 (10-lmg/ml) was negative in the Ames mutagenicity assay 

(Ishidate and Yoshikawa, 1980), and several other investigators have confirmed 

the lack of mutagenicity for H 2O2 (Stich et al., 1978; Kawachi et al., 1980). 

Johnson et al. (1981) reported that the intrapulmonary instillation of 

glucose oxidase, a generator of H2O 2 , increases lung permeability in rats. A 

greater increase in lung permeability was achieved by the addition of a combination of glucose oxidase and lactoperoxidase than by the glucose oxidaseH2O2-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 

H2O2 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 

hal i de- dependent pathway described for cell injury produced by H 2 O2 and lactoperoxidase/myeloperoxidase (MPO) (Klebanoff and Clark, 1975). Any source of 

H2O2 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 (Hailiwell, 1982). 

Carp and Janoff (1980) have shown that a H 2O2 generating system. with MPO 

and Cl will suppress the elastase inhibitory capacity of the protease inhibitor (BMPL) present in bronchial mucus. This antiprotease is capable of inhibiting 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 in vitro experiments, Suttorp and Simon (1982) demonstrated that 

H2O2 generated by glucose oxidase was cytotoxic to cultured lung epithelial 

cells (L2 cells) in a concentration-dependent fashion. Cytotoxicity was 

measured by determining 51Cr release from target cells. Cytotoxicity was 

prevented by the addition of catalase. It was stressed that there is no 

established identity between the L 2 cell line and the in situ type 2 pneumocytes from which they were derived. 

• ~ : 
9.5.3 Formic Acid 

Toxicological interest in formic acid has focused primarily on its role 

as the metabolic end product of inethanol and on any similarity the effects 

from inhalation exposure to formic acid vapor may have with methanol toxicity. 

Unfortunately, the concentrations (20 ppm) used in the two studies discussed 

below are over three orders of magnitude greater than the trace concentrations 

(<0.015 ppm) reported in the highest oxidant areas of Southern California (see 

Chapter 5). 

Zitting and Savolainen (1980) exposed rats to 20 ppm (0.8 pmol/L) formic 

acid vapor, 6 hr/day for 3 and 8 days. Tissue samples were analyzed for 

neurochemical effects as well as effects on drug-metabolizing enzymes in the 

liver and kidneys. Enzyme profiles were variable, depending on the sampling 

period during exposure. However, a general pattern developed. Cerebral 

glutathione concentration and acid proteinase activity increased after 3 and 

8 days of exposure, respectively, indicating possible lipid peroxidation 

associated with cerebral hypoxia. Decreased kidney ethoxycoumarin deethylase 

activity, cytochrome P-450 content, and glutathione concentrations were also 

consistent with changes due to lipid peroxidation. The liver, which is less 

sensitive to tissue hypoxia, showed only small increases in deethylase activity 

although associated with a reduction in glutathione. Since prolonged low-level 

cerebral hypoxia can potentially lead to demyelination and subsequent nerve 

degeneration, the authors repeated the study to look specifically at effects 

on glial cells (Savolainen and Zitting, 1980). Rats were exposed to 20 ppm 

formic acid vapor, 6 hr/day, 5 days/week for 2 or 3 weeks: Again, enzyme 

profiles were indicative of inetabolic responses to tissue hypoxia, providing 

evidence for potential central nervous system toxicity at high formic acid 

vapor concentrations. 

9.5.4 Complex­Pollutant 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 difficuit - to analyze because 

9-209 
of their chemical complexity. Variable concentrations of total oxidants, 

carbon monoxide, hydrocarbons, nitrogen oxides, sulfur oxides, and other 

unidentified complex pollutants have been reported. For this reason, the 

studies presented in this section differ from the more simplified combinations 

of 03 and one or two other nonreactive pollutants discussed under previous 

sections of the chapter. The effects described in animals exposed to UVirradiated exhaust mixtures are not necessarily uniquely characteristic of 0 3 , 

but most of them could have been produced by 0 3 . In most cases, however, the 

biological effects presented would be difficult to associate with any one 

pollutant. 

Research on ambient air and UV-irradiated or nonirradiated exhaust mixtures is summarized in Table 9-27. Long-term exposure of various species of 

animals to ambient California atmospheres have produced changes in the pulmonary 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 containing oxidant levels of 0.2,to 1.0 ppm produced histopathologic changes 

(Nakajima et al., 1972) and increased susceptibility to infection (Hueter et 

al., 1966) in mice. Both UV-irradiated and nonirradiated mixtures produced 

decreased spontaneous running activity (Hueter et al., 1966; Boche and Quilligan, 

1960) and decreased infant survival rate and fertility (Kotin and Thomas, 

1957; Hueter et al., 1966; Lewis, et al., 1967) in a number of experimental 

animals. Pulmonary changes were demonstrated in guinea pigs after short-term 

exposure to irradiated automobile exhaust (Murphy et al., 1963; Murphy, 1964) 

and in dogs after long-term exposure to both irradiated and nonirradiated 

automobile exhaust (Lewis et al., 1974; Orthoefer et al., 1976). Irradiation 

of the air-exhaust mixtures led to the formation of photochemical reaction 

products that were biologically more active than nonirradiated mixtures. The 

concentration of total oxidant as expressed by 0 3 ranged from 588 to 1568 

pg/m3 (0.30 to 0.80 ppm) in the irradiated exhaust mixtures, compared to only 

a trace or no oxidant detected in the nonirradiated mixtures. 

The description of effects following exposure of dogs for 68 months to 

automobile exhaust, simulated smog, oxides of nitrogen, oxides of sulfur, and 

their combination has been expanded in a monograph by Stara et al. (1980). 

The dogs were examined after 18 months (Vaughan et al., 1969), 36 months 

9-210 
a 

W 

C lo 4-

~ 

ro 

C 

O 

W 

10 

i ~ 

W E C a 

d 6 

U v 

C 

O 

U 

V 

C r 

yl ro O 

~ C O (n O .1•) 

O •r O 

Q i~ i 

w z ° 

v 

N 

w 

~ N 

X 

tr 

Z 

~ 

Z 

4 

1-- 

~ 

W 

..d 

O 

d 

x 

W 

~ 

C? 

0 

uN 

f-- 

W W W 

W 

N 

Ci 

W 

J 

m 

Q 

}•~ 

i 

ro ! 

iJ , 

C ' 

QI 

a 

~ 

Q 

a 

v_ 

u 

d 

o. 

N 

u 

N 

iJ 

V 

(U W 

W 

C7 

' O 

m 

L 

N 

N 

a 

O 

d 

c 

~ 

W 

d 

~ 

LO 

C ; 

M 

~ a, 

~ ~ 

i r1 rl E 

GJ 

a 

1~ 01 7 

L 

tn 

(0 

E ri rt U E 

(f1 

L ^ +J 

0 10 

O 

L 

U CO H 

d I 

c a c c 

ro +J 

01 

c 

1O 

m ro 

ai 

C Op Y Y 

c 

C 

c 

4.+ 

roO~ 3 r-1 • E 

w 

•E 

w 

3 N O '. Y 

of ol 

a n 

s 

v ro +J ro ai +J v•.- M ^ Uf c a 

7 Y E 

Ul •`.G 

+1 a 

Vl 

7 

c tn 

7 

o m ro ~ ro o ro ro o •~ o f i L cm L f i i f C-1 E 

) U 1 

}o) o 

O v _o 

4J o w 1 af 1 O 0 1 1 c U r E ai 

+J 

•- c•r al U 7 E Cl a T o O t7 a) ro 

Cl O -- O a 

? L i•YS 

N(0 Gl v -(y .^ ttl 

ro O C /1 E 

.1..7 a••) U C{- 

•O Rf H 

i 

. 

~

o• u, a a c 

i 10 iJ In •r 

a1 n. T•^ w o 

ro i0 +~ ~ u G 

^ ai 4- 

~ n S. O 

c 

tn o E Vf ro L X"O •r G/ •e- N +J ~ 10 O . TJ ~ • . 

N E O O G) Q) 

07 C+1 i uf 

Ci O Cl r- u E'O 

tn tn t0 •r 

U d E XG) a 

i E 0.0 +' 10 

+•~ .- 

0. ~ O rn ^avuro 

7 C•IJ Y N C 

ouro+~ Eal 

L•r 41 i— r• 0) Ql 

un.e.zm•r 

C O. i i 

a> 

U•r 

~ n O 10 I CO •r 

= t~ E 

G E i O Rf C 

ro.0 E u> E C 

C C, W•1•~ ro 

rO V• o u~ > 

C> 

O i 

+.> T rl ro 

^ U i • tn 

61 ij C i.~ `~./ r• 

C O•.- ~ 'O L 

•C 

C 

C! M G 

U n I E O•W 

U O 

IA 

iN-1 ~ C N ~ ro ~ O1 •^ N ~~ O S~.. CM17 ° W pa. 4Ef 1C0 o c 

W ~ 

y Ol •r •1•/ /~ I O 'S7 W i 

37 E ~ d+~ 

W^,+~ N 

E tA ~ } G 

O d-~ i • O 

N 

O 

^ N i 

'O ul T O 

N O 

+J a 

W L 7 i t0 U rt7 L•r U T+J • O vN 0/ C r••1 +~ Cl U 3 tll ~II c1 t.t GJ •r 

C c c d~ 

al .G C'O u ~ 

T 1 /1 a•r O ro U 

Q! 

c 

tll .G O r E 

i U•I^ •o >•r 

N N 

W C 

U O a- t-+ •r 01 V; 

•- E o o u,•~ 

• C 

s- ro a O 

ro ~ v i• on v 

Ir1 

...+ 

rt7 .rj C•r 

3 d ro ro+) c 

W 

v i 

r- Ol N•r 

O ~ Cl r- ,q 

i G U 1/1 C IE a 

C y vi^+•O C C 

O U G7 Ql •r •r 

E O 31 U u C 

C - O 

C. 

O i i r ttf 

~I-~ ^ •N 

W }SG U C V) 

O 

4.+ 

G 

L G r X R7 

u G N ~V • O i 

r'q E C 

"O N•r O C i.l 

~ 61 ro E ro a c 

n s C ~ G7 i ro 

!M 

'O 

O 01 S N rt7 

T UN 3.` 

i C c W 

97 41 

d4 d•i {t rtf 

i • r O u W 

ro tn •U •C •r r O 

Ol 61 C t0 •1-~ E 

i u 

•O •^ •E 1/1 'T7 •r 

O) ~ ro•r 

OJ 

N 

O Ql O O r N 

aJ .= U (0 L 

r• f.. 

•N 

r- CA•r W 10 E U sn tn +•> rv VI •r OJ 4J O tlJ 

U C 'Q i+J ~ 

r 

~ +j C•r y R! C V 

L 

i r tn y p••r ro 

•X 

U i-~ 

O.0 C 01 X y 

Z U 10 rtl N ~ tA 

y L% O ~ L•r.~•E 

~ v a.a E E u In v 

G! 

p 

N•C tD r y 

w•O ..p r•I G. 

O•eZ rN •• N ^ N M VI 

7 ~ 7 . Uf 3 7 - •• 7 

E 7 t~0 7 •N 7 ^ 7 Y 7 

•• C Gl C C C i C G! C 

dv++ T 4J E++ a

roi+a 3+J W 7 C tn c C T c c 

r aJ O 

J uf u 

• O 

N U 

M O 

rl U 

O 

rl u 

Q1 O 

r•1 U 

~o 0 0 >o c.~ o o¢ >o c> >o c) o x 

° U z z o U 2 z z C o z 2 o U 

o r` ~}• 

lt'f h ch 

OL O r-1 n 

~ G1 

N O ~ ~ --1 O 

OMOO 

rl O O sY 

M 1~ 

1 1 1 1 t t 1 

G 00 T O 

M X 

N s!• I- 01 N--r N 1 1 E 

M cf rl 11'f r•1 r-i O t0 M 

OriO O O1~ d• OOO 001~ N O 

~ 

0 010 0 O rI N 0 0 0 0 0 N O M N O 

rl O 

9-211 
a u 

c 

d L 

a 

w 

d C 

~ 

C, 

10 r 

L c t0./ 

7O O 

04.)o 

~ in 

v 

U 

N 

~ 

u 

V.d. 

w 

a 

V 

} 

d 

.] 

O 

tn 

d U 

a 

a. 

N 

a N 

c 

1I1 

Y 

r 

r 

O 

d 

ro 

c 0 

4j 

ro 

~ E 

C n 

a a. 

U v 

C 

O 

ti 

n 

~ 

d 

7 

G 

r 

~..1 

C 

O 

U 

v 

~ 

W 

a 

~ 

r 

x 

.-. 

z 

~ 

~ J J 

O 

0. 

W 

J 93- 

V 

LL. 

0 

~ W 

W 

W 

f` 

N+ 

LLJ 

Q1 

m 

9 

44 

~ 

ro 

m 

a 

y:. 

6 

E 

a.O.i 

4 

aa 

~ ~ 

~ N r0-1 n 

t0 O

^

i e- ~i t00 

U7 

d 

lD t% 

01 t0 

-1 N 

I- •• 

Q1 <t 

r-i 1- 

C 

L 

rl Q1 

ri 

.• Q1 

r-1 O 

' 01 

•• r-I 

ro m - Cp i0 

++ 

r 1 ++ 

a 

io 

c ro 

d 41 t0 

a 

ro ~.+ •,- 

io 

a w 

O L tn d R1 

X ~ ~ • 1n0 U ra- t rOf 42 

r°- ~ a3i m o ~ s m a3i .7 SJ Z mC.7 O C >J 

€ 

ro ~ rn 

'r" •i ~ •p, 

a 7 

C E 

L 

d f0 O • - 

E L 

a +7 a 

ul •. M C a Yl 

7 4- E•~ - 7 Ol t7f Of i 

N 4 proroo E i.L Of 0 2 0 G 0 Cf . 0 CI 

II1 

C I C 1 d•~ Uf N 

C •'• f0 >i }f d•~ O L Vl C 7 C 

ro r Olf" 

•• N~ ro d ro6 •r 7 0 O ~ N C X u`" o..>,i i~ ro o c0 ro• ~ w 

O >a' i

w ~ ro u 

o i•t i'Cy d O r•'- Of ro r a o •ci •I• I E vt i a 

N aH L 

o ua+ 

cz++> 

u•r • 

c++ a 

c•~ w 

r o 

c o 

r w 

ur e 

u 

N••- O • ~ U S. „O E d 

c a +~ 

• d L i•t 

c r w ro o 

O ro 0 

Eo Ot 

a a C 

C X••- 

~ - O+t 

M 

n+t d OI a Vf 'II • >>~ tT O u1 C rt1 i t0 7 V! 

c ro 3a a 

a d•~ •W 'O 

+t •~ s c q. 

u> ro•'- X L d n i U) d>> 41 L ttl 

r= tp C 

~ a c d 

n r O+t a 

d i C C a 

w o o c 

C N E 7 w 

C 

a~o o 

a••- •r 

+1 

u 

d 

U.0 c 

c d 

ar u 

Qt.0 

x C. d Uf Uf 

O f0 U'.^ V1 r• r L. p Of.0 +~ 4- L. >> C O d.1 ya i • ~ V eq C 

n'LY i•t RJ O O 

+. 7 

>T C n y N 

C U R! 

ro C I. 

4- 

a 

a r O 

w p U 

w •O u t7 

d a a C L L n 

w R1 t0 O'O N 

i t0 ~ Y 

ro w C O d 

~ p.N U L C L 

w L 

r^ n L C+~ w w O(O w w O 0. Q. C d (9 i ro L 

C C•r d a 

o••- c ~ 

~ d~ 

v u 

ro 

r 

'O Of 

..•. 

• • a a 

~ y~ a O•a o 

o d u s u 

E tn ro 

r "C a ~ a+ 3 

~ o c 

~Of c O 

~ 

u 

+J v, r 

c p 

u m o 0 

c s c G 

'0 44 C a•r» 

i- a U•~ •W C 

O C t7~ C d 

C. ~ 6 C O w O t0 U 

r 

N 

f0 

f0 • ~ - .0 U i•1 a.t O i 

w..- 

R7 U d i r d 

o r a ro rn 

R1 C. 

-o >,.c n+~ ' O U➢ L .c • ~ - a 

> 

o 

r~ ro•~ u- i 3 u. 

A Uf IA 

d•t to 

ro o 0 10 .4t +j i i u r o 7 

a da u ttl V1 •.- "O L ro r+J d•rbt +•t t tO ••- O! 

n d 

'O •c c'O - L N L L 

ro+J d-1 +J 

C C c Ln nc ~ a•~.caw occ ro .-awtn otoop 

t N d d ZI a oro 

u L u a a 

•atu 

Vl d a 

u ut++roa 

o a x 

~ vEE 

J O ^ L•+- L C a•~ L.0 • L.G O. O S. i•.- S •x q0 t0 O Z ~ i~ 'O i~ ~+ w w U it S a-t +~ Z Z n U E CL. O r1 M 

O Y 

uf 

L Y C L 

_ E a fT0 

en 

G a>> N •^ • ro 

L N Y ~

1

a n L E 1~A\ a d C 

1D a~i 

t0 

t 

ttY 

adi b= 

3'O M 

tOD ro.c 

1 V 

~' o•~ 

v Or•i I~ r~-1 

M u.C@ 

O r-i . ct tn N N i ro 

~ 

d 

c 

~ d 

x r .. .. 

E G N N 

v O O O O 

N S 

MO O tl O X X 

2 

C i U 

V• 

N NO NnZZtA OZZvt~ O ZS<..> O C.7 vZZN N O ~ + _ = S 

t..t 

2 

t N 

Lr)

t) r

0

10 N100 17 O tff tG •~ 0 0 ~ 0 

O r1UYr1 OOrlf.07r-OI OO O t00 O Mr-INC10 

rl --!' LO tD 

00000 

d' u7 O rl ~ rIN 

N N 

NO r-1rl c}' O . 

OMO00 OOOtON OOtf) ~ O~O-INO000 

9-212 
~ n ao rn ri ~ o rn o rt 

ro 

4-) ai ie 

N •n aJ 

N 

N G1 

Q7 

3 'roa 

>T O O 

0 

G 

0 

G 

0 

C 

~ 

V

G/ L O 

O Nro E L.% CN 

N 

'+-• 

CJ O r O 

U U •ro Li•r N 

~ v 

rr 

G1 

l7 

D7 

C 

U •r .0 

• C; U 

O -- ro 

Cl U r• X ~~ Nf + ) 

N O} 

tII 

(3) 

L 

\ 

O 

N 

n ti 

>•O r- C 

O G1 

i C J O v N T L•Rf 

U r C1 L'O L•r ro•r' 

c N rn _ 

a N r N N m L 

+J X 

= o 

U 

ro 

X O 

r• 

ro 

O1 i 41 C U n • C o f0 

L {• M. {• C c E CO C! 

1•-1 r r E 

rtJ. 

3 >> •• ~ N c0 +j ~r M C U N 

Nv y v N E 

~ C7 O 7 

N E 4- OZ •O O TJ 01 ' v Cf ••• ~ . 

tll . N u QI 

31 

N L. 

u 

O 

> 

i 10 O•r- 

>~ ~ d •r QL N f0 L ro C C71r• N N U 

o U rou>u E L d~ o~ E•r y• aJ n U ci L 

r-1 J A c ~ O O. T ~ c t O d' N> 

G 

ro n Z -N L; ro 

v 

u 

C 

N 

i 

a~ 

wN 

C 

•D 

a 

0 

C 

41 

C 

O 

u 

N 

W 

C 

~ 

X 

~ 

Z 

f- 

~ 

J 

J 

0 

X 

~ d_ ~ 

V 

O 

~ 

F- U 

W 

W 

n N 

rn 

W 

m 

Q 

t-- 

b C y 1p rL O 3 c u 

N O O 

O .r+l 

X ~ 0 L 

W L. n :3 'O 

.c 

~ 

C 

ro 

4J 

7 

O 

6. 

ro 

C 

O 

4J 

ro 

L 

C E 

d , n 

U n 

C v 

O u 

u 

~ 

..Ni 

~ 

U 

W 

uN 

'O 

N 

> L 

N N 

.G 

O 

N 

N 

.~ 

G1 0 

N 

C" 

~ I 

~4 

M 

~ a 01 

ri 

M 

o ~ 

r

cn

• i o0 

~ ^ J 

o ~o 

rn 

-a 

c 

+1 rl ra m 

d 

a.~ C 

A T N O 

L L 

na .0 N ^ 

L. > 

E f 6L N 

Of ~ 

i 

OG1 4) 61 

C N 

E 

N 

7 

•7 ~0 O 

C 

oe•r c 

C •r r p1 C 

C 

. ~ ro u N•r C C1 

 d G1 rtl N O u i ro •• a•> O ~- •u 

+~ ro 

i t 

~ tm 7 a1 

LC L U d f0 C N A I(0 aj Rl Gl > u 

• C L L 

N Gl GJ d V i i - r N N C7 ~ 

' •tA. 10 'C Q G/ 61 

7 4- 0I C •• N 

a> al A a> u v x ~ s A d 

LU d O 4i~ - N L E O •7 r• 'rop d 

.o.. ~ ~-~ ~ t~l1 ~ +~ 4~- N 

f0 rt7 N•r r x 

y 7•r 

M r 

L 1 C! 

~ v- - E s N N ro .c •a 3,-I i 

L 7 w SJ i - O 

d L ro m 

i••X 

L 

- i-~ G7 A 61 O 

.- c o o c L° U `n ro ~ '9~•• 

•> 

X 

o> 4•> - C) •r 10 L L ~ C) 10 N 

7 i0 7 ~0 CI E L ~ C C U i.~[') i- C.'O 'O E r• 

• 7 7 .N 

O 

C •r 

ro t~ n 

N 

,a

a, +' >cio ' o ° ° ° c ~ v o b N•o c" > v x 

ro d.- ~ d 3_ ao o c.i +~ ro 

ro 

m N •o 

++ ro a2 

 G/ ~ uf 7 O N Z L ro""'? r ld ro S-. r•+ 

G) ~ N 

C Cl ~A ~ 7 f.. Op ri 

C L- L+~ ~ L C ~ Q O d O W 

`-•~ U w ~ 0 d-~ u ro - Q ~ >'0 Ja O 

Y Y 

; ro 3 b N \ O N \ V 

\s- a~ A i 

tc - 3 •C L 3 .0  L I 

~ 

M 

c!• 1- N 

M 

N(-I N 

N 

C1 Gl 'O 

•O C 

N L 

41 C 

- X_ N 

7 O 

M NOO r- M N N 

O Z Z f O r• ~ t~i f N i•~ O Z 

 CD 

 N . 

u 

O u tD 

tL Q 

I 

OD 11'1Nd'N 

N O i~ N O 

O u7 at N O 

1 1 1 11 

I M ri e--I r/ O O 1l O 1- M O O 

I 00000 OONrI O rIN 

9-2213 
r, 

~ 

ro 

tm 

ro +J 

E 

o ro ~ ~ ~ L 

C 

• c 

U 

c 

c 

m v- c o u cO 

m ~ r-1 

+~ 

01 

m 

C Y N X 

~ O 

r- L 

o a 

~o u 

cz~ 

y 

d 

rnn 

L •^ 

~+ •U VOi VOf •C C1 7 7 • c •U 

7 c N T~ E ro Il v 

41 N•~ 

C Z 7 O U y 

~ N 

W (a Xp II 

C ~ c t7) L a co 

~ °_ 

 cn 

Z N C C 

~ +.) v d 11 c J 

~ 

U 

~ 

L 

3 

+~ 

O 4- tn 

i 

Z E 

d 61 7 c r ll p o ro ai J d m CL > )C C.7 

Z 07 C U 7 C• O 

c.) 

Vi 

.c 

O 

0. 

' c V1 

O 

O C 

E O 

~ C Yl U C p 

i c L 

u ~ 

d

tn i v~i 3 

V ro•- 

..s O ~ 

aroi c XCro7 ~ DO 

N C O 11 = L+• 

w 

(1)U 

cro om 

N 

08u v M U _ L>1 

C 

N L U ^ vl C•^ p c 

~ Vpl ~+~ L YG! ~ 

~ o. e+~o sp. 'c 3•a 

cc W~~ 

rt o o c' 

Q 

~ ~ 

N Ol C 

~ U 

+1 Il 'O O 

d Kt. c 

U O 3 O 

C 4- • O •••r +~ 

L 

m 

L C n U 

c f0 > m o

> 

11 4- ro T7 N +J 

II O C 7 M tq 

CD 

 Cf 

Uf 

m 

O N 'O 

r O v v 

O~J 

^ N 

~ O x O •'~• ~ ~ 4.i 

U N X 

Nu/O L 

ro uniC 4- 

C V1 61 

O 

~ ~N M C O O L 

L ro 

ro >f z i~ N 

~ E O N ~ ro•- a 

c O 

ai a ~ ~ ~ •L II c9 

v N 0 ~ O 

C¢Z t%fi U O rl ri M .O V 

9-214 
(Lewis et. al. , 1974), 48 to 61 months (Bloch et al. , 1972, 1973; Lewis et 

al., 1974), and 68 monthp (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+SOx ). See Table 9-27 for exposure 

concentrations. 

No specific cardiovascular effects were reported during the course of 

exposures (Bloch et al., 1971, 1972, 1973) or 3 years after exposure (Giilespie, 

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 i n, ambi ent ai r. However, prolyl 

hydroxylase levels were reported to have increased in the lungs of dogs exposed 

to I and I+SO x , when compared to control air and the nonirradiated exhaust 

alone or in combination with SOx . 

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 x , when coinpared to their respective controls receiving clean air and SO xalone. Two-years 'after exposure ceased, pulmonary 

function was remeasured by Gillespie (1980). These measurements were made in 

a different laboratory than the one used during exposure, but consistency 

among measurements of the control. group and, another set of dogs of similar age 

at the new 1 aboratory i ndi cated that thi s di fference di d not have a major 

impact on the findings. Arterial partial pressure of CO 2increased in the 

group exposed to I+SO xand total deadspace increased in the the I and I+SOx 

groups. The diffusing capacity for carborr monoxide (DLco) was similar in ali 

exposure groups, but when normalized for total lung capacity (TLC), the DLco/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 animais were measured 2 years later, 

increases were reported in the I+SO xgroup. llnfortunately, the sample size of 

dogs exposed to I was too small (n = 5) to permit meaningful comparisons. In 

9-215 
general, pulmonary function changes were found to be similar in all groups 

exposed to automobile exhaust alone or supplemented with SO x . Exposure to 

these mixtures with or without UV-irradiation produced lung alterations normally associated with injury to the airway and parenchyma. 

The functional abnormalities mentioned above showed relatively good 

correlation with structural changes reported by Hyde et al. (1978). After 32 

to 36 months of recovery in clean air, morphologic examination of the lungs by 

light microscopy, scanning electron microscopy, and transmission electron 

microscopy revealed a number of exposure-related effects. The displaced 

volume of the fixed right lung was larger in the I-exposed group. Both the I 

and I+SOx groups showed random enlargement of alveolar airspaces centered in 

respiratory bronchioles and alveolar ducts. Small hyperplastic lesions were 

observed at the junction of the terminal bronchiole and the first-order respiratory bronchiole. Foci of ciliary loss associated with squamous metaplasia 

were also observed in the intrapulmonary bronchi of the I+SOx group. However, 

because these was no significant difference in the magnitude of these lesions, 

oxidant gases and SOx 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 o.f diesei 

exhaust particle extracts collected under smog-chamber conditions was evaluated 

by theSalmonella typhimurium plate=incorporation test (Ames et al., 1975). 

The authors demonstrated that the irradiation of propylene, S0 2 , N0, and NO 2 

produced 03 and a mutagenic moiety. In baseline studies on diesel exhaust, in 

which 03 was neither added nor produced, the mutagenicity of each sample was 

similar under dark or UV-light conditions. When 0 3 was introduced into the 

smog chamber, the mutagenicity of the organic compounds was reduced. 

The behavioral effect of a nonirradiated reaction mixture was examined by 

Stinson and Loosli (1979). Voluntary wheel-running activity was recorded 

during a continuous 2-week exposure to synthetic smog containing 588 pg/m 3 

(0.3 ppm) of 03 , 1 ppm of NO2 , and 2 ppm of S0 2 , or to each component separately. 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 2 and S02 alone had only moderate effects, the authors 

concluded that 03 had the major influence on depression of activity. 

9-216 
More recently, Raub et al. (1983b) reported pulmonary function changes in 

hamsters exposed 23 hr/day for 4 weeks to a nonirradiated reaction mixture of 

trans-2-butene, 0 3 , and S0 2 . Decreases in the nitrogen washout slope and 

increases in the diffusing capacity indicated a significant compensatory 

change in distribution of ventiiation in the lungs of exposed animals. Animals 

compromised by the presence of elastase-induced emphysema were unable to 

respond to this pulmonary insult in the same manner as animals without impaired 

lung function. 

9.6 SUMMARY 

9.6.1 Introduction 

The biological effects of 0 3 have been studied extensively in animals and 

a wide array of toxic effects have been ascribed to 0 3 inhalation. Although 

much has been accomplished to improve the existing data base, refine the concentration-response relationships and interpret better the mechanisms of 0 3 

effects, many of the present data were not accumulated with the idea that 

quantitative comparisons to man 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 3 , the 

qualitative as well as quantitative similarities between the toxicity of 0 3 to 

animals and man must be considered more carefully in the future. Significant 

advances have -been made in understanding the : toxicity of 0 3 through appropriate 

animal models. This summary highlights the significant results of selected 

studies that will provide useful data for better predicting and assessing, in 

a scientifically sound manner, the possible human responses to 0 3 . 

Summary figures and tables are presented in the following sections. The 

practical purpose of this presentation of the data is to help the reader focus 

on what types of effects or responses have been reported, what concentrations 

have been tested (1.0 ppm and lower), and as a convenient list of references 

with each of the bioiogical parameters measured. Studies were-selected for 

inclusion in these figures and tables on the basis of specific criteria presented below: 

1. Studies have been cited when the reported effects are clearly due to 0 3 

exposure. Effects due to mixtures of 0 3 with other pollutants have been 

summarized in a separate figure and table. Studies involving exercise, 

9-217 
diet deficiencies, or other possible modifiers of response to 0 3 have not 

been included. 

2. Cited studies report the effects of 03 exposure over a broad range of 

animal species and strains and for varying lengths of time. Specific 

details on animal species, exposure duration, and observed biological 

effects can be obtained from the tables within the body of this chapter. 

3. Each closed symbol on the figures represents one or more studies conducted 

at that particular concentration that caused effects. Specific references 

can be found in the accompanying tables. 

4. Each open circle represents one or more studies that used the given 

concentration, but reported no significant effects. No-effect levels are 

also indicated by brackets in the accompanying tables. 

5. Only pulmonary function effects were divided by short-term (<14 days) and 

long-term exposures to follow the discussion in the text. 

In order to keep this section brief,and concise, it was necessary to be somewhat 

selective in determining what and how this information would be presented. A 

number of important factors, such as the specific length of exposure, were not 

included. Also, the parameter selected to illustrate a specific response was 

usually broad and very general. For example, the category "decreases in 

macrophage function" includes such diverse endpoints as measurements of lysosomal and phagocytic activity, macrophage mobility, or chemotactic response. 

These responses may or may not be related to one another. Thus, care must be 

taken in how these data are used and interpreted. The only appropriate use is 

to gain an overview of the broad array of the effects of ozone and the concentrations which did and did not cause these effects. 

9.6.2 Regional Dosimetry in the Respiratory Tract 

The amount of 03 acting at a given site in the lung is related to the 

airway luminal concentration at that level. As a result, 0 3 does not immediately 

interact with cellular components of the respiratory tract. Instead, it first 

comes into contact with the mucous or surfactant layer lining the airway. It 

should be noted that 0 3 is quite reactive chemically. Reactions with components 

• , 
of this layer cause an increase in total absorption of 0 3 in the upper airways 

and in a reduction of the amount of 03 reaching sensitive tissues. The site 

at which uptake and subsequent interaction occur and the local dose (quantity 

of 0 3 absorbed per unit area per time), along with cellular sensitivity, will 

determine the type and extent of the injury. Also, the capacity for responding 

to a specific dose may vary between animals and humans because of dissimilarities 

in detoxification systems, pharmacokinetics, metabolic rates, genetic makeup, 

or other factors. Thus, along with the above, a knowledge of the complex 

process of gas transport and absorption is crucial to understanding the effects 

of 03 and other oxidants in humans. 

The animal studies that have been conducted on ozone absorption are 

beginning to indicate the quantity and site of 0 3 uptake in the respiratory 

tract. Experiments on the nasopharyngeal removal of 0 3 in animals suggest 

that the fraction of 0 3 uptake depends inversely on flow rate, that uptake is 

greater for nose than for mouth breathing, and that tracheal and chamber 

concentrations are positively correlated. Only one experiment measured 0 3 

uptake in the lower respiratory tract, finding 80 to 87 percent uptake by the 

lower respiratory tract of dogs (Yokoyama and Frank, 1972). At present, 

however, there are no reported results for human nasopharyngeal or lower 

respiratory tract absorption. Caution must be used in estimating nasopharyngeal 

uptake for normal respiration based upon experiments employing unidirectional 

fl ows: 

To further an understanding of 0 3 absorption, mathematical models have 

been developed to simulate the processes involved and to predict 03 uptake by 

various regions and sites within the respiratory tract. The model of Aharonson 

et al. (1974) has been used to analy,ze nasopharyngeal uptake data. Applied to 

03 data, the model indicates that the average mass transfer coefficient in the 

nasopharyngeal region increases with increasing air flow, but the actual 

percent uptake decreases. 

Three models have been developed to simulate lower respiratory uptake 

(McJilton et al., 1972; Miller et al., 1978b, 1985). These models are very 

similar in their treatment of 0 3 in.the airways (taking into account convection, 

diffusion, wall losses, arid ventilatory patterns) and in their use of morphological data to define the dimensions of the airways and liquid lining. The 

model.s differ in their treatment of the mechanism of absorption. Both of the 

models of Miller and co-workers take into account chemical reactions of 0 3 

with constituents of the liquid lining, whereas the model of McJilton et al. 

9-219 
does not. The models of Miller et al. differ in their treatment of chemical 

reactions, as well as in the fact that the newer model includes chemical reactions of 03 in additional compartments, such as tissue and blood. 

Tissue dose is predicted by the models of Miller et al. to be relatively 

iow in the trachea, to increase to a maximum between the junction of the 

conducting airways and the gas-exchange region, and then to decrease distally. 

This is not only true for animal simulations (guinea pig and rabbit) but it 

is also characteristic of the human simulations (Miller et al., 1978b; 1985). 

A comparison of the results of M-iller and co-workers with morphological 

data (that shows the centriacinar region to be most affected by 0 3 ) indic«tes 

qualitative agreement between predicted tissue doses and observed effects in 

the pulmonary region. However, comparisons in the tracheobronchial region 

indicate that dose-effect correlations may be improved by considering other 

expressions of dose such as total absorption by an airway and by further 

partitioning of the mucous layer compartment in mathematical models. Further 

research is needed to define toxic mechanisms, as well as to refine our knowledge of important chemical, physical, and morphological parameters. 

At present, there are few experimental results that are useful in judging 

the validity of the modeling efforts. Such results are needed, not only to 

understand better the absorption of 0 3 and its role in toxicity, but also to 

support and to lend confidence to the modeling efforts. With experimental 

confirmation, models which further our understanding of the role of 0 3 in the 

respiratory tract will become practical tools. 

The consistency and similarity of the human and animal lower respiratory 

tract dose curves obtained thus far lend strong support to the feasibility of 

extrapolating to man the results obtained on animals exposed to 0 3 . In the 

past, extrapolations have usually been qualitative in nature. With a.dditional 

research in areas which are basic to the formulation of dosimetry models, 

quantitative dosimetric differences among species can be determined. If in 

addition, more information is obtained on species sensitivity to a given dose, 

significant advances can be made in quantitative extrapolations and in making 

inferences about the likelihood of effects of 0 3 in man. Since animal studies 

are the only available approach for investigating the full array of potential 

disease states induced by exposure to 0 3 , quantitative use of animal data is 

in the interest of better establishing 0 3 levels to which man can safely be 

exposed. 

9-220 
9.6.3 Effects of Ozone on the Respiratory Tract 

9.6.3.1 Morphol o gical Effects. The morphological changes which follow 

exposure to less than 1960 pg/m 3 (1.0 ppm) 03 are very similar in all species 

of laboratory mammals studied. Of the many specific cell types found in the 

respiratory System, two types, ciliated cells and type 1 alveolar epithelial 

cells, are the cells most damaged morphologically following 0 3 inhalation. 

Ciliated cells are found in the conducting airways, e.g., trachea, bronchi, 

and nonrespiratory bronchioles. Ciliated cells function in the normal clearance 

of the airways and the removal of inhaled foreign material. Following 0 3 

exposure of experimental animals, damaged ciliated cells have been reported in 

all of these conducting airways (Schwartz et al., 1976; Castleman et al., 

1977). In rats, damage to ciliated cells appears most severe at the junction 

of the conducting airways with the gas exchange area (Stephens et al., 1974a; 

Schwartz et al., 1976). Damage to type 1 aiveolar epithelial cells is limited 

to those cells located near this junction, i.e., the centriacinar or proximal 

alveolar region of the pulmonary acinus (Stephens et al., 1974b; Schwartz et 

al., 1976; Castleman et al., 1980; Barry et al., 1983; Crapo et al., 1984). 

Type 1 alveolar cells form most of the blood-air barrier where gas exchange 

occurs. Severely damaged ciliated and type 1 alveolar epithelial cells are 

shed (sloughed) from the tissue surface and are replaced by multiplication of 

other cell types less damaged by 0 3 (Evans et al., 1985). This process has 

been most extensively studied in the centriacinar region where nonciliated 

bronchiolar cells and type 2 alveolar epithelial cells become more numerous 

(Evans et al., 1976a,b,c; Lum et al., 1978). Some of these nonciliated bronchiolar and type 2 cells differentiate into ciliated and type 1 cells, respectively. Cell multiplication in bronchioles may be more than that required for 

replacement of damaged ciliated cells, and nonciliated bronchiolar cells may 

become hyperplastic (Castleman et al., 1977; Ibrahim et al., 1980; Eustis et 

al., 1981) and sometimes appear as nodules (Zitnik et al., 1978; Moore and 

Schwartz, 1981; Fujinaka et al., 1985). Inflammatory changes characterized by 

a variety of leukocytes with alveolar macrophages predominating, intramural 

edema, and fibrin are also seen in the centriacinar region (Stephens et al., 

1974a; Schwartz et al., 1976; Castleman et al., 1977; Fujinaka et al., 1985). 

The damage to ciliated and centriacinar type 1 alveolar epithelial cells 

and the inflammatory changes tend to occur soon after exposure to concentrations 

9-221 
of 03 as low as 392 pg/m 3 (0.2 ppm). Damage to centriacinar type 1 alveolar 

epithelium in rats has been well documented as early as 2 hours after exposure 

to 03 concentrations of 980 pg/m 3 (0.5 ppm) (Stephens et al., 1974a). In the 

same publication the authors report damage to centriacinar type 1 alveolar 

epithelial cells after 2 hours exposure to 392 pg/m 3 (0.2 ppm) 03 , but this 

portion of their report is not documented by published micrographs (Stephens 

et al., 1974a). Loss of cilia from cells in the rat terminal bronchiole 

occurs following exposure to 980 pg/m 3 (0.5 ppm) 03 for 2 hours (Stephens et 

al., 1974a). Damage to ciliated cells has been seen following exposure of 

both rats and monkeys to 392 pg/m3 (0.2 ppm) 0 3 , 8 hr/day for 7 days (Schwartz 

et al., 1976; Castleman et al., 1977). Centriacinar inflammation has been 

reported as early as 6 hours after exposure to 980 pg/m3 (0.5 ppm) 0 3 (,Stephens 

et al., 1974b) and 4 hours after exposure to 1568 pg/m3 (0.8 ppm) 0 3 (Castleman 

et al., 1980). 

During long-term exposures, the damage to ciliated cells and to centriacinar 

type 1 cells and centriacinar inflammation continue, though at a reduced rate. 

Damage to cilia has been reported in monkeys following 90-day exposure,to 980 

Ng/m3 (0.5 ppm) 03 , 8 hr/day (Eustis et al., 1981) and in rats exposed to 980 

pg/m3 (0.5 ppm) 03 , 24 hr/day for 180 days (Moore and Schwartz, 1981). Damage 

to centriacinar type 1 cells was reported following exposure of young rats to 

490 pg/m3 (0.25 ppm) 0 3 , 12 hrs/day for 42 days (Barry et al., 1983; Crapo et 

al., 1984). Changes in type 1 cells were not detectable after 392 pg/m 3 (0.2. 

ppm) 03 , 8 hr/day for 90 days but were seen in rats exposed to 980 pg/m 3 (0.5 

ppm) for the same period (Boorman et al., 1980). Centriacinar inflammatory 

changes persist during 180-day exposures of rats to 980 pg/m3 (0.5 ppm) 03 , 24 

hr/day (Moore and Schwartz, 1981) and one-year exposures of monkeys to 1254 

ag/m3 (0.64 ppm) 03 , 8 hr/day (Fujinaka et al., 1985). 

Remodeling of distal airways and centriacinar regions oc,curs following 

long-term exposures to 0 3 . Rats develop respiratory bronchioles between the 

terminal bronchiole to alveolar duct junction seen in control rats (Boorman et 

al., 1980; Moore and Schwartz, 1981). In monkeys, distal airway remodeling 

results in increased volumes of respiratory bronchioles which have thicker 

walls and a smaller internal diameter (Fujinaka et al., 1985). The walls of 

centriacinar alveoli are also thickened (Schwartz et al., 1976; Boorman et 

al., 1980; Barry et al., 1983; Crapo et al., 1984; Last et al., 1984a). Studies 

of the nature of these thickened interalveolar septa and bronchiolar walls 

9-222 
revealed increases in inflammatory cells, fibroblasts, and amorphous extracellular matrix (Last et al., 1984a; Fujinaka et al., 1985). Three studies 

provide morphoiogical evidence of mild fibrosis (i.e., local increase of 

collagen) in centriacinar interalveolar septa following exposure to < 1960 

pg/m3 (< 1 ppm) of 0 3 (Last et al., 1979; Boorman et al., 1980; Moore and 

Schwartz, 1981). Changes in collagen location or amounts, or both, which occur 

with the remodeling of the distal airways, were reported in two of those studies 

(Boorman et al., 1980; Moore and Schwartz, 1981). 

While morphometry of small pulmonary arteries is not commonly studied in 

0 3-exposed animals, pulmonary artery walls thickened by muscular hyperplasia 

and edema were reported in rabbits exposed to 784 pg/m 3 (0.4 ppm) 03 , 6 hr/day, 

5 days/week for 10 months (P'an et al., 1972). Thickened intima and media in 

pulmonary arterioles were reported in monkeys exposed to 1254 pg/m 3 (0.64 ppm) 

0 3 , 8 hr/day for 1 year (Fujinaka et al., 1985). 

Several of the effects of 0 3 inhalation persisted after the 0 3 inhalation 

ended and the animals breathed only filtered air several days or weeks. Lungs 

from rats exposed to 1568 pg/m 3 (0.8 ppm) 0 3 for 72 hours appeared normal 6 

days after the end of the exposure (Plopper et al., 1978). However, incomplete 

reso7ution of the nonciliated bronchiolar epithelial hyperplasia was reported 

in monkeys 7 days after 50 hours exposure to 1568 pg/m 3 (0.8 ppm) 0 3 (Castleman 

et al., 1980) and in mice 10 days after a 20-day exposure to 1568 pg/m 3 (0.8 

ppm) 0 3 , 24 hr/day (Ibrahim et al., 1980). Centriacinar inflammation and 

distal airway remodeling were still apparent 62 days after a 180-day exposure 

to 980 pg/m3 (0.5 ppm) 0 3 , 24 hr/day (Moore and Schwartz, 1981). 

While not all species of laboratory mammals have been studied following a 

single 0 3 exposure regimen or using the same morphological techniques because 

investigators have asked different biological questions, there is a striking 

similarity of morphological effects in the respiratory system of all species 

studied. The cell types most damaged are the same. One of these cells, the 

type 1 alveolar epithelial ce11, has a wide distribution in the pulmonary 

acinus and yet is damaged only in one specific location in all species studied. 

The other, the ciliated cell, appears damaged wherever it is located in the 

conducting airways. Damage to these cells is seen within hours after exposure 

to concentrations'of 0 3 much lower than l ppm and continues during exposures 

of weeks or roonths. Hyperplasia of other cell types is reported to start 

early in the exposure period, to continue throughout a long-term exposure, and 

9-223 
when studied, to persist following postexposure periods of days or weeks. 

Centriacinar inflammation is also seen early and is reported throughout long 

exposure periods. Duration of centriacinar inflammation during postexposure 

periods has been studied less often and appears dependent upon length of the 

exposure period. 

Other effects which have been reported in fewer studies or in a more 

limited number of species include distal airway remodeling and thickened pulmonary arteriolar walls. Remodeling of distal airways has only been reported 

in rats and monkeys after long-term exposures. In rats, remodeling of distal 

airways has been reported to persist for several weeks after the 0 3 exposure 

has ended. Thickened pulmonary arteriolar walls have been reported only 

twice, once after long-term exposure of rabbits and once after long-term 

exposure of monkeys. 

Studies on the morphologic effects of 03 exposures of experimental animals 

are summarized in Figure 9-4 and Table 9-28 (see Section 9.6.1 for criteria 

used to summarize the studies). 

9.6.3.2Pulmonary Function . One of the limitations of animal studies is 

that many pulmonary function tests comparable to those conducted after acute 

exposure of human subjects are difficult to interpret. Methods exist, however, 

for obtaining similar measurements of many variables pertinent to understanding the effects of ozone on the respiratory tract, particularly after longer 

exposure periods. A number of newer studies reported here reflect recent 

advances in studying the effects of 0 3 on pulmonary function in small animals: 

Changes in lung function following ozone exposure have been studied in 

mice, rats, guinea pigs, rabbits, cats, dogs, sheep, and monkeys. Short-term 

exposure for 2 hr to concentrations of 431 to 980 pg/m 3 (0.22 to 0.5 ppm) 

produces rapid, shallow breathing and increased pulmonary resistance during 

exposure (Murphy et al., 1964; Yokoyama, 1969; Watanabe et al., 1973; Amdur 

et al., 1978). The onset of these effects is rapid and the abnormal breathing 

pattern usually disappears within 30.min`,after cessation of exposure. Other 

changes in lung function measured following short-term ozone exposures lasting 

3 hr to 14 days are usually greatest 1 day following exposure and disappear by 

7 to 14 days following exposure. These effects are associated with premature 

closure of the small, peripheral airways and include increased residual volume, 

closing volume, and closing capacity (Inoue et al., 1979). 

Studies of airway reactivity following short-term ozone exposure of 1 to 

2 hr duration in experimental animals show that 0 3 increases the reactivity of 

9-224 
0.0 

0.1 

E 

a 

a 

c 

•+-. 0 

~ 

~ 

~. 

c 

~ 

c 

0 

~ 

d 

c 

0 

N 

X 

0.2 

0.3 

0.4 

0.5 

0.6 

0.7 

0.8 

0.9 

1.0 

~ OrG.~~y

gLe 

 Ge~g ~~'t~°~ °a~,~.r~ °r,~c1 

s<~,~e° r~< ~~~~s0 s~ <8~ r6aQ~`~ ~s 

Os~Q 10

p 

 Q<°~`<°rG~``o 0rti<~ec` ~5'~s~ e`~ ~'~`~o<'c'e<`°`a r1 ~o G O s 

Figure 9-4. Summary of morphological effects in experimental animals 

exposed to ozone. See Table 9-28 for reference citations of studies 

summarized here. 

§-225 
TABLE 9-28. SUMMARY TABLE: MQRPHOLOGICAL EFFECTS OF OZONE 

IN EXPERIMENTAL ANIMALS 

Effect/response 0 3 concentration, ppm References 

Damaged ciliated [0.2], 0.5, 0.8 Boorman et al. (1980) 

and type 1 cells 0.2, 0.5, 0.8 Schwartz et al. (1976) 

0.2, 0.35 Castleman et al. (1977) 

0.25 Barry et al. (1983) 

0.25 Crapo et al. (1984) 

0.26, 0.50, 1.0 Boatman et al. (1974) 

0.5 Stephens et al. (1974b) 

0.5 Moore and Schwartz (1981) 

0.5 Evans et al. (1985) 

0.5, 0.8 Eustis et al. (1981) 

0.5, 0.8 Mellick et al. (1975, 1977) 

0.54, 0.88 Stephens et al. (1974a) 

0.8 Castleman et al. (1980) 

0.8 Plopper et al. (1978) 

0.85 Stephens et al. (1978) 

Proliferation of non- 0.2, 0.35 Castleman et al. (1977) 

ciliated bronchiolar 0.35, 0.50, 0.70, Evans et al. (1976b) 

and type 2 cells 0.75, 1.0 

0.5 Evans et al. (1985) 

0.5 Zitnik et al. (1978) 

0.5 Moore and Schwartz (1981) 

0.5, 0.8 Eustis et al. (1981) 

0.54, 0.88 Freeman et al. (1974) 

0.64 Fujinaka et al. (1985) 

0.7 Evans et al. (1976a) 

0.8 Castleman et al. (1980) 

0.8 Lum et al. (1978) 

0.8 Ibrahim et al. (1980) 

1.0 Cavender et al. (1977) 

Centriacinar [0.21, 0.5 1 0.8 Boorman et al. (1980) 

inflammation 0.2 Plopper et al. (1979) 

0.2, 0.5, 0.8 Schwartz et al. (1976) 

0.25 Barry et al. (1983) 

0.25 Crapo et al. (1984) 

0.35 Castleman et al. (1977) 

0.5 Stephens et al. (1974b) 

0.5 Moore and Schwartz (1981) 

0.5, 0.8 Mellick et al. (1975, 1977) 

0.5, 0.8 Brummer et al. (1977) 

0.5, 0.8 Last et al. (1979) 

0.54, 0.88 Stephens et al. (1974a) 

0.54, 0.88 Freeman et al. (1974) 

0.64 Fujinaka et al. (1985) 

0.8 Castleman et al. (1980) 

1.0 Freeman et al. (1973) 

9-226 
TABLE 9-28. SUMMARY TABLE: MORPHOLOGICAL EFFECTS OF OZONE 

IN EXPERIMENTAL ANIMALS (continued) 

Effect/response 0 3 concentration, ppm References 

Distal airway 

remodeling 

Thickened pulmonary 

arteriolar walls 

[0.21, 0.5, 0.8 

0.2, 0.5, 0.8 

0.5 

0.64, 0.96 

0.64 

1.0 

0.4 

0.64 

Boorman et al. (1980) 

Schwartz et al. (1976) 

Moore and Schwartz (1981) 

Last et al. (1984a) 

Fujinaka e -t al. (1985) 

Freeman et al. (1973) 

P'an et al. (1972) 

Fujinaka et al. (1985) 

the lungs to a number of stimuli. Mild exercise, histamine aerosol inhalation, 

and breathing air with reduced oxygen or elevated carbon dioxide concentrations 

caused rapid, shallow breathing in conscious dogs immediately following 2-hr 

exposures to 1100 to 1666 pg/m 3 (0.56 to 0.85 ppm) of 0 3 (Lee et al., 1979, 

1980). Aerosolized ovalbumin caused an increased incidence of anaphylaxis in 

mice preexposed to 980 or 1568 pg/m 3 (0.5 or 0.8 ppm) of 0 3 continuously for 3 

to 5 days (Osebold et al., 1980). In addition, increased airway sensitivity 

to histamine or cholinomimetic drugs administered by aerosol or injection has 

been noted in several species after exposure to 980 to 5880 pg/m 3 (0.5 to 3.0 

ppm) of 0 3 (Easton and Murphy, 1967; Lee et al., 1977; Abraham et al., 1980, 

1984a,b; Gordon and Amdur, 1980; Gordon et al., 1981, 1984; Roum and Murlas, 

1984). The mechanism responsible for 0 3-induced bronchial reactivity is stili 

uncertain but may involve more than one specific factor. Ozone has been shown 

to cause increased sensitivity of vagal sensory endings in the dog airway (Lee 

et al., 1977, 1979, 1980). Ozone exposure may also enhance the airway responsiveness to bronchoconstrictors by altering sensitivity of the airway smooth 

muscl e. di-rectly .o-r _through re-1 eased cel l u-1 ar medi.ators (Gor_don et al.. , 1981, 

1984; Abraham et al., 1984a,b). In some species, increased airway hyperreactivity may be explained by increased transepithelial permeability or decreased 

thickness of the airway mucosa (Osebold et al., 1980; Abraham et al., 1984b). 

Ozone exposure may also decrease airway hyperreactivity by causing mucous 

hypersectetion, thereby limiting the airway penetration of inhaled bronchoconstrictors (Abraham et al., 1984a). 

9-227 
The time course of airway hyperreactivity after exposure to,980 to 5880 

pg/m (0.5 to 3.0 ppm) of 0 3 suggests a possible association with inflammatory 

cells and pulmonary inflammation (Holtzman et al., 1983a,b; Sielczak et al., 

1983; Fabbri et al., 1984; 0'Byrne et al., 1984a,b; Murlas and Roum, 1985). 

However, the time course of responsiveness is variable in different species 

and the relationships between airway inflammation and reactivity at different 

concentrations of 0 3 are not well understood. Additional studies that demonstrate increased collateral resistance following 30 min local exposure of 0 3 

or histamine in sublobar bronchi of dogs (Gertner et al., 1983a,b,c,1984) 

suggest that other mechanisms, along with amplification of reflex pathways, 

may contribute to changes in airway reactivity depending not only on the 

concentration of 0 3 in the airways but also on the extent of penetration of 

ozone into the lung periphery. 

The effects of short-term exposures to 0 3 on pulmonary function and 

airway reactivity in experimental animals are summarized in Figure 9-5 and 

Table 9-29 (see Section 9.6.1 for criteria used in developing this summary). 

Exposures of 4 to 6 weeks to ozone concentrations of 392 to 490 pg/m 3 

(0.2 to 0.25 ppm) increased lung distensibility at high lung volumes in young 

rats (Bartlett et al., 1974; Raub et al., 1983a). Similar increases in lung 

distensibility were found in older rats exposed to 784 to 1568 pg/m 3 (0.4 to 

0.8 ppm) for up to 180 days (Moore and Schwartz, 1981; Costa et al., 1983; 

Martin et al., 1983). Exposure to 0 3 concentrations of 980 to 1568 pg/m 3 (0.5 

to 0.8 ppm) increased pulmonary resistance and caused impaired stability of 

the small peripheral airways in both rats and monkeys ( Wegner, 1982; Costa 

et al., 1983; Yokoyama et al., 1984; Kotlikoff et al., 1984). The effects in 

monkeys were not completely reversed by 3 months following exposure; lung 

distensibility had also decreased in the postexposure period, suggesting the 

development of lung fibrosis which has also been suggested morphologically and 

biochemically. 

The effects of long-term exposures to ozone on pulmonary function and 

airway reactivity in experimental animals are summarized in Figure 9-6 and 

Table 9-30 (see Section 9.6.1 for criteria used in developing this summary). 

9.6.3.3 Biochemical Effects The lung is metabolically active, and- several 

key steps in metabolism have been studied after 0 3 exposure. Since the procedures for such studies are invasive, this research has been conducted only in 

animals. Effects, to be summarized below, have been observed on antioxidant, 

metabolism, oxygen consumption, proteins, lipids, and xenobiotic metabolism. 

9-228 
T 

Tv ~AdJ6 

O (° TA ~OoJJ / 

G 

JG 

,00

~~O n~iP 

O 6 o 

.~ 

°OJ 

s 

J JJ ~ 

d 

J6+ JC 

G ° 

i 

'a~ • ~Jif~ 

~ 

~G~e ~rJC N 

v9 

°Od 

dv 

~ 

0--------------~ 

O O O O O O wwdd •suoi;eilua3uoo ouozo 
TABLE 9-29. SUMMARY TABLE: EFFECTS ON PULMONARY FUNCTION 

OF SHORT-TERM EXPOSURES TO OZONE IN EXPERIMENTAL ANIMALS 

Effect/response 03 concentration, ppm References 

Increased breathing 0.22, 0.41, 0.8 Amdur et al. (1978) 

frequency 0.34, 0.68, 1.0 Murphy et al. (1964) 

0.5 Yokoyama (1969) 

Decreased tidal volume 0.34, 0.68, 1.0 Murphy et al. (1964) 

Decreased lung [0.221, 0.41, 0.8 Amdur et al. (1978) 

compliance 0.26, 0.5, 1.0 Watanabe et al. (1973) 

1.0 Yokoyama (1974) 

Increased residual 

volume (RV), 

closing capacity 

(CC), and closing 

volume (CV) 

0.24 - 1.0 Inoue et al. (1979) 

Decreased diffusion 0.26, 0.5, 1.0 Watanabe et al. (1973) 

capacity 

Increased pulmonary 

resistance 

Increased airway 

reactivity 

[0.221 Amdur et al. (1978) 

0.26, 0.5, 1.0 Watanabe et al. (1973) 

0.5 Yokoyama (1969) 

1.0 Yokoyama (1974) 

[0.1]-0.8 Gordon and Amdur (1980) 

[0.11-0.8, 1.0 Gordon et al. (1981, 1984) 

0.5, 1.0 Abraham et al. (1980, 1984a,b) 

0.7 Lee et al. (1977) 

1.0 Holtzman et al. (1983a,b) 

The lung contains several compounds (e.g. ; vitamin E, sulfhydryls, glutathione) and enzymes (e.g., glutathione peroxidase, glutathione reductase, 

glucose-6-phosphate dehydrogenase, and superoxi_de dismutase) that function as 

antioxidants, thereby defending the lung against oxidant toxicity from the 

oxygen in air, from oxidants produced during metabolic processes, and from 

oxidizing air pollutants such as ozone. Obviously, this protection is only 

partial for 03 since exposure to ozone causes numerous effects on lung structure, function, and biochemistry. Acute exposure to high ozorie levels (2920 

pg/m3 , 2 ppm) typically decreases antioxidant metabolism, whereas repeated 

exposures to lower levels (between 272 and 1568 pg/m 3 , 0.2 and 0.8 ppm) increases this metabolism (DeLucia et al., 1975b). In rats maintained on normal 

9-230 
~ 

7~ 

~ 

~ dA O~ y 

~ rJ 

r~ d 

IP 0 CI~~~~ 

v 

dJ V~ O~O 

G p 

. dr ddd 

Z dr 

d`d JO 

7 O' 

i7eA✓ 

v 

7 ~ 

A 

dd dd 

vJ 

I 

Ir- L 0 

UN le 

~ ~ 

O6 

c. ~ 

x~ 

O N ;? ~ 

E ~ t 

ti Gd 

~

•E N ~ 

cc~ 

0=E 

v9 a9 7 

~ Ey 

~ K ~ 

O O y 

cc cO' 

E~ o 

3 C Cc 

y ~ v 

~ W V 

O O ~ 

7 ~ y 

~ ~ d ii a ~ 

p ~ N r°f at tq 

wdd •suolzejtueq.uoa euozo_ 

9-231 
TABLE 9-30. SUMMARY TABLE: EFFECTS ON PULMONARY FUNCTION 

OF LONG-TERM EXPOSURES TO OZONE IN EXPERIMENTAL ANIMALS 

Effect/response 03 concentration, ppm References 

Increased lung volume [0.081, [0.12], 0.25 Raub et al. (1983a) 

0.2 Bartlett et al. (1974) 

[0.21, 0.8 Costa et al. (1983) 

0.4 Martin et al. (1983) 

Increased pulmonary 

resistance 

Decreased lung 

compliance 

Decreased inspiratory 

flow 

0.2, 0.8 Costa et al. (1983) 

0.5, 1.0 Yokoyama et al., 1984 

0.64 Wegner (1982) 

0.64 Kotlikoff et al., 1984 

0.5, 0.8 Eustis et al. (1981) 

0.64 Wegner (1982) 

[0.08], 0.12, 0.25 Raub et al. (1983) 

Decreased forced 

expiratory volume 

(FEV ) and flow 

(FEF~ 

0.2, 0.8 

0.64 

Costa et al. (1983) 

Wegner (1982) 

diets, this response has been observed after a week of continuous or intermittent exposure to 392 pg/m3 (0.2 ppm) 0 3 (Mustafa, 1975; Mustafa and Lee, 1976; 

Plopper et al., 1979). Similar responses are seen in monkeys and mice, but at 

higher concentrations (980 pg/m3 , 0.5 ppm) (Fukase et al., 1978; Mustafa and 

Lee, 1976). 

The effects of 0 3 on oxygen consumption have been studied since oxygen 

consumption is a fundamental parameter of cellular metabolism, reflecting 

energy production by cells. As with antioxidant metabolism, acute exposure to 

high ozone levels (> 3920 pg/m 3 ; > 2 ppm) decreases metabolism (and thus, 

oxygen consumption); repeated exposure to lower levels (> 1568 pg/m3 , 0.8 ppm) 

increases oxygen consumption (Mustafa et al.-, 1973; Schwartz et al., 1976; 

Mustafa and Lee, 1976)., Effects in rats on normal diets have been observed 

after a short-term exposure to ozone 1 evel s as 1 ow as 392 pg/m 3(0.2 ppm) 

(Schwartz et al., 1976; Mustafa et al., 1973; Mustafa and Lee, 1976). Monkeys 

are affected at a higher level of ozone (980 pg/m3 , 0.5 ppm). 

9-232 
Simiiar patterns of response for both antioxidant metaboiism and oxygen 

consumption are observed after exposure to ozone. A 7-day exposure to ozone 

produces linear concentration-related increases in activities of glutathione 

peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and 

succinate oxidase (Mustafa and Lee, 1976; Chow et al., 1974; Schwartz et al., 

1976; Mustafa et al., 1973). Rats on a vitamin E-deficient diet experience an 

increase in enzyme activities at 196 pg/m 3 (0.1 ppm) ozone as compared to 

392 pg/m 3

(0.2 ppm) in animals on normai diets (Chow et al., 1981; Mustafa and 

Lee, 1976; Mustafa, 1975). Research on these enzymes has shown that there is 

no significant difference in effects from continuous versus intermittent 

exposure; this, along with concentration-response data, suggests that the concentration of ozone is more important than duration of exposure in causing 

these effects (Chow et al., 1974; Schwartz et al., 1976; Mustafa and Lee, 

1976). 

Duration of exposure still plays a role, however. During exposures up to 

1 or 4 weeks, antioxidant metabolism and 0 2 consumption generally do not 

change on the first day of exposure; by about day 2, increases are observed and 

by about day 4 a plateau is reached (Mustafa and Lee, 1976; DeLucia et al., 

1975a). Recovery from these effects occurs by 6 days post-exposure (Chow 

et al., 1976b). This plateauing of effects in the presence of exposure does 

not result in long-term tolerance: If rats are re-exposed after recovery is 

observed, the increase in enzyme activities is equivalent to that observed in 

animals exposed for the first time (Chow et al., 1976b). 

The influence of age on responsiveness is also similar for antioxidant 

metabolism and oxygen consumption (Elsayed et al., 1982a; Tyson et al., 1982; 

Lunan et al., 1977). Suckling neonates (5 to 20 days old) generally exhibited 

a decrease in enzyme activities; as the animals grew older (up to about 180 days 

old), enzyme activities generally increased with age. Species differences may 

exist in this response (Mustafa and Lee, 1976; Mustafa et al., 1982; Chow 

et al., 1975; DeLucia et al:, 1975a). Studies in which monkeys have been 

compared to rats did not include a description of appropriate statistical 

considerations applied (if any); thus, no definitive conclusions about responsiveness of monkeys versus rats can be made. 

The mechanism responsible for the increase in antioxidant metabolism and 

oxygen consumption is not known. The response is typically attributed, however, 

to concurrent morphological changes, principally the loss of type 1 cells and 

an increase in type 2 cells that are richer in the enzymes measured. 

9-233 
hlonooxygenases constitute another class of enzymes investigated after 

ozone exposure. These enzymes function in the metabolism of both endogenous 

(e.g., biogenic amines, hormones) and exogenous (xenobiotic) substances. The 

substrates acted upon are either activated or detoxified, depending on the 

substrate and the enzyme. Acute exposure to 1470 to 1960 pg/m 3 (0.75 to 

1 ppm) ozone decreased cytochrome P-450 levels and enzyme activities related 

to both cytochrome P-450 and P-448. The health impact of these changes is 

uncertain since only a few elements of a complex metabolic system were measured. 

The activity of lactate dehydrogenase is increased in lungs of vitamin Edeficient rats receiving a short-term exposure to 196 pg/m 3 (0.1 ppm) ozone 

(Chow et al., 1981). Higher levels caused a similar response in rats, but not 

in monkeys, on normal diets (Chow et al., 1974, 1977). This enzyme is frequently used as a marker of cellular damage because it is released upon cytotoxicity. 

It i s not known, however, whether the i ncrease i n thi s enzyme i s a di rect 

reflection of cytotoxicity or whether it is an indicator of an increased 

number of type 2 cells and macrophages in the lungs. 

An increase in a few of the measured•activities of lysosomal enzymes has 

been shown in the lungs of rats exposed to > 1372 pg/m3 (0.7 ppm) ozone (Dillard 

et al., 1972; Castleman et al., 1973a;'Chow et al., 1974). This response is 

most likely the result of an increase in inflammatory cells in the lungs 

rather than an induction of enzymes, since lysosomal enzymes in alveolar 

macrophages decrease after in vivo or in vitro exposure to ozone (Hurst et al., 

1970; Hurst and Coffin, 1971). 

As discussed previously, long-term exposure to high 0 3 concentrations 

causes mild lung fibrosis (i.e., local increase of collagen in centriacinar 

interalveolar septa). This morphological change has been correlated with 

biochemical changes in the activity of prolyl hydroxylase (an enzyme that 

catalyzes the production of hydroxyproline) and in hydroxyproline content (a 

component of collagen-that is present in excess in f7brosis) (Last et al., 

1979; Bhatnagar et al., 1983). An increase in collagen synthesis has been 

observed, with 980 pg/m 3 (0.5 ppm) 03 being the minimally effective concentration tested (Hussain et al., 1976a,b; Last et al., 1979). During a prolonged 

exposure, prolyl hydroxylase activity increases by day 7 and returns to control 

levels by 60 days of exposure. When a short-term exposure ceases, prolyl 

hydroxylase activity returns to normal by about 10 days post-exposure, but 

hydroxyproline levels remain elevated 28 days post-exposure. Thus, the product 

9-234 
of the increased synthesis, collagen, remains relatively stable. One study 

(Costa et al., 1983) observed a small decrease in collagen levels of rats at 

392 and 1568 pg/m 3 (0.2 and 0.8 ppm) 0 3 after an intermittent exposure for 62 

days. 

The effects of 03 on increasing collagen content may be progressive; 

i. e. , after a 6-week i ntermi ttent exposure of rats to 0.64 or 0. 96 ppm 03 

ceased, collagen levels 6 week post-exposure were elevated over the levels 

immediateiy after exposure (Last et al., 1984b). Also, there appears to be 

little difference between continuous and intermittent exposure in increasing 

collagen levels in rat lungs (Last et al, 1984b). Thus, the intermittent clean 

air periods were not sufficient to permit recovery. 

Although the ability of 0 3 to initiate peroxidation of unsaturated f•atty 

acids in vitro is well established, few in vivo studies of lung lipids have 

been conducted. Generally, ozone decreases unsaturated fatty acid content of 

the lungs (Roehm et al., 1972) and decreases incorporation of fatty acids into 

lecithin (a saturated fatty acid) (Kyei-Aboagye et al., 1973). These alterations, however, apparently do not alter the surface-tension-lowering properties 

of lung lipids that are important to breathing (Gardner et al., 1971; Huber 

et al., 1971). 

One of the earliest demonstrated effects of ozone was that very high 

concentrations caused mortality as a result of pulmonary edema. As more 

sensitive techniques were developed, iower levels (510 pg/m3 , 0.26 ppm) were 

observed to increase the protein content of the lung (Hu et al., 1982). Since 

some of the excess protein could be attributed to serum proteins, the interpretation was that edema had occurred. This effect was more pronounced several 

hours after exposure ceased. At higher concentrations, a loss of carriermediated transport from the air side of the lung to the blood side was observed 

(Williams et al., 1980). These changes imply an effect on the barrier function 

of the lung, which regulates fluxes of various substances with potential 

physiological activities across the alveolar walls. 

The biochemical effects observed in experimental animals exposed to 0 3 

are summarized in Figure 9-7 and Table 9-31 (see Section 9.6.1 for criteria 

used in developing this summary). 

9.6.3.4 Host Defense Mechanisms. Reports over the years have presented 

substantial evidence that exposure to ozone impairs the antibacterial activity 

of the lung, resulting in an impairment of the lung's ability to kill inhaled 

9-235 
• 

0 ---- ----------0----------0 

~ 

0-------0--- 

o•-------- 

0----------0-------~~ 

0---* • 0-0 0 

y 

~0 

E d ~' 

~ a 

~ y 

O = O 

Ey C 

~ O o. *' c0 X

'~ 

C y 

N v O C 

C ~ 

t O 

v ` 

E r~ 

~ 

v~ O~ 

~ <p 

O ~ 

~ (D 

eo N 

E~;~; 

y os 

^ 0 N 

c) -a ~ 

i y E 

Q, °c.E 

u. ~ N 

.oJ 

ro 

~~+I O~ 

~

~ 

~ ~J 

o~ J 

~ 

.~ot 

~ 

J p 

m~o ~~ 

0

~ O 

~ 

y00 ~if 1 • JJy u 

OO JJ ~00 

J►0, 

G~ GtA ~' oo Or 

d+GOi •,~ G• ~ 10~J d i 

o~ ~O 9t4 Ov c~ o~ J 

e-~ % 

~py ~OOSm10 

J 

s'y o 

J 

p~ p T~J~JI 

~ O 

~ 

d 

Ja d'G yj~, ~ei 

~J % ~O tG .O ~ Go f o~o m~ ~G 

O' ~T Ky 65 

GoJc, ~oo✓ 

~ ui 

m~, 

00 

vJ 

I 

O ~ N M qt O (D N 00 Of O 

wdd •uoi;ea}uaouo:) auozp 

; : 
TABLE 9-31. SUMMARY TABLE: BIOCHEMICAL CHANGES 

IN EXPERIMENTAL ANIMALS EXPOSED TO OZONE 

Effect/response 0 3 concentration, ppm References 

Increased 0 2 [0.1], 0.2 

consumption [0.1], 0.2, 0 

0.2, 0.5, 0.8 

0.2, 0.5, 0.8 

0.45 

0.8 

0.8 

35, 0.5, 0.8 

Mustafa (1975) 

Mustafa and'Lee (1976) 

Mustafa et ai. (1973) 

Schwartz et al. (1976) 

Mustafa et al. (1982) 

Chow et al. (1976b) 

Eisayed et al. (1982a) 

Increased lysosomal 

enzyme activities 

Increased lung 

hydroxyproline 

and prolyl 

hydroxylase 

activity 

Altered mucus 

glycoprotein 

secretions 

Increased alveolar 

protein and 

permeability 

changes 

Increased LDH 

activity 

Increased NADPH - 

cytochrome c 

reductase 

activity 

[0.2], [0.5], 0.8 

0.7, 0.8 

0.7, 0.8 

[0.2], 0.5, 0.8 

0.2, 0.8 

0.45, 0.8 

0.5, 0.64, 0.96 

0.5 

0.8 

[0.2], [0.4], 0.5, 0.6, 0.8 

0.5, 0.6, 0.8 

0.6, 0.8 

[0.1] 

[0.5], 0.8 

0.8 

0.2, 0.35, 0.8, 

0.2, 0.5, 0.8 

0.2, 0.5, 0.8 

Chow et al. (1974) 

Dillard et al. (1972) 

Castleman et al. (1973a,b) 

Hussain et al. (1976a,b) 

Costa et al. (1983) 

Bhatnagar'et al. (1983) 

Last et ai. (1979, 1984b) 

Last and Greenberg (1980) 

Hesterberg and Last (1981) 

Last and Kaizu (1980) 

Last and Cross (1978) 

Last et al. (1977) 

Chow et al. (1981) 

Chow et al. (1977) 

Chow and Tappel (1973) 

Mustafa and Lee (1976) 

Schwartz et al. (1976) 

DeLucia et al. (1972, 1975a,b) 

[0.1], 0.26, 0.51, 1.0 Hu et al. (1982) 

[0.25], 0.5, 1.0 Alpert et al. (1971a) 

0.6, 1.0 Williams et ai. (1980) 

1.0 Reasor et al. (1979) 

Increased GSH 

metabolism 

[0.1] 

0.1, 0.2 

0.2, 0.35, 0.5, 

0.2, 0.5, 0.8 

0.2, 0.5, 0.8 

0.2, 0.5, 0.8 

0.2, 0.5, 1.0 

0.32 

0.45 

0.5 

Chow et ai. (1981) 

Plopper et al. (1979) 

0.8 Mustafa and Lee (1976) 

Chow et al. (1974) 

DeLucia et al. (1972, 1975a,b) 

Schwartz et al. (1976) 

Fukase et al. (1975) 

Moore et al. (1980) 

Mustafa et al. (1982) 

Chow et al. (1975) 

9-237 
TABLE 9-31. SUMMARY TABLE: BIOCHEMICAL CHANGES 

IN EXPERIMENTAL ANIMALS EXPOSED TO OZONE (continued) 

Effect/response 0 3 concentration, ppm References 

Increased NPSH 

Decreased 

unsaturated 

fatty acids 

0.5, 1.0 Fukase et al. (1978) 

0.7, 0.75, 0.8 Chow and Tappel (1972, 1973) 

0.8 Elsayed et al. (1982a,b; 

1983) 

0.8 Chow et al. (1976b) 

0•9 Tyson et al. (1982) 

0.9 Lunan et al. (1977) 

0.1, 0.2 Plopper et'al. (1979) 

0.2, 0.5, 0.8 DeLucia et al: (1975b) 

0.45 Mustafa et al. (1982) 

0.8 Chow et al. (1976b) 

0.5• Roehm et al., 1972 

microorganisms. Suppression of this biocidal defense of the lung can lead to 

microbial proliferation within the lung, resulting in mortality. The mortality 

response is concentration-related and is significant at concentrations as low 

as 157 to 196 pg/m3 (0.08 to 0.1 ppm) (Coffin et al., 1967; Ehrlich et al., 

1977; Miller et al., 1978a; Aranyi et al., 1983). The biological basis for 

this response appears to be that ozone or one of its reactive products can 

impair or suppress the normal bactericidal functions of the pulmonary defenses, 

which results in prolonging the life of the infectious agent, permitting its 

multiplication and ultimately, in this animal model, resulting in death. Such 

infections can occur because of 0 3 effects on a complex host defense,system 

involving alveolar macrophage functioning, lung fluids, and other immune 

factors. 

The data obtained in various experimental animal studies indicate that 

short-term ozone exposure can reduce the effectiveness of several vital defense 

systems including (1) the ability of the lung to inactivate bactcria and 

viruses (Coffin et al., 1968; Coffin and Gardner, 1972b; Goldstein et al., 

1974a, 1977; Warshauer et al se,e 1974; Bergers Qet al; 1983. Schwartz and Christman, 

1979; Ehrlich et al., 1979); (2) the mucociliary transport system (Phalen 

et al., 1980; Frager et al., 1979; Kenoyer et al., 1981; (3) the immunological 

system (Campbell and Hilsenroth, 1976; Fujimaki et al., 1984; Thomas et al., 

9-238 
1981b; Aranyi et al., 1983; and (4) the pulmonary macrophage (Dowell et al., 

1970; Goldstein et al., 1971a,b, and 1977; Hadley et al., 1977; McAllen et al., 

1981; - Wi tz - et al., 1983; Hurst et al: ;--1970; Hurst and Coff-in,--1971; Amoruso 

et al., 1981). Studies have also indicated that the activity level of the 

test subject and the presence of other airborne chemicals are important variables that can influence the determination of the lowest effective concentration of the pollutant (Gardner et al., 1977; Aranyi et al., 1983; Ehrlich, 

1980, 1983; Grose et al., 1980, 1982; Phalen et al.; 1980; Goldstein et al., 

1974a; Illing et al., 1980). 

Ciliated cells are damaged by 0 3 inhalation, as demonstrated by major 

morphoiogical changes in these cells, including necrosis and sloughing, or by 

the shortening of the cilia in cells attached to the bronchi. Sufficient 

ciliated cell damage should result in decreased transport of viable and nonviable particles from the lung. Rats exposed to 784, 1568, 1960, or 2352 

Pg/m3 (0.4, 0.8, 1.0, or 1.2 ppm) for times as short as 4 hr have decreased 

short-term clearance of particles from the lung (Phalen et al., 1980; Frager 

et al., 1979; Kenoyer et al., 1981). Short-term clearance is mostly due to 

mucus transport of particles, and the decreased short-term clearance is an 

anticipated functional result predicted from morphological observations. The 

mucous glycoprotein production of the trachea is also altered by 0 3 exposure. 

Mucous glycoprotein biosynthesis, as measured ex vivo in cultured tracheal 

explants from exposed rats, was inhibited by short-term continuous exposure to 

1568 pg/m3 (0.8 ppm) of 0 3 for 3 to 5 days (Last and Cross, 1978; Last and 

Kaizu, 1980; Last et al., 1977). Glycoprotein synthesis and secretion recovered 

to control values after 5 to 10 days of exposure and increased to greater than 

controi values after 10 days of, exposure. With this increase in production of 

mucus, investigators have found that the velocity of the tracheal mucus was 

significantly reduced following a 2 hr exposure to 1960 pg/m 3 (1.0 ppm) (Abraham 

at .al., 1980). 

A probl,em remains in assessing-the relevance of these animal data to 

humans. Green (1984) reviewed the literature and compared the host antibacterial 

defense systems of the rodent and man and found that these two species had 

defenses that are very similar and thus provide a good basis for a qualitative 

extrapolation. Both,defenses consist of an aerodynamic filtration system, a 

fluid layer lning the respi i 

. _. 

'ratory membranes, a transport mechanism - for removing 

foreign particles, microorganisms, and pulmonary cells, and immune secretions 

9-239 
of lymphocytes and plasma cells. In both rodents and humans, these components 

act in concert to maintain the lung free of bacteria. 

If the animal models are to be used to reflect the toxicological response 

occurring in humans, then the endpoint for comparison of such studies should 

be morbidity rather than mortality. A better index of 0 3 effect in humans 

might be the increased prevalence of infectious respiratory illness in the 

community. Such a comparison may be proper since both mortality from respiratory infections (animals) and morbidity from respiratory infections (humans) 

can result from a loss in pulmonary defenses (Gardner, 1984). Whether the 

microorganisms used in the various animal studies are comparable to the organisms responsible for the respiratory infections in a community still requires 

further investigation. 

Ideally, studies of pulmonary host defenses should be performed in man, 

using epidemiological or volunteer methods of study. Unfortunately, such 

studies have not been reported yet. Attention must therefore be paid to the 

results of host-defense experiments conducted with animals. 

In the area of host defense of the lung against infection, present knowledge of the physiology, metabolism, and function have come primarily from the 

study of various animal systems, but it is generally accepted that the basic 

mechanisms of action of these defense cells and systems function similarly in 

both animals and man. There are also human data to support this statement, 

especially in such areas as immunosuppression, ciliostasis, and alveolar 

macrophages. The effects seen in animals represent alterations in basic 

biological systems. One can assume that similar alterations in basic defense 

mechanisms could occur in humans since they possess equivalent pulmonary 

defense systems. It is understood, however, that different exposure levels 

may be required to produce similar responses in humans. The concentration of 

03 at which effects become evident can be influenced by a number of factors, 

such as preexisting disease, virulence of the infeftious agent, dietary factors, 

concurrent exposure to other pollutants, exercise, or the presence of other 

environmental stresses, or a combination of these. Thus, one could hypothesize 

that humans exposed to 0 3 could experience effects on host defense mechanisms. 

At the present time, however, one cannot predict the exact concentration at 

which effects may occur in man nor the severity of , the effects. 

The effects of 03 on host defense mechanisms'in experimental animals are 

summarized in Figure 9-8 and Table 9-32 (see Section 9.6.1 for-criteria used 

in developing this summary). 

a ER 
qN 

N 

•R O 

V y 

(D ~ 

. O Ew. ca 

C V 

~ ~ d ~ 

-a ~ 

~ 

S L 

O ,0 

~ c N M 

O , 

wo 

N~ 

O 

O ~ ~ 

-4- CD 

@ CO ai 

U. 

t 

c~o £ 'a 

E c . O ,N 

tn c E 

4)E 06E7 

C6 ~a~ H 

` Q ~ 

5 x ~C _CM 

p -7 N trf a It! (A CO O~ O 

p O C O O O C O O O e- 

.~ 

uo 

'ru 

i 

G~uG 3e,p uG 

i 

o• .o 

• ?~v~ v~~~ . 

~G 

a~ Adv 

~J• Jrm~J ~~ 

du~ uG u9A 

~~ot v 6~,,• ~~~9 i 

. uo'oo d9 

~ 

G~ ~m~~i 

O 

wdd 'uoilei;uaouoo auozo 

9-241 
TABLE 9-32. SUMMARY TABLE: EFFECTS OF OZONE ON HOST DEFENSE 

MECHANISMS IN EXPERIMENTAL ANIMALS 

Effect/response 0 3 concentration, ppm References 

Delayed mucociliary [0.1] Grose et al. (1980) 

clearance; accelerated 0.4, 0.8, 1.0 Kenoyer et al. (1981) 

alveolar clearance, [0.5] Friberg et al. (1972) 

ciliary beating [0.5], 1.0 Abraham et al. (1980) 

frequency 0.8 Phalen et al. (1980) 

1.2 Frager et al. (1979) 

Inhibited bactericidal 0.4 Coffin and Gardner (1972b) 

activity 0.4 Goldstein et al. (1972b) 

0.5 Friberg et al. (1972) 

0.62 Goldstein et al. (1971b) 

0.7 Bergers et al. (1983) 

0.7 Warshauer et al. (1974) 

0.99 Goldstein et al. (1971a) 

Altered macrophage 0.1, 1.0 Gardner et al. (1971) 

membrane 0.5 Dowell et al. (1970) 

0.5 Hadley et al. (1977) 

0.5, 1.0 Goldstein et al. (1977) 

Decreased macrophage 0.25, 0.5 Hurst et al. (1970) 

function Hurst and Coffin (1971) 

0.5 Alpert et al. (197 ~b) 

0.5, 0.67 Coffin et al. (1968) 

0.5, 0.67 Coffin and Gardner (1972b) 

0.8 Schwartz and Christman (1979) 

1.0 Shingu et al. (1980) 

1.0 McAllen et al. (1981) 

Altered no. of defense 0.2 Plopper et al. (1979) 

cells 0.2, 0.35, 0.5, 0.8 Dungworth et al. (1975b) 

0.2, 0.35 Castleman et al. (1977) 

0.2, 0.5, 0.8 Boorman et al. (1977, 1980) 

0.25 Barry et al. (1983) 

0.5 Zitnik et al. (1978) 

0. 5, 0. e 88 Stephens et al .,(1974a) 

0.5 Last et al. (1979) 

0.5, 0.88 Brummer et al. (1977) 

0.5, 0.8 Eustis et al. (1981) 

0.54, 0.88 Freeman et al. (1974) 

0.8 Castleman et al. (1980) 

1.0 Freeman et al. (1973) 

1.0 Cavender et al. (1977) 

Increased suscepti- 0.08 Coffin et al. (1967) 

bility to infection 0.08, 0.1 Miller et al. (1978a) 

0.1 Ehrlich et al. (1977) 

9-242 
TABLE 9 - 32. SUMMARY TABLE: EFFECTS OF OZONE ON HOST DEFENSE 

MECHANISMS IN EXPERIMENTAL ANIMALS (continued) 

Effect/response 0 3 concentration, ppm References 

Increased suscepti- 0.1 Aranyi et al. (1983) 

biiity (cont'd) 0.1, 0.3 Illing et al. (1980) 

[0.2], 0.4, 0.7 Bergers et al. (1983) 

0.3 Abraham et al. (1982) 

0.5 Wolcott et al. (1982) 

[0.64] [Sherwood et al. (1984)] 

0.7, 0.9 Coffin and Blommer (1970) 

1.0 Thomas et al. (1981b) 

Altered immune 0.1 Aranyi et al. (1983) 

activity 0.5, 0.8 Osebold et al. (1979, 1980) 

0.5, 0.8 Gershwin et al. (1981) 

0.59 Campbell and Hilsenroth 

(1976) 

0.8 Fujimaki et al. (1984) 

9.6.3.5Tolerance . Examination of responses to short-term, repeated exposures 

to 0 3 clearly indicates that with some of the parameters measured, animals 

have an increased capacity to resist the effects of subsequent exposure. This 

tolerance persists for varying times, depending on the degree of development 

of the tolerance. Previous exposure to low concentrations of 0 3 will protect 

against the effects of subsequent exposure to lethal doses and the development 

of lung edema (Stokinger et al., 1956; Fairchild, 1967; Coffin and Gardn -er ; 

1972a; Chow, 1984). The prolongation of mucociliary clearance reported for 0 3 

can also be eliminated by pre-exposure to a lower concentration (Frager et 

al., 1979). This effect is demonstrated for a short period of time and is 

lost as soon as the mucus secretion rate returns to normal. However, not all 

of the toxic effects of 0 3 , such as reduced functioning activity of the pulmonary 

defense system (Gardner et al., 1972); hyperplasia of the type 2 cells (Evans a 

et al., 1971, 1976a,b); increased susceptibility to respiratory disease (Gardner 

and Graham, 1977); loss of pulmonary enzymatic activity (Chow, 1976, Chow 

et al., 1976b); and inflammatory response (Gardner et al., 1972) can be totally 

prevented by prior treatment with low levels of 0 3 . 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 0 3 exposures. 

These investigators suggest that during continuous exposure to 03 the injured 

9-243 
cells attempt to initiate early repair of the specific lesion. The repair 

phase results in a reduction of the effect first observed but lasts only for a 

short time since.the recovered cells are as sensitive to re-exposure to 0 3 as 

the pre-exposed counterpart (Plopper et al., 1978). This information is an 

important observation because it implies that the decrease in susceptibility 

to 03 persists only as long as the exposure to 0 3 continues. The biochemical 

studies of Chow et al. (1976b) support this conclusion. 

At this time, there are a number of hypotheses proposed to explain the 

mechanism of this phenomenon (Mustafa and Tierney, 1978; Schwartz et al., 

1976; Mustafa et al., 1977; Berliner et al., 1978; Gertner et al., 1983b; 

Bhatnagar et al., 1983). Evidence by Nambu and Yokoyama (1983) indicates that 

although the pulmonary antioxidant system (glutathione peroxidase, glutathione 

reductase, and glucose-6-phosphate dehydrogenase) may play an active role in 

defending the lung against ozone, it does not explain the mechanism of tolerance in that the development of tolerance does not coincide with the described 

biochemical enhancement of the antioxidant system in the lungs of rats. 

From this literature, it would appear that tolerance, as seen in animals, 

may not be the result of any one single biological process, but instead may 

result from a number of different events, depending on the specific response 

measured. Tolerance does not imply complete or absolute protection, because 

continuing injury does still occur, which could potentially lead to nonreversible pulmonary changes. 

Tolerance may not be long-lasting. During 0 3 exposure, the increase in 

antioxidant metabolism reaches a plateau and recovery occurs a few days after 

exposure ceases. Upon re-exposure, effects observed are similar to those that 

occurred during the primary exposure (Chow et al., 1976b). 

9.6.4Extrapulmonary Effects of Ozone 

It is still believed that 0 3 , on contact with respiratory system tissue, 

immediately reacts and thus is not absorbed or transported to extrapulmonary 

sites to any significant degree. However, several studies suggest that possibly 

products formed by the interaction of 0 3 and respiratory system fluids or 

tissue can produce effects in lymphocytes, erythrocytes, and serum, as well as 

in the parathyroid gland, the heart, the liver, and the CNS. Ozone exposure 

also produces effects on animal behavior that may be caused by pulmonary 

consequences of 0 3 , or by nonpulmonary (CNS) mechanisms. The mechanism by 

9-244 
which 03 causes extrapulmonary changes is unknown. Mathematical models of 03 

dosimetry predict that very iittle 0 3 penetrates to the blood of the alveolar 

capillaries. Whether these effects result from 0 3 or a reactio.n product of 0 3 

which penetrates to the blood and is transported is the subject of speculation. 

9.6.4.1. Central Nervous System and Behavioral Effects. Ozone significantly 

affects the behavior of rats during exposure to concentrations as low as 

235 pg/m3 (0.12 ppm) for 6 hr. With increasing concentrations of 0 3 , further 

decreases in unspecified motor activity and in operant learned behaviors have 

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 03 exposure concentrations (490 pg/m3, 0.25 ppm), an increase in activity is observed after exposure 

ends. Higher 0 3 concentrations (980 pg/m 3 , 0.5 ppm) produce a decrease in 

rodent activity that persists for several hours after the end of exposure 

(Tepper et al., 1982, 1983). 

The mechanism by which behavioral performance is reduced is unknown. 

Physically active responses appear to- enhance the effects of 0 3 , although this 

may be the result of an enhanced minute volume that increases the e,ffective 

concentration delivered to the lung. Several reports indicate that it is 

unlikely that_animals have reduced physiological capacity to respond, prompting Weiss et al. (1981) to.suggest that 03 impairs the inclination to respond. 

Two studies indicate that mice will respond to remove themselves from an 

atmosphere containing greater than 980 pg/m 3 (0.5 ppm) (Peterson and Andrews, 

1963, Tepper et al., 1983). These studies suggest that the aversive.effects 

of, 0 3 may be due to lung irritation. It is unknown whether lung.irritation, 

odor, or a direct effect on the CNS causes change in rodent behavior at lower 

0 3 concentrations. 

9.6.4.2 Cardiovascular Effects. Studies on the effects of 0 3 on,the cardiovascular system are few, and to date there are no reports of attempts to confirm these studies. The exposure of rats to'0 3 alone or in combination with . 

cadmium (1176 pg/m , 0.6 ppm 0 3 3 ) resulted in measurable increases in systolic 

pressure and heart rate (Revis et al., 1981). No additive or antagonistic 

response was observed with the combined exposure. Pulmonary capillary blood 

flow and Pa0 2 decreased 30 min following exposure of dogs to 588 pg/m3 (0.3 

ppm) of 03 (Friedman et al., 1983). The decrease in pulmonary capillary blood 

flow persisted for as long as 24 hr following exposure. 

9-245 
9.6.4.3 Hematological and Serum Chemistry Effects. The data base for the 

effects of 0 3 on the hematological system is extensive and indicates that 0 3 

or one of its reactive products can cross the blood-gas barrier, causing 

changes in the circulating erythrocytes (RBC) as well as significant differences in various components of the serum. 

Effects of 0 3 on the circulating RBCs can be readily identified by examining either morphological and/or biochemical endpoints. These cells are 

structually and metabolically well understood and are available through relatively 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 pg/m3 (0.75 ppm) of 0 3 4 hr/day for 

4 days (Clark et al., 1978). A single 4-hr exposure to 392 pg/m 3 (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 

following a 4-hr exposure to 1666 pg/m 3 (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 pg/m 3 (0.75 ppm) of 0 3 

for 4 days also had a decreased level of glutathione (GSH) and decreased 

acetylcholinesterase (AChE) activity, an enzyme bound to the RBC membranes. 

The RBC GSH activity remained significantly lower 4 days postexposure (Clark 

et al., 1978). 

Animals deficient in vitamin E are more sensitive to 0 3 . The RBCs from 

these animals, after being exposed to 0 3 , 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 pg/m 3 (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-64D), superoxide dismutase, 

or catalase activities. At a lower level (980 pg/m 3 , 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 Ng/m3 (0.5 ppm). These effects were not observed in vitamtn E-supplemented 

rats. Mice on a vitamin E-supplemented diet and those on a deficient diet 

9-246 
showed an increase in G-6-PD activity after an exposure of 627 }ag/m3 (0.32 ppm) 

of 0 3 for 6 hr. Decreases observed in AChE activity occurred in both groups 

(Moore et al., 1980). 

Other blood changes are attributed to 0 3 . Rabbits exposed for 1 hr to 

392 }ag/m3 (0.2 ppm) of 0 3 showed a significant drop in total blood serotonin 

(Veninga, 1967). Six- and 10-month exposures of rabbits to 784 pg/m 3 (0.4 ppm) 

of 0 3 produced an increase in serum protein esterase and in serum trypsin 

i nhi bi tor. Thi s l atter effect may be a resui t of thi ckeni ng of the smal 1 

pulmonary arteries. The same exposure caused a significant decrease in albumin 

levels and an increase in alpha and gamma globulins (P'an and Jegier, 1971, 

1976; P'an et al., 1972; Jegier, 1M). Chow et al. (1974) reported that the 

serum lysozyme level of rats increased significantly after 3 days of continuous 

exposure to 0 3 but was not affected when the exposure was intermittent (8 hr/day, 

7 days). The 03 concentration in both studies was 1568 pg/m 3 (0.8 ppm) of 0 3 . 

Short-term exposure to low concentrations of 03 induced an immediate 

change in the serum creatine phosphokinase level in mice. In this study, the 

0 3 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 3 have also been seen following in vitro exposure of RBCs from humans (Freeman 

and Mudd, 1981; Menzel et al., 1975b; Verweij and Van Steveninck, 1981). A 

common effect observed by a number of investigators.is that 0 3 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 Varr Steveninck, 

1980). It has been postulated that this inhibition of ATPase could be related 

to the spherocytosis and increased fragility of RBCs seen in animal and human 

cells. 

Although these in 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 3 reaching the RBC cannot be determined with any accuracy. 

9.6.4.4 Cytogenetic and Teratogenic Effects. Uncertainty still exists regarding possible reproductive, teratogenic, and mutational effects of exposure to 

ozone. Based on various in vitro data, a number of chromosomal effects of 

ozone have been described for isolated cultured cell lines, human lymphocytes, 

9-247 
and microorganisms (Fetner, 1962; Hamelin et al., 1977a,b, Hamelin and Chung, 

1975a,b, 1978; Scott and Lesher, 1963; Erdman and Hernandez, 1982; Guerrero 

et al., 1979; Dubeau and Chung, 1979, 1982). The interpretation, relevance, 

and predictive values of such studies to human health are questionable since 

(1) the concentrations used were many-fold greater than what is found in the 

ambient air (see Chapter 10); (2) extrapolation of in vitro exposure concentrations to human exposure dose is not yet possible; and (3) direct exposure 

of isolated cells to ozone is highly artifactual since it bypasses all the 

defenses of the host that would normally be functioning in protecting the 

individual from the inhaled gas. Furthermore, the direct exposure of isolated 

cells in yitro to ozone may result in chemical reactions between ozone and 

culture media that might not occur in vivo. 

Important questions still exist regarding in vivo cytogenetic effects of 

ozone in rodents and humans. Zelac et al. (1971a,b) reported chromosomal 

abnormalities in peripheral leukocytes of hamsters exposed to 0 3 (0.2 ppm). 

Combined exposures to ozone and radiation (227-233 rads) produced an additive 

effect on the number of chromosome breaks in peripheral leukocytes. These 

specific findings were not confirmed by Gooch et al. (1976) or by Tice et al. 

(1978), but sufficient differences in the various experimental protocols make 

a direct comparison difficult. The latter group did report significant increases 

in the number of chromatid deletions and achromatic lesions resulting from 

exposure to 0.43 ppm ozone. 

Because the volume of air inspired during pregnancy is significantly 

enhanced, the pregnant animal may be at greater risk to low levels of ozone 

exposure. Early studies on the possible teratogenic effects of ozone have 

suggested that exposures as low as 0.2 ppm can reduce infant survival rate and 

cause unlimited incisor growth (Brinkman et al., 1964; Veninga, 1967). Kavlock 

et al. (1979, 1980) found that pregnant rats exposed to 1.0 and 1.49 ppm ozone 

showed a significant increase in embryo resorption rate, slower growth, slower 

development of righting reflexes, and delayed grooming and rearing behavior, 

but no increase in neonatal mortality was observed. 

9.6.4.5 Other Extrapulmonary Effects. A series of studies was conducted to 

show that 03 increases drug-induced sleeping time in a number of species of 

animals (Gardner et al., 1974; Graham, 1979; Graham et al., 1981, 1982a,b, 

1983, 1985). At 1960 Ng/m 3 (1.0 ppm), effects were observed after 1, 2, and 3 

days of exposure. As the concentration of 03 was reduced, increasing numbers 

of daily 3-hr exposures were required to produce a significant effect. At the 

9-248 
lowest concentration studied (196 pg/m 3 , 0.1 ppm), the increase was observed 

at days 15 and 16 of exposure. It appears that this effect is not specific to 

the strai n of mouse or to the three speci es of ani mal s tested, but i t i s 

sex-specific, with females being more susceptible. Recovery was complete 

within 24 hr after exposure. Aithough a number of inechanistic studies have 

been conducted, the reason for this effect on pentobarbital-induced sleeping 

time is not known. It has been hypothesized that some common aspect related 

to liver drug metabolism is quantitatively reduced (Graham et al., 1983). 

Several investigators have attempted to elucidate the involvement of the 

endocrine system in 0 3 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 3 could be increased by use of various thyroid 

blocking agents or by thyroidectomy. To follow up these findings, Clemons and 

Garcia (1980a,b) and Clemons and Wei (1984) investigated the effects of a 

24-hr exposure to 1960 pg/m3 (1.0 ppm) of 0 3 on the hypothalamo-pituitary-thyroid 

system of rats. These three organs regulate the function of each other through 

various hormonal feedback mechanisms. Ozone caused decreases in serum concentration of thyroid stimulating hormone (TSH), in circulating thyroid hormones 

(T3 and T4 ) and in protein-bound iodine. No alterations were observed in many 

other hormone levels measured. d Thyroidectomy prevented the effect of 0 3 on 

TSH and T4 and hypophysectomy prevented effects on T 4 , unless the animals were 

supplemented with T4 in their drinking water. The thyroid gland itself was 

altered (e.g., edema).by -0 3 . The authors hypothesyzed that 0 3 alters serum 

binding of these hormones. 

The extrapulmonary effects of ozone in experimental animals are summarized 

in Figure 9-9 and Table 9-33. Criteria used in developing the summary were 

presented in Section 9.6.1. 

9.6.5 Interaction of Ozone With Other Pollutants. 

Combined exposure studies in laboratory animals have produced varied 

results, depending upon the pollutant combination evaluated and the measured 

variables. Additive and/or possibly synergistic effects of 0 3 exposure in 

combination with Na2 have been described for increased susceptibility to 

bacterial infection (Ehrlich et al., 1977, 1979; Ehrlich, 1980, 1983), morphological lesions (Freeman et al., 1974), and increased antioxidant metabolism 

(Mustafa et al., 1984). Additive or possibly synergistic effects from exposure 

9-249 
v Jm ~m ~pJ `~T ~ mG 

~ 

J ✓ 

O 

J~ 

a+J~ ~py m+I 

m ~J /dy J~ , O /O ✓p Ey 

~ Jm 

im ~2 

v 

E 

N 

~ 

'O 

7 

N 

~ 

O 

N C C 

.c *' eo 

o~ 

~ o 

o ~ 

N C 

C~ 

~ O 

d i 

~ 

OM 

OM 

E c~ 

7 01 

C.:E e0 b. X ~ 

a~ o 

~ 4' 00 ~0 ~0 

E 

~+ O 

Q~ ~ ~w 

V 

~ •~ G 

o~aGE 

LL ~ y 

wdd •uoijei;uaauoa auozp 

9-250! 
TABLE 9-33. SUMMARY TABLE: EXTRAPULMONARY EFFECTS OF OZONE 

IN EXPERIMENTAL ANIMALS 

Effect/response 03 concentration, ppm References 

CNS effects 0.05, 0.5 Konigsberg and Bachman (1970) 

0.1 - 1.0 Weiss et al. (1981) 

0.12 - 1.0 Tepper et al. (1982) 

0.2, 0.3, 0.5, 0.7 Murphy et al. (1964) 

0.5 Tepper et al. (1983) 

0.5 Reynolds and Chaffee (1970) 

0.5 Xintaras et al. (1966) 

0.6 Peterson and Andrews (1963) 

1.0 Fietcher and Tappel (1973) 

1.0 Trams et al. (1972) 

Hematological effects 0.06, 0.12, 0.48 Calabrese et al. (1983a) 

0.2 Brinkman et al. (1964) 

0.2, 1.0 Veninga (1967, 1970) 

Veninga et al. (1981) 

0.25, 0.32, 0.5 Moore et al. (1980; 1981a,b) 

0.4 Jegier (1973) 

0.4 P'an and Jegier (1972, 1976) 

0.5 Menzel et al. (1972) 

0.64 Larkin et al. (1983) 

0.75 Clark et al. (1978) 

0.8 Chow and Kaneko (1979) 

0•8 Chow et al. (1974) 

0.85 Menzel et al. (1975a) 

0.86 Schlipkoter and Bruch (1973) 

1.0 Dorsey et al. (1983) 

1.0 Mizoguchi et al. (1973) 

1.0 Christiansen and Giese (1954) 

Chromosomal, reproduc- 0.1 Brinkman et al. (1964) 

tive, teratological 0.2 Veninga (1967) 

effects 0.24, 0.3 Zelac et al. (1971a) 

0.43 Tice et al. (1978) 

0.44 Kavlock et al. (1979) 

1.0 Kavlock et al. (1980) 

Liver effects 0.1, 0.25, 0.5, 1.0 Graham (1979) 

Graham et al. (1981, 1982a,b) 

0.82 Veninga et al. (1981) 

1.0 Gardner et al. (1974) 

Endocrine system 0.75 Atwal and Wilson (1974) 

effects 0.75 Atwal et al. (1975) 

0.75 Atwal and Pemsingh (1981, 1984) 

0.75 Pemsingh and Atwal (1983) 

1.0 Clemons and Garcia (1980a,b) 

1.0 Clemons and Wei (1984) 

9-251 
to 03 and H2SO4 have also been reported for host defense mechanisms (Gardner 

et al., 1977; Last and Cross, 1978; Grose et al., 1982), pulmonary sensitivity 

(Osebold et al. 1980), and collagen synthesis (Last et al., 1983), but not 

for morphology (Cavender et al., 1977; Moore and Schwartz, 1981). Mixtures of 

03 and (NH4) 2 SO4 had synergistic effects on collagen synthesis and morphometry, 

including percentage of fibroblasts (Last et al., 1983, 1984a). 

Combining 03 with other particulate pollutants produces a variety of 

responses, depending on the endpoint measured. Mixtures of 03, Fe2(SO4)3' 

H2504 , and (NH4) 2SO4 produced the same-effect on clearance rate as exposure to 

03 alone. However, when measuring changes in host defenses, the combinat;on 

of 03 with NO2 and ZnSO4 or 03 with S02 and (NH4 ) 2SO4 produced enhanced effects 

that can not be attributed to 03 only. 

However, since these issues are complex, they must be addressed experimentally using exposure regimens for combined pollutants that are more representative of ambient ratios of peak concentrations, frequency, duration, and time 

intervals between events. 

The interactive effects of 03 with other pollutants are summarized in 

Figure 9-10 and Table 9-34. 

9.6.6 Effects of Other Photochemical Oxidants 

There have been far too few controlled toxicological studies with the 

other oxidants to permit any sound scientific evaluation of their contribution 

to the toxic action of photochemical oxidant mixtures. When the effects seen 

after exposure to 0 3 and PAN are examined and compared, it is obvious that the 

test animals must be exposed to concentrations of PAN much greater than those 

needed with 03 to produce a similar effect on lethality, behavior modification, 

morphology, or significant alterations in host pulmonary defense system (Campbell 

et al., 1967; Dungworth et al., 1969; Thomas et al., 1979, 1981a). The concentrations of PAN required to produce these effects are many times greater than 

what has been measured in the atmosphere (0.047 ppm). 

Similarly, most of the investigations reporting H2O 2 toxicity have involved 

concentrations much higher than those found in the ambient air, or the investigations were conducted by using various in vitro techniques for exposure. Very 

limited information is available on the health significance of inhalation exposure to gaseous H2O 2 . Because H2O2 is highly soluble, it is generally assumed 

that it does not penetrate into the alveolar regions of the lung but is instead 

9-252 
d m 

tA 

~ N ~ 

m~ 

N ~ 

0 

C~ 

N ~ 

~ N 

m 0 

a y 

m 

~ ~ N 

~ ~ t 

0 00 

~ g' •N 

N ~ ~ 

®~ E 

® E 

s 

E ~3 ~ 

E~~ 

N ~ ~ 

® E ® 

~ v c L N O 

~ C ~ 

m PO4 ~+ 

U. ® tS 

0 ---------- 0-C ---------- 0 

• 

C3 

® 

0-------- 0---- 

~ 

Zo +tOr'~~yJ 

d d 

J'GO~ rO~J 

.it v 

~ • 9'r`i~v ~I 

d~ 

G ~ J O~ 

O~ OJ ~ 

a rJ'OOJ► 

O G 

.09~ 

GO O~ •~ ~O 

~i G~0 Ot' Gv 

• ~i O~ `i O,l 

. O 

! . •~J1G0~ 

ca 

~ 

0 G GO~~ 

~ O i 

.o 

Zio. di,~ 

o m 

0 

~9~ s 

O~ 

Or JG 

1 

tV M tt tA 00 Gy ® 

p O C O O O O O O ® r 

wdd •uopei;uaauoo auozp 

9-253 
TABLE 9-34. SUMMARY TABLE: INTERACTION OF OZONE 

WITH OTHER POLLUTANTS IN EXPERIMENTAL ANIMALS 

Effect/response Pollutant concentrations References 

Increased [0.25 ppm 03 Freeman et al. (1974) 

pulmonary + 2.5 ppm NO2 1 

lesions [0.5 ppm 0 3 Moore and Schwartz (1981) 

+ 1 mg/m3 H2 SO4 1 

[0.5 ppm 03 Cavender et al. (1978) 

+ 10 mg/m3 H2 SO4 

0.64, 0.96 ppm 0 3 Last et al. (1984a) 

+ 5 mg/m3 (NH4)2 SO4 

0.9 ppm 03 Freeman et al. (1974) 

+ 0.9 ppm NO2 

1.2 ppm 03 Last et al. (1983) 

+ 5 mg/m3 (NH4)2SO4 

Increased 0.5 ppm 03 Osebold et al. (1980) 

pulmonary + 1 mg/m3 H2 SO4 

sensitivity 

Increased anti- 0.45 ppm 03 Mustafa et al. (1984) 

oxidant metabolism + 4.8 ppm NO2 

and 02 consumption 

Altered mucus 0.5 ppm 03 Last and Cross (1978); 

secretion + 1.1 mg/m3 H2 SO4 Last and Kaizu (1980) 

Increased collagen [0.51, [0.81, 1.5 ppm 03 Last et al. (1983) 

synthesis + 5 mg/m3 (NH4 ) 2 SO4 

0.5 ppm 03 Last et al. (1983) 

+ 1 mg/m3 H2 SO4 

0.64, 0.96 ppm 0 3 Last et al. (1984a) 

+ 5 mg/m3 (NH4 ) 2 SO4 

Increased 0.05 ppm 03 Ehrlich et al. (1977, 1979); 

susceptibility to + 3760 pg/m3 (NH4) 2 SO4 Ehrlich (1980) 

respiratory 0.05 ppm 03 Ehrlich et al. (1983) 

infections + 100-400 pg/m3 NO2 

+ 1.5 mg/m3 ZnSO4 

0.1 ppm 03 Gardner et al. (1977) 

+ 0.9 mg/m3 H2 SO4 

(sequential exposure) 

0.1 ppm 03 Grose et al. (1982) 

+ 4.8 mg/m3 H2 SO4 

0.1 ppm 03 Ehrlich (1980) 

+ 940 Ng/m3 NO2 

0.1 ppm 03 Aranyi et al. (1983) 

+ 13.2 mg/m3 S02 

+ 1.0 mg/m3 (NH4)2SO4 

9-254 
TABLE 9-34. SUMMARY TABLE: INTERACTION OF OZONE 

WITH OTHER POLLUTANTS (continued) 

Effect/response Pollutant concentrations References 

Altered upper 

respiratory 

clearance 

mechanisms 

[0.1 ppm 03 

+ 1.1 mg/m3 H2SO4 1 

(sequential exposure) 

0.4 ppm 03 

+ 7.0 ppm NO 2 

0.5 ppm 0 3 

+ 3 mg/m3 H2 SO4 

[0.8 ppm 03 

+ 3.5 mg/m3 

[Fe2 (SO4 )3 

• H2SO4 

• (NH4)2SO411 

Grose et al. (1980) 

Goldstein et al. (1974b) 

Last and Cross (1978) 

Phalen et al. (1980) 

deposited on the surface of the upper airways (Last et al., 1982). Unfortunately, there have not been studies designed to look for possible effects in 

this region of the respiratory tract. 

A few in vitro studies have reported cytotoxic, genotoxic, and biochemical 

effects of H2O2when using isolated cells or organs (Stewart et al., 1981; 

Bradley et al., 1979; Bradley and Erickson, 1981; Speit et al., 1982; MacRae 

and Stich, 1979). Although these studies can provide useful data for studying 

possible mechanisms of action, it is not yet possible to extrapolate these 

responses to those that might occur in the mammalian system. 

Field and epidemiological studies have shown that human health effects 

from exposure to ambient mixtures of oxidants and other airborne pollutants 

can produce human health effects (Chapter 11). Few such studies have been 

conduc.ted with laboratory animals, because testing and measuring of such 

mixtures is not only complicated, but extremely costly. In these studies, the 

investigators attempted to simulate the photochemical reaction products produced under natural conditions and to define the cause-effect relationship. 

Exposure to complex mixtures of oxidants plus the various components 

found in UV-irradiated auto exhaust indicates that certain effects, such as 

histopathological changes, increase in susceptibility to infection, a variety 

of altered pulmonary functional activities were observed in this oxidant 

atmosphere which was not reported in the nonirradiated exhaust (Murphy et al., 

9-255 
1963; Murphy, 1964; Nakajima et 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, fertility, and certain pulmonary functional abnormalities (Hueter et al., 1966; 

Boche and Quilligan, 1960; Lewis et al., 1967). 

Dogs exposed to UV-irradiated auto exhaust containing oxidants either 

with or without SOx showed significant pulmonary functional abnormalities that 

had relatively good correlation with structural changes (Hyde et al., 1978; 

Gillespie, 1980; Lewis et al., 1b74). There were no significant differences 

in the magnitude of the response in these two treatment groups, indicating 

that oxidant gases and SO xdid not interact in any synergistic or additive 

manner. 

9-256 
9.7 REFERENCES 

Abraham, W. M..; Januszkiewicz, A. J.; Mingle, M.; Welker, M.; Wanner, A.; 

Sackner, M. A. (1980) Sensitivity of bronchoprovocation and tracheal 

mucous velocityin detecting airway responses to 0 3 . J. Appl. Physiol.: 

Respir. Environ. Exercise Physiol. 48: 789-793. 

Abraham, W. M.; Lauredo, I.; Sielczak, M.; Yerger, L.; King, M. M.; Ratzan, K. 

(1982) Enhancement of bacterial pneumonia in sheep by ozone exposure. Am. 

Rev. Respir. Dis. Suppl. 125(4 pt. 2): 148. 

Abraham, W.; Chapman, G. A.; Marchette, B. (1984a) Differences between inhaled 

and intravenous carbachol in detecting 0 3 -induced airway effects. Environ. 

Res. 35: 430-438. 

Abraham, W. M.; Delehunt, J. C.; Yerger, L.; Marchette, B.; Oliver; W., Jr. 

(1984b) Changes in airway permeability and responsiveness after exposure 

to ozone. Environ. Res. 34: 110-119. 

Aharonson, E. F.; Menkes, H.; Gurtner, G.; Swift, D. L.; Proctor, D. F. (1974) 

Effect of respiratory airflow rate on removal of soluble vapors by the 

nose. J. Appl. Physiol. 37: 654-657. 

Altman, P. L.; Dittmer, D. S., eds. (1971) Lung volumes and other ventilatory 

variables during pregnancy: man. In: Respiration and circulation. Bethesda, 

MD: Federation of American Societies for Experimenta1 Biology; pp. 61-63. 

Alpert, S. M.; Lewis, T. R. (1971) Ozone tolerance studies uti1izing unilateral lung exposure. J. Appl. Physiol. 31: 243-246.- 

Alpert, S. M.; Schwartz, B. B.; Lee, S. D.; Lewis, T. R. (1971a) Alveolar 

protein accumulation: a sensitive indicator of low level oxidant toxicity. Arch. Intern. Med. 128: 69-73. 

Alpert, S. M. ; Gardner, D. E. ; Hurst, D. J. ; Lewis, T. R. ; Coffin, D. L. 

(1971b) Effects of exposure to ozone on defensive mechanisms of the lung. 

J. Appl. Physiol. 31: 247-252. 

Amdur, M. 0.; Ugro, V.; Underhill, D. W. (1978) Respiratory response of guinea 

pigs to ozone alone and with sulfur dioxide. Am. Ind. Hyg. Assoc. J. 39: 

958-961. 

American Thoracic Society. (1962) Chronic bronchitis, asthma, and pulmonary 

emphysema. Am. Rev. Respir. Dis. 85: 762-768. 

Ames, B. N.; McCann, J.; Yamasaki, E. (1975) Methods for detecting carcinogens 

and mutagens with the Salmonella/mammalian- microsome mutagenicity test. 

Mutat. Res. 31: 347-364. 

Amoruso, M. A.; Witz, G.; Goldstein, B. D. (1981) Decreased superoxide anion 

radical production by rat alveolar macrophages following inhalation of 

ozone or nitrogen dioxide. Life Sci. 28: 2215-2221. 

9-257 
Aranyi, C.; Fenters, J.; Ehrlich, R.; Gardner, D. (1976) Scanning electron 

microscopy of alveolar macrophages after exposure to oxygen, nitrogen 

dioxide, and ozone. EHP Environ. Health Perspect. 16: 180. 

Aranyi, C.; Vana, S. C.; Thomas, P. T.; Bradof, J. N.; Fenters, J. D.; Graham, 

J. A.; Miller, F. J. (1983) Effects of subchronic exposure to a mixture 

of 03, S02 , and (NH4 ) 2 SO4 on host defenses of mice. J. Toxicol. Environ. 

Health 12: 55-71. 

Astarita, G. (1967) Mass transfer with chemical reaction. New York, NY: Elsevier 

Publishing Co.; p. 187. 

Atwal, 0. S.; Pemsingh, R. S. (19h) Morphology of microvascular changes and 

endothelial regeneration in experimental ozone-induced parathyroiditis. 

III. Some pathologic considerations. Am. J. Pathol. 102: 297-307. 

Atwal, 0. S.; Pemsingh, R. S. (1984) Occurrence of mallory body-like inclusions 

in parathyroid chief cells of ozone-treated dogs. J. Pathol. 142: 169-174. 

Atwal, 0. S.; Wilson, T. (1974) Parathyroid gland changes following ozone 

inhalation: a morphologic study. Arch. Environ. Health 28: 91-100.' 

Atwal, 0. S.; Samagh, B. S.; Bhatnagar, M. K. (1975) A possible autoimmune 

parathyroiditis following ozone inhalation. II. A histopathologic, ultrastructural, and immunofluorescent study. Am. J. Pathol. 80: 53-68. 

Bailey, P.S. (1978) Ozonization in organic chemistry. V. 1, olefinic compounds. 

New York: Academic Press. 

Ballew, M.; Calabrese, E. J.; Moore, G. S. (1983) The effect of dietary vitamin I 

on ozone-induced oxidative changes in guinea pig erythrocytes. J. Environ. 

Sci. Health A18: 597-610. 

Barr, B. C. (1984) Terminal airway remodeling in chronic ozone exposure. Diss. 

Abstr. Int. 44: 3724. 

Barry, B. E.; Miller, F. J.; Crapo, J. D. (1983) Alveolar epithelial injury 

caused by inhalation of 0.25 ppm of ozone. In: Lee, S. D.; Mustafa, 

M. G.; Mehlman, M. A., eds. International symposium on the biomedical 

effects of ozone and related photochemical oxidants; March 1982; Pinehurst, 

NC. Princeton, NJ: Princeton Scientific Publishers, Inc; pp. 299-309. 

(Advances in modern environmental toxicology: v. 5). 

Bartlett, D., Jr.; Faulkner, C. S. II; Cook, K. (1974) Effect of chronic ozone 

' exposure on lung elasticity in young rats. J. App1. Physiol. 37: 92-96. 

Bergers, W. W. A.; Gerbrandy, J. L. F.; Stap, J. G. M. M.; Dura, E. A. (1983) 

Influence of air polluting components viz ozone and the open air factor 

on host-resistance towards respiratory infection. In: Lee, S. D.; Mustafa, 

M. G.; Mehlman, M. A., eds. International symposium on the biomedical effects of ozone and related photochemical oxidants; March 1982; Pinehurst, 

NC. Princeton, NJ: Princeton Scientific Publishers, Inc.; pp. 459-467. 

(Advances in modern environmental toxicology: v. 5). 

9-258 
Berliner, J. A.; Kuda, A.; Mustafa, M. G.; Tierney, T. F. (1978) Pulmonary 

morphologic studies of ozone tolerance in the rat (a possible mechanism 

for tolerance). Scanning Electron Microsc. 2: 879-884. 

Bhatnagar, R. S.; Hussain, M. Z.; Sorensen, K. R.; Mustafa, M. G.; von Dohlen, 

F. M.; Lee, S. D. (1983) Effect of ozone on lung collagen biosynthesis. 

In: Lee, S. D.; Mustafa, M. G.; Mehlman, M. A., eds. International 

symposium on the biomedical effects of ozone and related photochemical 

oxidants; March 1982; Pinehurst, NC. Princeton, NJ: Princeton 

Scientific Publishers, Inc.; pp. 311-321. (Advances in modern environmental toxicology: v. 5). 

Bils, R. F. (1970) Ultrastructural alterations of alveolar tissue of mice. 

III. Ozone. Arch. Environ. Health 20: 468-480. 

Bloch, W. N., Jr.; Miller, F. J.; Lewis, T. R. (1971) Pulmonary hypertension 

in dogs exposed to ozone. Cincinnati, OH: U.S. Environmentai Protection 

Agency, Air Pollution Control Office. 

Bloch, W. N., Jr.; Lewis, T. R.; Busch, K. A. Orthoefer, J. G.; Stara, J. F. 

(1972) Cardiovascular status of female beagles exposed to air pollutants. 

Arch. Environ. Health 24: 342-353. 

Bloch, W. N., Jr.; Lassiter, S.; Stara, J. F. Lewis, T. R. (1973) Blood 

rheology of dogs chronically exposed to air pollutants. Toxicol. Appl. 

Pharmacol. 25: 576-581. 

Boatman, E. S. Sato, S.; Frank, R. (1974) Acute effects of ozone on cat 

lungs. II. Structural. Am. Rev. Respir. Dis. 110: 157-169. 

Boatman, E. S. Ward, G.; Martin, C. J. (1983) Morphometric changes in rabbit 

lungs before and after pneumonectomy and exposure to ozone. J. Appl. 

Physiol.: Respir. Environ. Exercise Physiol. 54: 778-784. 

Boche, R. D.; Quilligan, J. J., Jr. (1960) Effects of synthetic smog on spontaneous activity of mice. Science (Washington, DC) 131: 1733-1734. 

Boorman, G. A.; Schwartz, L. W.; McQuillen, N. K.; Brummer, M. E. G. (1977) 

Pulmonary response following long-term intermittent exposure to ozone: 

structural and morphometric changes. Am. Rev. Respir. Dis. 115: 201. 

Boorman, G. A.; Schwartz, L. W.; Dungworth, D.. L. (1980) Pulmonary effects of 

prolonged ozone insult in rats: morphometric evaluatiun of the central 

acinus. Lab. Invest. 43: 108-115. 

Boushey, H. A.; Holtzman, M. J.; Sheller, J. R.; Nadel, J. A. (1980) Bronchial 

hyperreactivity. Am. Rev. Respir. Dis. 121: 389-413. 

Bowes, C.; Cumming, G.; Horsfield, K.; Loughhead, J.; Preston, S. (1982) Gas 

mixing in a model of the pulmonary acinus with asymmetrical alveolar ducts. 

J. Appl. Physiol.: Respir. Environ. Exercise Physiol. 52: 624-633. 

9-259 
Bradley, M. 0.; Erickson, L. C. (1981) Comparison of the effects of hydrogen 

peroxide and X-ray irradiation on toxicity, mutation, and DNA damage/ 

repair in mammalian cells (V-79). Biochim. Biophys. Acta 654: 135-141. 

Bradley, M. 0.; Hsu, I. C.; Harris, C. C. (1979) Relationships between sister 

chromatid exchange and mutagenicity, toxicity, and DNA damage. Nature 

(London) 282: 318-320. 

Brain, J. D. (1980) Macrophage damage in relation to the pathogenesis of lung 

diseases. EHP Environ. Health Perspect. 35: 21-28. 

Brain, J. D.; Sorokin, S. P.; Godleski, J. J. (1977) Quantification, origin, 

and fate of pulmonary macrophages. In: Brain, J. D.; Proctor, D. F.; 

Reid, C. M., eds. Respiratory defense mechanisms. New York, NY: Marcel 

Dekker, Inc.; pp. 849-891 (Lung biology in health and disease: v. 5). 

Brain, J. D.; Golde, D. W.; Green, G. M.; Massaro, D. J., Valberg, P. A.; 

Ward, P. A.; Werb, Z. (1978) Biological potential of pulmonary macrophages. 

Am. Rev. Respir. Dis. 118: 435-443. 

Brinkman, R.; Lamberts, H. B.; Veninga, T. S. (1964) Radiomimetic toxicity of 

ozonised air. Lancet (7325): 133-136. 

Brummer, M. E. G.; Schwartz, L. W.; McQuillen, N. K. (1977) A quantitative 

study of lung damage by scanning electron microscopy: inflammatory cell 

response to high-ambient levels of ozone. Scanning Electron Microsc. 2: 

513-518. 

Byers, D. H.; Saltzman, B. E. (1959) Determination of ozone in air by neutral 

and alkaline iodide procedures. In: Ozone chemistry and technology: 

proceedings of the first international ozone conference; November 1956; 

Chicago, IL. Washington, DC: American Chemical Society; pp. 93-101. 

(Advances in chemistry series: v. 21). 

Calabrese, E. J.; Moore, G. S. (1980) Does the rodent model adequately predict 

the effects of ozone induced changes to human erythrocytes? Med. Hypotheses 6: 505-507. 

Calabrese, E. J.; Moore, G.; Brown, R. (1979) Effects of environmental oxidant 

stressors on individuals with a G-6-PD deficiency with particular reference 

to an animal model. EHP Environ. Health Perspect. 29: 49-55. 

Calabrese, E. J.; Moore, G. S.; Williams, P. (1982) Effect of inethyl oleate 

ozonide, a possible ozone intermediate, on normal and G-6-PD deficient 

erythrocytes. Bull. Environ. Contam. Toxicol. 29: 498-504. 

Calabrese, E. J.; Moore, G. S.; Grunwald, E. L. (1983a) Ozone-induced decrease 

of erythrocyte survival in adult rabbits. In: Lee, S. D.; Mustafa, M. G.; 

Mehlman, M. A., eds. International symposium on the biomedical effects of 

ozone and related photochemical oxidants; March 1982; Pinehurst, NC. 

Princeton, NJ: Princeton Scientific Publishers, Inc.; pp. 103-117. (Advances in modern environmental toxicology: v. 5). 

9-260 
Caiabrese, E. J.; Moore, G. S.; Williams, P. S. (1983b) An evaluation of the 

Dorset sheep as a predictive animal model for the response of G-6-PD 

deficient human erythrocytes to a proposed systemic toxic ozone intermediate, methyl oleate hydroperoxide. Vet. Hum. Toxicol. 25: 241-246. 

Caiabrese, E. J.; Moore, G. S.; Weeks, B. L.; Stoddard, A. (1983c) The effect 

of ozone exposure upon hepatic and serum ascorbic acid levels in male 

Sprague-Dawley rats. J. Environ. Sci. Health A18: 79-93. 

Calabrese, E. J.; Williams, P. S.; Moore, G. S. (1983d) An evaluation of the 

Dorset sheep as a predictive animal model for the response of glucose-6- 

phosphate dehydrogenase-deficient human erythrocytes to a proposed systemic 

toxic ozone intermediate, methyl oleate ozonide. Ecotoxicol. Environ. 

Safety 7: 416-422. 

Campbell, K. I.; Hilsenroth, R. H. (1976) Impaired resistance to toxin in 

toxoid-immunized mice exposed to ozone or nitrogen dioxide. Clin. Toxicol. 

9: 943-954. 

Campbel 1, K. I.; C1 arke, G. L. ; Emi k, L. 0. ; Pl ata, R. L. (1967) The atmospheric contaminant peroxyacetyl nitrate. Arch. Environ. Health 15: 739-744. 

Campbel l, K. I. ; Emik, L. 0. ; Ciarke, G. L. ; Plata, R. L. (1970) Inhalation 

toxicity of peroxyacetyl nitrate: depression of voluntary activity in 

mice. Arch. Environ. Health 20: 22-27. 

Carp, H.; Janoff, A. (1980) Inactivation of bronchial mucous proteinase inhibitor by cigarette smoke and phagocyte-derived oxidants. Exp. Lung Res. 

1: 225-237. 

Case, G. D.; Dixon, J. S.; Schooley, J. C. (1979) Interactions of blood metalloproteins with nitrogen oxides and oxidant air pollutants. Environ. Res. 

20: 43-65. 

Castleman, W. L.; Dungworth, D. L.; Tyler, W. S. (1973a) Cytochemically detected alterations of lung acid phosphatase reactivity following ozone 

exposure. Lab. Invest. 29: 310-319. 

Castleman, W. L.; Dungworth, D. L.; Tyler, W. S. (1973b) Histochemically 

detected enzymatic alterations in rat lung exposed to ozone. Exp. Mol. 

Pathol. 19: 402-421. 

Castleman, W. L.; Dungworth, D. L.; Tyler, W. S. (1975) Intrapulmonary airway 

morphology in three species of monkeys: A correlated scanning and transmission electron microscopic study. Am. J. Anat. 142: 107-121. 

Castleman, W. L.; Tyler, W.. S.; Dungworth, D. L. (1977) Lesions in respiratory 

bronchioles and conducting airways of monkeys exposed to ambient levels 

of ozone. Exp. Mol. Pathol. 26: 384-400. 

Castleman, W. L.; Dungworth, D. L.; Schwartz, L. W.; Tyler, W. S. (1980) Acute 

respiratory bronchiolitis: an ultrastructural and autoradiographic study 

of e.pithelial cell injury and renewal in rhesus monkeys exposed to ozone. 

Am. J. PathoI. 98: 811-840. 

9-261 
Cavender, F. L.; Steinhagen, W. H.; Ulrich, C. E.; Busey, W. M.; Cockrell, B. 

Y.; Haseman, J. K.; Hogan, M. D.; Drew, R. T. (1977) Effects in rats and 

guinea pigs of short-term exposures to sulfuric acid mist, ozone, and 

their combination. J. Toxicol. Environ. Health 3: 521-533. 

Cavender, F. L.; Singh, B.; Cockrell, B. Y. (1978) Effects in rats and guinea 

pigs of six-month exposures to sulfuric acid mist, ozone, and their 

combination. J. Toxicol. Environ. Health 4: 845-852. 

Chan, P. C.; Kindya, R. J.; Kesner, L. (1977)+Stulies on the mechanism of ozone 

inactivation of erythrocyte membrane (Na + K)-activated ATPase. J. Biol. 

Chem. 252: 8537-8541. 

Chaney, S. G. (1981) Effects of ozone on leukocyte DNA. Research Triangle 

Park, NC: U.S. Environmental Protection Agency, Health Effects Research 

Laboratory; EPA report no. EPA-600-81-031. Available from: NTIS, 

Springfield, VA; PB81-179277. 

Chang, H.-K.; Farhi, L. E. (1973) On mathematical analysis of gas transport in the 

lung. Respir. Physiol. 18: 370-386. 

Chiappino, G.; Vigliani, E. C. (1982) Role of infective, immunological, and 

chronic irritative factors in the development of silicosis. Br. J. Ind. 

Med. 39: 253-258. 

Chow, C. K. (1976) Biochemical responses in lungs of ozone-tolerant rats. 

Nature (London) 260: 721-722. 

Chow, C. K. (1983) Influence of dietary vitamin E on susceptibility to ozone 

exposure. In: Lee, S. D., Mustafa, M. G.; Mehlman, M. A., eds. International symposium on the biomedical effects of ozone and related photochemical oxidants; March 1982; Pinehurst, NC. Princeton, NJ: Princeton 

Scientific Publishers, Inc.; pp. 75-93. (Advances in modern environmental 

toxicology: v. 5). 

Chow, C. K. (1984) Glutathione peroxidase system in the lungs of ozone-tolerant 

and non-tolerant rats. In: Bors, W.; Saran, M.; Tait, D., eds. Oxygen 

radicals in chemistry and biology; proceedings of.the third international 

conference; July 1983; Neuherberg, West Germany. Berlin, West Germany: 

Walter de Gruyter & Co.; pp. 707-712. 

Chow, C. K.; Kaneko, J. J. (1979) Influence of dietary vitamin E on the red , 

cells of ozone-exposed rats. Environ. Res. 19: 49-55. 

Chow, C. K.; Tappel, A. L. (1972) An enzymatic protective mechanism against 

lipid peroxidation damage to lungs of ozone-exposed rats. Lipids 7: 

518-524. 

Chow, C. K.; Tappel, A. L. (1973) Activities of pentose shunt and glycolytic 

enzymes in lungs of ozone-exposed rats. Arch. Environ. Health 26: 205-208. 

Chow, C. K.; Dillard, C. J.; Tappel, A. L. (1974) Glutathione peroxidase 

system and lysozyme in rats exposed to ozone or nitrogen dioxide. Environ. 

Res. 7: 311-319. 

9-262 
Chow, C. K.; Mustafa, M. G.; Cross, C. E.; Tarkington, B. K. (1975) - Effects of 

ozone exposure on the lungs and the erythrocytes of rats and monkeys: 

relative biochemical changes. Environ. Physiol. Biochem. 5: 142-146. 

Chow, C. K.; Aufderheide, W. M.; Kaneko, J. J.; Cross, C. E.; Larkin, E. C. 

(1976a) Effect of dietary vitamin E on the red blood cells of ozone-exposed 

rats. Fed. Proc. Fed. Am. Soc. Exp. Biol. 35: 741. 

Chow, C. K.; Hussain, M. Z.; Cross, C. E.; Dungworth, D. L. Mustafa, M. G. 

(1976b) Effect of low levels of ozone on rat lungs. I. Biochemical responses during recovery and reexposure. Exp. Mol. Pathol. 25: 182-188. 

Chow, C. K.; Cross, C. E.; Kaneko, J. J. (1977) Lactate dehydrogenase activity 

and isoenzyme pattern in lungs, erythrocytes, and plasma of ozone-exposed 

rats and monkeys. J. Toxicol. Environ. Health 3: 877-884. 

Chow, C. K.; Plopper, C. G.; Dungworth, D. L. (1979) Influence of dietary 

vitamin E on the lungs of ozone-exposed rats: a correlated biochemical 

and histological study. Environ. Res. 20: 309-317. 

Chow, C. K.; Plopper, C. G.; Chiu, M.; Dungworth, D. L. (1981) Dietary vitamin E 

and pulmonary biochemical and morphological alterations of rats exposed 

to 0.1 ppm ozone. Environ.'Res. 24: 315-324. 

Christensen, E.; Giese, A. C. (1954) Changes in absorption spectra of nucl.eic 

acids and their derivatives following exposure to ozone and ultraviolet 

radiation. Arch. Biochem. Biophys. 51: 208-216. 

Christman, C. A. Schwartz, L. W. (1982) Enhanced phagocytosis by alveolar 

macrophages induced by short-term ozone insult. Environ. Res. 28: 241-250. 

Clark, R. A.; Klebanoff, S. J. (1975) Neutrophil-mediated tumor cell cytotoxicity: role of the peroxidase system. J. Exp. Med. 141: 1442-1447. 

C1 ark, K. W. ; Posi n, C. I. ; Buckl ey, R. D. (1978) Bi ochemi cal , response of 

squirrel monkeys to ozone. J. Toxicol. Environ. Health 4: 741-753. 

Claxton, L. D.; Barnes, H. M. (1981) The mutagenicity of diesel-exhaust 

particle extracts collected under smog-chamber conditions using the 

Salmonella typhimurium test system. Mutat. Res. 88: 255-272. 

Clemons,: G. K.; Garcia, J. F. (1980a) Endocrine aspects of ozone exposure in 

.rats...._ Arch. .,.Toxi col_. .. Suppl ._... (4) :,_ .301.-304,,._. 

Clemons, G. K.; Garcia, J. F. (1980b) Changes in thyroid function after shortterm ozone exposure in rats. J. Environ. Pathol. Toxicol. 4: 359-369. 

Clemons, G. K.; Wei, D. (1984) Effect of short-term ozone exposure on exogenous 

thyroxine levels in thyroidectomized and hypophysectomized rats. Toxicol. 

Appl. Pharmacol. 74: 86-90. 

9-263 
Coffin, D. L.; Blommer, E. J. (1970) Alteration of the pathogenic role of 

streptococci group C in mice conferred by previous exposure to ozone. In: 

Silver, I. H., ed. Aerobiology: proceedings of the third international 

symposium; September 1969; Sussex, United Kingdom. New York, NY: Academic 

Press, Inc.; pp. 54-61. 

Coffin, D. L.; Gardner, D. E. (1972a) The role of tolerance in protection of 

the lung against secondary insults. In: MEDICHEM: first international 

symposium of occupational physicians of the chemical industry; April; 

Ludwigshafen, West Germany. Ludwigshafen, West Germany: Badische 

Anilin-und Soda-Fabrik (BASF); pp. 344-364. 

Coffin, D. L.; Gardner, D. E. (1972b) Interaction of biological agents and 

chemical air pollutants. Ann. Occup. Hyg. 15: 219-235. 

Coffin, D. L.; Blommer, E. J.; Gardner, D. E.; Holzman, R. (1967) Effect of 

air pollution on alteration of susceptibility to pulmonary infection. In: 

Proceedings of the third annual conference on atmospheric contamination 

in confined spaces; May; Dayton, OH. Wright-Patterson Air Force Base, OH: 

Aerospace Medical Research Laboratories; pp. 71-80; report no. 

AMRL-TR-67-200. Available from: NTIS, Springfield, VA; AD-835008. 

Coffin, D. L.; Gardner, D. E.; Holzman, R. S.; Wolock, F. J. (1968) Influence 

of ozone on pulmonary cells. Arch. Environ. Health 16: 633-636. 

Cortesi, R.; Privett, O.S. (1972) Toxicity of fatty ozonides and peroxides. 

Lipids 7: 715-721. 

Cosio, M. G.; Hale, K. A.; Niewoehner, D. E. (1980) Morphologic and morphometric effects of prolonged cigarette smoking on the small airways. Am. 

Rev. Respi r.. Di s. 122: 265-271. 

Costa, D. L. ; Kutzman, R. S. ; Lehmann, J. R. ; Popenoe, E. A. ; Drew, R. T. 

(1983) A subchronic multi-dose ozone study in rats. In: Lee, S. D.; 

Mustafa, M. G.; Mehlman, M. A., eds. International symposium on the 

biomedical effects of ozone and related photochemical oxidants; March 

1982; Pinehurst, NC. Princeton, NJ: Princeton Scientific Publishers, 

Inc.; pp. 369-393. (Advances in modern environmental toxicology: v. 5). 

Crapo, J. D.; Barry, B. E.; Chang, L.-Y.; Mercer, R. R. (1984) Alterations in 

lung structure caused by inhalation of oxidants. J. Toxicol. Environ. 

Health 13: 301-321. 

Cross, C. E. ; DeLuci a, A. J. ; Reddy, A. K. ; Hussai n, M. Z. ; Chow, C. K. ; 

Mustafa, M. G. (1976) Ozone interactions with lung tissue: biochemical 

approaches. Am. J. Med. 60: 929-935. 

Curran, S. F.; Amoruso,.M. A.; Goldstein, B. D.; Berg, R. A. (1984) Degradation 

of soluble collagen by ozone or hydroxyl radicals. FEBS Lett. 176: 155-160. 

Dawber, T. R. (1980) Lipids- and atherosclerotic disease. In: The Framingham 

study. The epidemiology of atherosclerotic disease. Cambridge, MA: 

Harvard University Press; pp. 121-141. 

9-264 
DeLucia, A. J.; Hoque, P. M.; Mustafa, M. G.; Cross, C. E. (1972) Ozone interaction with rodent lung: effect on suifhydryls and sulfhydryl-containing 

enzyme activities. J. Lab. Clin. Med. 80: 559-566. 

DeLucia, A. J. ; Mustafa, M. G. ; Cross, C. E. ; Plopper, C. G. ; Dungworth, 

D. L.; Tyler, W. S. (1975a) Biochemical and morphological alterations in 

the lung following ozone exposure. In: Rai, C.; Spieiman, L. A., eds. 

Air: I. pollution control and ciean energy; 1973; New Orleans, LA; 

Detroit, MI; Philadelphia, PA; Vancouver, British Columbi.a. New York, 

NY: American Institute of Chemical Engineers; pp. 93-100. (AIChE symposium series: v. 71, no. 147). 

DeLucia, A. J.; Mustafa, M. G.; Hussain, M. Z.; Cross, C. E. (1975b) Ozone 

interaction with rodent lung. III. Oxidation of reduced glutathione and 

formation of mixed disulfides between protein and nonprotein sulfhydryls. 

J. Clin. Invest. 55: 794-802. 

Dillard, C. J.; Urribarri, N.; Reddy, K.; Fletcher, B.; Taylor, S.; de Lumen, 

B.; Langberg, S.; Tappel, A. L. (1972) Increased lysosornal enzymes in 

lungs of ozone-exposed rats. Arch. Environ. Health 25: 426-431. 

Dixon, J. R.; Mountain, J. T. (1965) Role of histamine and related substances 

in development of tolerance to edemagenic gases. Toxicol. Appl. Pharmacol. 

7: 756-766. 

Dohan, F. C.; Everts, G. S.; Smith, R. (1962) Variation in air pollution and 

the incidence of respiratory disease. J. Air Pollut. Control Assoc. 

12: 418-436. 

Donovan, D. H.; Menzel, D. B. (1975) Dietary and environmental modification of 

mouse lung lipids. J. Am. Oil Chem. Soc. 52: 122A. 

Donovan, D. H.; Menzel, D. B. (1978) Mechanisms of lipid peroxidation: iron 

catalyzed decomposition of fatty acid hydroperoxides as the basis of 

hydrocarbon evolution in vivo . Experientia 34: 775-776. 

Donovan, D. H.; Williams, S. J.; Charles, J. M.; Menzel, D. B. (1977) Ozone 

toxicity: effect of dietary vitamin E and polyunsaturated fatty acids. 

Toxicol. Lett. 1: 135-139. 

Dorsey, A. P.; Morgan, D. L.; Menzel, D. B. (1983) Filterability of erythrocytes from ozone-exposed mice. In: Abstracts of the third international 

corigress on toxicology; September; San Diego,.,CA. Toxicol._ Lett. 

18(suppl. 1): 146. 

Doty, R. L. (1975) Determination of odour preferences in rodents: a methodological review. In: Moulton, D. G.; Turk, A.; Johnson, J. W., eds. Methods 

in olfactory research. New York, NY: Academic Press; pp. 395-406. 

Dowell, A. R.; Lohrbauer, L. A.; Hurst, D.; Lee, S. D. (1970) Rabbit alveolar 

macrophage damage caused by in vivo ozone inhalation. Arch. Environ. 

Health 21: 121-127. 

9-265 
Downey, J. E.; Irving, D. H.; Tappel, A. L. (1978) Effects of dietary antioxidants on in vivo lipid peroxidation in the rat as measured by pentane 

production. Lipids 13: 403-407. 

Dubeau, H.; Chung, Y. S. (1979) Ozone response in wild type and radiationsensitive mutants of Saccharomyces cerevisiae. Mol. Gen. Genet. 176: 

393-398. 

Dubeau, H.; Chung, Y. S. (1982) Genetic effects of ozone: induction of point 

mutation and genetic recombination in Saccharomyces cerevisiae. Mutat. 

Res. 102: 249-259. 

Dubick, M. A.; Critchfield, J. W.; Last, J. A.; Cross, L. E.; Rucker, R. B. 

(1983) Ascorbic acid turnover in the mouse following acute ozone exposure. 

Toxicology (Ireland) 27: 301-313. 

Dumelin, E. E.; Dillard, C. J.; Tappel, A. L. (1978a) Breath ethane and pentane 

as measures of vitamin E protection of Macaca radiata against 90 days of 

exposure to ozone. Environ. Res. 15: 38-43. 

Dumelin, E. E.; Dillard, C. J.; Tappel, A. L. (1978b) Effect of vitamin E and 

ozone on pentane and ethane expired by rats. Arch. Environ. Health 33: 

129-135. 

Dungworth, D. L. (1976) Short-term effects of ozone on lungs of rats, mice, and 

monkeys. EHP Environ. Health Perspect. 16: 179. 

Dungworth, D. L.; Clarke, G. L.; P1ata, R. L. (1969) Pulmonary lesions produced 

in A-strain mice by long-term exposure to peroxyacetyl nitrate. Am. Rev. 

Respir. Dis. 99: 565-574. 

Dungworth, D. L.; Cross, C. E.; Gillespie, J. R.; Plopper, C. G. (1975a) The 

effects of ozone on animals. In: Murphy, J. S.; Orr, J. R., eds. Ozone 

chemistry and technology: a review of the literature 1961-1974. Philadelphia, PA: The Franklin Institute Press; pp. 29-54. 

Dungworth, D. L.; Castleman, W. L.; Chow, C. K.; Mellick, P. W.; Mustafa, M. 

G.; Tarkington, B.; Tyler, W. S. (1975b) Effect of ambient levels of 

ozone on monkeys. Fed. Proc. Fed. Am. Soc. Exp. Biol. 34: 1670-1674. 

Easton, R. E.; Murphy, S. D. (1967) Experimental ozone preexposure and histamine. Arch. Environ. Health 15: 160-166. 

Eglite, M. E. (1968) A contribution to the hygienic assessment of atmospheric 

ozone. Hyg. Sanit. 33: 18-23. 

Ehrlich, R. (1963) Effect of air pollutants on respiratory infection. Arch. 

Environ. Health 6: 638-642. 

Ehrlich, R. (1980) Interaction between environmental pollutants and respiratory 

infections. EHP Environ. Health Perspect. 35: 89-100. 

9-266 
Ehrlich, R. (1983) Changes in susceptibility to respiratory infection caused 

by exposures to photochemicai oxidant pollutants. In: Lee, S. D.; Mustafa, 

M. G.; Mehlman, M. A., eds. International symposium on the biomedical 

effects of ozone and reiated photochemical oxidants; March 1982; Pinehurst, 

NC. Princeton, NJ: Princeton Scientific Publishers, Inc.; pp. 273-285. 

(Advances in modern environmental toxicology: v. 5). 

Ehrl i ch, R. ; Fi ndl ay, J. C. ; Fenters, J. D. ; Gardner, D. E. (1977) Heal th 

effects of short-term inhalation of nitrogen dioxide and ozone mixtures. 

Environ. Res. 14: 223-231. 

Ehrlich, R.; Findlay, J. C.; Gardner, D. E. (1979) Effects of repeated exposures 

to peak concentrations of nitrogen dioxide and ozone on resistance to 

streptococcal pneumonia. J. Toxicol. Environ. Health 5: 631-642. 

Elsayed, N. M.; Mustafa, M. G.; Postlethwait, E. M. (1982a) Age-dependent 

pulmonary response of rats to ozone exposure. J. Toxicol. Environ. Health 

9: 835-848. 

Elsayed, N. M.; Hacker, A.; Mustafa, M.; Kuehn, K.; Schrauzer, G. (1982b) 

Effects of decreased glutathione peroxidase activity on the pentose 

phosphate cycle in mouse lung. Biochem. Biophys. Res. Commun. 104: 

564-569. 

Elsayed, N. M.; Hacker, A. D.; Kuehn, K.; Mustafa, M. G.; Schrauzer, G. N. 

(1983) Dietary antioxidants and the biochemical response to oxidant 

inhalation. II: Influence of dietary selenium on the biochemical effects 

of ozone exposure in mouse lung. Toxicol. Appl. Pharmacol. 71: 398-406. 

Emi k, L. 0. ; P1 ata, R. L. (1969) Depressi on of runni ng acti vi ty i n mi ce by 

exposure to polluted air. Arch. Environ. Health 18: 574-579. 

Emik, L. 0.; Plata, R. L.; Campbell, K. I.; Clarke, G. L. (1971) Biologicai 

effects of urban air pollution: Riverside summary. Arch. Environ. Heaith 

23: 335-342. 

Engel, L. A.; Macklem, P. T. (1977) Gas mixing and distribution in the lung. 

In: Widdicombe, J. G., ed. Respiratory physiology II. Baltimore, MD: 

University Park Press; pp. 37-82 (International review of physiology: 

v. 14). 

Erdman, H. E.; Hernandez, T. (1982) Adult toxicity and dominant lethals induced 

by ozone at specific stages in spermatogenesis.-i.n Drosophil.a virilis .. 

Environ. Mutagen. 4: 657-666. 

Eustis, S. L.; Schwartz, L. W.; Kosch, P. C.; Dungworth, D. L. (1981) Chronic 

bronchiolitis in nonhuman primates after prolonged ozone exposure. Am. J. 

Pathol. 105: 121-137. 

Evans, M. J. (1984) Oxidant gases. EHP Environ. Health Perspect. 55: 85-95. 

Evans, M. J.; Mayr, W.; Bils, R. F.; Loosli, C. G. (1971) Effects of ozone on 

cell renewal in pulmonary alveoli of aging mice. Arch. Environ. Health 

22: 450-453. 

9-267 
Evans, M. J.; Johnson, L. V.; Stephens, R. J.; Freeman, G. (1976a) Renewal of 

the terminal bronchiolar epithelium in the rat following exposure to NO 2 

or 03 . Lab. Invest. 35: 246-257. 

Evans, M. J.; Johnson, L. V.; Stephens, R. J.; Freeman, G. (1976b) Cell renewal 

in the lungs of rats exposed to low levels of ozone. Exp. Mol. Pathol. 

24: 70-83. 

Evans, M. J.; Stephens, R. J.; Freeman, G. (1976c). Renewal of pulmonary 

epithelium following oxidant injury. In: Bouhuys, A., ed. Lung cells in 

disease: proceedings of a Brook Lodge conference; April; Augusta, MI. New 

York, NY: Elsevier/North-Holland Biomedical Press; pp. 165-178. 

Evans, M. J.; Dekker, N. P.; Cabral=Anderson, L. J.; Shami, S. G. (1985) 

Morphological basis of tolerance to ozone. Exp. Mol. Pathol. 42: 366-376. 

Fabbri , L. M. ; Ai zawa, H. ; A1 pert, S. E. ; Wal ters , E. H. ; 0' Byrne, P. M. ; 

Gold, B. D.; Nadel, J. A.; Holtzman, M. J. (1984) Airway hyperresponsiveness and changes in cell counts in br•onchoalveolar lavage after ozone 

exposure in dogs. Am. Rev. Respir. Dis. 129: 288-291. 

Fairchild, E. J., II. (1963) Neurohumoral factors in injury from inhaled irritants. Arch. Environ. Health 6: 85-92. 

Fairchild, E. J., II. (1967) Tolerance mechanisms: determinants of lung responses 

to injurious agents. Arch. Environ. Health 14: 111-126. 

Fairchild, E. J., II; Graham, S. L. (1963) Thyroid influence on the toxicity of 

respiratory irritant gases, ozone and nitrogen dioxide. J. Pharmacol. 

Exp. Ther. 139: 177-184. 

Fairchild, E. J., II; Murphy, S. D.; Stokinger, H. E. (1959) Protection by sulfur 

compounds against the air pollutants ozone and nitrogen dioxide. Science 

(Washington, DC) 130: 861-862. 

Fairchild, E. J., II; Graham, S. L.; Hite, M.; Killens, R.; Scheel, L. D. (1964) 

Changes in thyroid 1 131 activity in ozone-tolerant and ozone-§usceptible 

rats. Toxicol. Appl. Pharmacol. 6: 607-613. 

Fairchild, G. A. (1974) Ozone effect on respiratory deposition of vesicular 

stomatitis virus aerosols. Am. Rev. Respir. Dis. 109: 446-451. 

Fairchild, G. A. (1977) Effects of ozone and sulfur dioxide on virus growth in 

mice. Arch. Environ. Health 32: 28-33.. 

Fetner, R. H. (1962) Ozone-induced chromosome breakage in human cell cultures. 

Nature (London) 194: 793-794. 

Fetner, R. H. (1963) Mitotic inhibition induced in grasshopper neuroblasts by 

exposure to ozone. Brooks Air Force Base, TX: USAF School of Aerospace 

Medicine; document no. SAM-TOR 63-39. Available from: NTIS, Springfield, 

VA; AD-489014. 

~ .: 
Filser, S. G.; Bolt, H. M.; Muliawan, H.; Kappus, H. (1983) Quantitative 

evaluation of ethane and n-pentane as indicators of lipid peroxidation in 

vivo. Arch. Toxicol. 52: 135-147. 

Fletcher, B. L.; Tappel, A. L. (1973) Protective effects of dietary a-Tocopheroi in rats-exposed to toxic levels of ozone and nitrogen dioxide: 

Envi ron. Res. 6: 165-175. 

Fletcher, C. M.; Gilson, J. G.; Hugh-Jones, P.; Scadding, J. G. (1959) Terminology, definitions, and classification of chronic pulmonary emphysema and 

related conditions: a report of the conclusions of a CIBA guest symposium. 

Thorax 14: 286-299. 

Frager, N. B.; Phalen, R. F.; Kenoyer, J. L. (1979) Adaptations to ozone in 

reference to mucociliary clearance. Arch. Environ. Health 34: 51-57. 

Freeman, B. A.; Mudd, J. B. (1981) Reaction of ozone with sulfhydryls of human 

erythrocytes. Arch. Biochem. Biophys. 208: 212-220. 

Freeman, B. A.; Sharman, M. C.; Mudd, J. B. (1979) Reaction of ozone with 

phospholipid vesicles and human erythrocyte ghosts. Arch. Biochem. 

Biophys. 197: 264-272. 

Freeman, G. ; Crane, S. C. ; Furiosi , N. J. ; Stephens, R. J. ; Evans, M. J. ; 

Moore, W. D. (1972) Covert reduction in ventilatory surface in rats 

during prolonged exposure to subacute nitrogen dioxide. Am. Rev. Respir. 

Dis. 106: 563-579. 

Freeman, G.; Stephens, R. J.; Coffin, D. L.; Stara, J. F. (1973) Changes in 

dogs' lungs after long-term exposure to ozone: light and electron microscopy. Arch. Environ. Health 26: 209-216. 

Freeman, G.; Juhos, L. T.; Furiosi, N. J.; Mussenden, R.; Stephens, R. J.; 

Evans, M. J. (1974) Pathology of pulmonary disease from exposure to 

interdependent ambient gases (nitrogen dioxide and ozone). Arch. Environ. 

Health 29: 203-210. 

Friberg, L.; Holma, B.; Rylander, R. (1972) Animal lung reactions after 

inhalation of lead and ozone. Environ. Physiol. Biochem. 2: 170-178. 

Friedman, M.; Gallo, J. M.; Nicho"ls, H. P.; Bromberg, P. A. (1983) Changes in 

inert gas rebreathing parameters after ozone exposure in dogs. Am. Rev. 

Respir. Dis. 128: 851-856. 

Fujimaki , H. ;. Ozawa, .-M..;.- Imai ,...T. ;.. Sh.imizu, F. (1984)-- Effect--of short-term 

exposure to 0 3 on: antibody response in mice. Environ. Res. 35: 490-496. 

Fujinaka, L. E.; Hyde, D. M.; Plopper, C. G.; Tyler, W. S.; Dungworth, D. L.; 

Lollini, L. 0. (1985) Respiratory bronchiolitis following long-term 

ozone exposure in Bonnet monkeys: a morphometric study. Exp. Lung Res. 

8: 167-190. 

Fukase, 0.; Isomura, K.; Watanabe, H. (1975) Effect of ozone on glutathione 

in vivo . Taiki Osen Kenkyu 10: 58-62. 

9-269 
Fukase, 0.; Watanabe, H.; Isomura, K. (1978) Effects of exercise on mice 

exposed to ozone. Arch. Environ. Health 33: 198-200. 

Gardner, D. E. (1981) Effects of environmental chemicals on host defense 

responses of the lung. In: Dean, J. H.; Padarathsingh, M., eds. Biological relevance of immune suppression as induced by genetic, therapeutic 

and environmental factors: second in a series of technology assessment 

workshops; November 1979; Williamsburg, VA. New York, NY: Van Nostrand 

Reinhold Co.; pp. 275-287. 

Gardner, D. E. (1982a) Use of experimental airborne infections for monitoring 

altered host defenses. EHP Environ. Health Perspect. 43: 99-107. 

Gardner, D. E. (1982b) Effects of gases and airborne particles on lung infections. In: McGrath, J. J.; Barnes, C. D. eds. Air pollution - physiological effects. New York, NY: Academic Press, Inc.; pp.• 47-79. 

Gardner, D. E. (1984) Oxidant-induced enhanced sensitivity to infection in 

animal models and their extrapolation to man. In: Miller, F. J.; Menzel, 

D. B., ed. Fundamentals of extrapolation modeling of inhaled toxicants. 

Washington, DC: Hemi.sphere Publ. Co.; pp. 423-439. 

Gardner, D. E.; Ehrlich, R. (1983) Introduction to host defense sessions. In: 

Lee, S.D.; Mustafa, M.G.; Mehlman, M.A., eds. International symposium on 

biomedical effects of ozone and related photochemical oxidants; March 

1982; Pinehurst, NC. Princeton, NJ: Princeton Scientific Publishers, 

Inc.; pp. 255-261. (Advances in modern environmental toxicology: v. 5). 

Gardner, D. E.; Graham, J. A. (1977) Increased pulmonary disease mediated 

through altered bacterial defenses. In: Sanders, C. L.; Schneider, R. P.; 

Dagle, G. E.; Ragen, H. A., eds. Pulmonary macrophage and epithelial 

cells: proceedings of the sixteenth annual Hanford biology symposium; 

September 1976; Richland, WA. Washington, DC: Energy Research and Development Administration; pp. 1-21. (ERDA symposium series: 43). Available 

from: NTIS, Springfield, VA; CONF-760927. 

Gardner, D. E.; Pfitzer, E. A.; Christian; R. T.; Coffin, D. L. (1971) Loss of 

protective factor for alveolar macrophages when exposed to ozone. Arch. 

Intern. Med. 127: 1078-1084. 

Gardner, D. E. ; Lewis, T. R. ; Alpert, S. M. ; Hurst, D. J. ; Coffin, D. L. 

(1972) The role of tolerance in pulmonary defense mechanisms. Arch. 

Environ. Health 25: 432-438. 

Gardner, D. E.; Illing, J. W.; Miller, F. J.; Coffin, D. L. (1974) The effect 

of ozone on pentobarbital sleeping time in mice. Res. Commun. Chem. 

Pathol. Pharmacol. 9: 689-700. 

Gardner, D. E.; Miller, F. J.; Illing, J. W.; Kirtz, J. M. (1977) Increased 

infectivity with exposure to ozone and sulfuric acid. Toxicol. Lett. 1: 

59-64. 

Gelmont, D. ; Stei n, R. A. ; Mead, J. F. (1981) The bacteri al ori gi n of rat 

breath pentane. Biochem. Biophys. Res. Commun. 102: 932-936. 

9-270 
Gershwin, L. J.; Osebold, J. W.; Zee, Y. C. (1981) Immunoglobulin E-containing 

celis in mouse lung following allergen inhalation and ozone exposure. 

Int. Arch. Allergy Appl. Immunol. 65: 266-277. 

Gertner, A.; Bromberger-Barnea, B.; Dannenberg, A. M., Jr.; Traystman, R.; 

Menkes, H. (1983a) Responses of the lung periphery to 1.0 ppm ozone. J. 

Appl. Physiol. Respir. Environ. Exercise Physiol. 55: 770-776. 

Gertner, A.; Bromberger-Barnea, B.; Traystman, R.; Berzon, D.; Menkes, H. 

(1983b) Responses of the lung periphery to ozone and histamine. J. Appl. 

Physiol.: Respir. Environ. Exercise Physiol. 54: 640-646. 

Gertner, A.; Bromberger-Barnea, B.; Traystman, R.; Menkes, H. (1983c) Effects 

of ozone in peripheral lung reactivity. J. "Appl. Physiol.: Respir. Environ. 

Exercise Physioi. 55: 777-784. 

Gertner, A.; Bromberger-Barnea, B.; Kelly, L.; Traystman, R.; Menkes, H. (1984) 

Local vagal responses in the lung periphery. J. Appi. Physiol.: Respir. 

Environ. Exercise Physiol. 57: 1079-1088. 

Gillespie, J. R. (1980) Review of the cardiovascular and pulmonary function 

studies on beagles exposed for 68 months to auto exhaust and other air 

pollutants •In: Stara, J. F.; Dungworth, D. L.; Orthoefer, J. C.; 

Tyler, W. S., eds. Long-term effects of air pollutants in canine species. 

Cincinnati, OH: U.S. Environmental Protection Agency, Environmental Criteria 

and Assessment Office; pp. 115-154; EPA report no. EPA 600/8-80-014. Available from: NTIS, Springfield, VA; PB81-144875: 

Giri, S. N.; Benson, J.; Siegel, D. M.; Rice, S. A.; Schiedt, M. (1975) Effects 

of pretreatment with anti-inflammatory drugs on ozone-induced lung damage in 

rats. Proc. Soc. Exp. Biol. Med. 150: 810-814. 

Giri, S. N.; Hollinger, M. A.; Schiedt, M. J. (1980) The effects of ozone and 

paraquat on PGF 2 and PGE2 levels in plasma and combined pleural effusion 

and lung lavage of rats. Environ. Res. 21: 467-476. 

Goldstein, B. D. (1973) Hydrogen peroxide in erythrocytes. Detection in rats 

and mice inhaling ozone. Arch. Environ. Health 26: 279-280. 

Goldstein, E. (1984) An assessment of animai models for testing the effect of 

photochemical oxidants on pulmonary susceptibility to bacterial infection. 

J. Toxicol. Environ. Health 13: 415-421. 

Goldstein, B. D.; Balchum, O. J. (1974) Modification of the response of rats 

to lethal levels of ozone by enzyme-inducing agents. Toxicol. Appl. 

Pharmacol. 27: 330-335. 

Goldstein, B. D.; McDonagh, E. M. (1975) Effect of ozone on cell membrane 

protein fluorescence. I. In vitro studies utiiizing the red cell membrane. Envi ron. Res. 9: 179-186. 

Goldstein, B. D.; Pearson, B.; Lodi, C.; Buckley, R. D.; Balchum, 0. J. (1968) 

The effect of ozone on mouse blood in vivo. Arch. Env_iron. Health 16: 

648-650. 

9-271 
Goldstein, E.; Tyler, W. S.; Hoeprich, P. D.; Eagle, C. (1971a) Ozone and the 

antibacterial defense mechani5ms of the murine lung. Arch. Intern. Med. 

127: 1099-1102. 

Goldstein, E.; Tyler, W. S.; Hoeprich, P. D.; Eagle, C. (1971b) Adverse influence of ozone on pulmonary bactericidal activity of murine lungs. Nature 

(London) 229: 262-263. 

Goldstein, B. D.; Levine, M. R.; Cuzzi-Spada, R.; Cardenas, R.; Buckley, R. 

D.; Balchum, 0. J. (1972a) p-aminobenzoic acid as a protective agent in 

ozone toxicity. Arch. Environ. Health 24: 243-247. 

Goldstein, E.; Eagle, M. C.; Hoeprich, P. D. (1972b) Influence of ozone on pulmonary defense mechanisms of silicotic mice. Arch. Environ. Health 24: 444-448. 

Goldstein, B. D.; Lai, L. Y.; Cuzzi-Spada, R. (1974a) Potentiation of complement-dependent membrane damage by ozone. Arch. Environ. Health 28: 

40-42. 

Goldstein, E.; Warshauer, D.; Lippert, W.; Tarkington, B. (1974b) Ozone and 

nitrogen dioxide exposure. Arch. Environ. Health 28: 85-90. 

Goldstein, B. D.; Solomon, S.; Pasternack, B. S.; Bickers, D. R. (1975) Decrease 

in rabbit lung microsomal cytochrome P-450 levels following ozone exposure. 

Res. Commun. Chem. Pathol. Pharmacol. 10: 759-762. 

Goldstein, B. D.; Hamburger, S. J.; Fal . k, G. W.; Amoruso, M. A. (1977) Effect 

of ozone and nitrogen dioxide on the agglutination of rat alveolar macrophages by concanavalin A. Life Sci. 21: 1637-1644. 

Goldstein, E.; Gibson, J. B.; Tassan, C.; Lippert, W. (1978a) Effect of 

levamisole on alveolar macrophage function. Am. Rev. Respir. Dis. 117: 

273. 

Goldstein, E. ; Bartlema, H. C. ; van der Ploeg, M. ; van Dui jn, P. ; van der 

Stap, J. G. M. M.; Lippert, W. (1978b) Effect of ozone on lysosomal 

enzymes of alveolar macrophages engaged in phagocytosis and killing of 

inhaled Staphylococcus aureus. J. Infect. Dis. 138: 299-311. 

Gooch, P. C.; Creasia, D. A.; Brewen, J. G. (1976) The cytogenetic effect of 

ozone: inhalation and in vitro exposures. Environ. Res. 12: 188-195. 

Gordon, T.; Amdur, M. 0. (1980) Effect of ozone on respiratory response of 

guinea pigs to histamine. J. Toxicol. Environ. Health 6: 185-195. 

Gordon, T.; Taylor, B. F.; Amdur, M. 0. (1981) Ozone inhibition of tissue 

cholinesterase in guinea pigs. Arch. Environ. Health 36: 284-288. 

Gordon, T.; Venugopalan, C. S.; Amdur, M. O.;.Drazen, J. M. (1984) Ozone-induced 

airway hyperreactivity in the guinea pig. J: Appl. Physiol.: Respir. 

Environ. Exercise Physiol. 57: 1034-1038. 

Gotto, A. M., Jr. (1979) Status report: plasma lipids, lipoproteins, and coronary 

artery disease. In: Paoletti, R.; Gotto, A. M., Jr., eds. Atherosclerosis 

reviews: v. 4. New York, NY: Raven Press; pp. 17-28. 

9-272 
Graham, J. A. (1979) Alteration of hepatic xenobiotic metabolism by ozone [dissertation]. Durham, NC: Duke University. Available from: University 

Microfilms, Ann Arbor, MI: publication no. 80-09646. 

Graham, J. A.; Menzel, D. B.; Miller, F. J.; Illing, J. W.; Gardner, D. E. 

(1981) Influence of ozone on pentobarbital-induced sleeping time in mice, 

rats, and hamsters. Toxicol. Appl. Pharmacol. 61: 64-73. 

Graham, J. A.; Menzel, D. B.; Miller, F. J.; Illing, J. W.; Gardner, D. E. 

(1982a) Effect of ozone on drug-induced sleeping time in mice pretreated 

with mixed-function oxidase inducers and inhibitors. Toxicol. Appl. 

Pharmacol. 62: 489-497. 

Graham, J. A.; Miiler, F. J.; Gardner, D. E.; Ward, R.; Menzel, D. B. (1982b) 

Influence of ozone and nitrogen dioxide on hepatic microsonal enzymes in 

mice. J. Toxicoi. Environ. Health 9: 849-856. 

Graham, J. A.; Menzel, D. B.; Miller, F. J.; Illing, J. W.; Ward, R.; Gardner, 

D. E. (1983) Influence of ozone on xenobiotic metabolism. In: Lee, 

S. D.; Mustafa, M. G.; Mehlman, M. A., eds. International symposium on 

the biomedical effects of ozone and related photochemical oxidants; March 

1982; Pinehurst, NC. Princeton, NJ: Princeton Scientific Publishers, 

Inc.; pp. 95 ~117. (Advances in modern environmental toxicology: v. 5). 

Graham, J. A. ; Menzel , D. B. ; Mol e, M. L. ; Mi 11 er, F. J. ; Gardner, D. E. 

(1985) Influence of ozone on pentobarbital pharmacokinetics in mice. 

Toxicol. Lett. 24: 163-170. 

Green, G. M. (1984) Similarities of host defense mechanisms°against pulmonary 

disease in animals and man. J. Toxicoi. Environ. Health 13: 471-478. 

Gregory, A. R.; Ripperton, L. A.; Miller, B. (1967) Effect of neonatal thymectomy on the development of ozone tolerance in mice. Am. Ind. Hyg. Assoc. 

J. 28: 278-282. 

Grose, E. C.; Gardner, D. E.; Miller, F. J. (1980) Response of ciliated epithelium to ozone and sulfuric acid..Environ. Res. 22: 377-385. 

Grose, E. C.; Richards, J. H.; Illing, J. W.; Miller, F. J.; Davies, D. W.; 

Graham, J. A.; Gardner, D. E. (1982) Pulmonary host defense responses to 

inhalation of sulfuric acid and ozone. J. Toxicol. Environ. Health 10: 

351-362. 

Gross, E. A.; Swenberg, J. A.; Fields, S.; Popp, J. A. (1982) Comparative 

morphometry of the nasal. cavity in rats and mice. J. Anat. 135: 83-88. 

Gross, P.; Scheel, L. D.; Stokinger, H. E. (1965) Ozone toxicity studies; 

destruction of alveolar septa--a precursor of emphysema? In: Mitchell, 

R. S., ed. The pathogenesis of the chronic obstructive bronchopulmonary 

disease: seventh annual conference on research in emphysema; June 1964; 

Aspen, C0. Basel, Switzerland: S. Karger, AG; pp. 376-381. (Progress 

in research in emphysema and chronic bronchitis: v. 2; Medicina 

Thoracalis: v. 22, nos. 1-3). 

9-273 
Gross, S.; Melhorn, D. K. (1972) Vitamin E red cell lipids and red cell stability in prematurity. Ann. N. Y. Acad. Sci. 203: 141-162. 

Guerrero, R. R.; Rounds, D. E.; Olson, R. S. Hackney, J. D. (1979) Mutagenic 

effects of ozone on human cells exposed in vivo and in vitro based on 

sister chromatid exchange analysis. Environ. Res. 18: 336-346. 

Hadley, J. G.; Gardner, D. E.; Coffin D. L.; Menzel, D. B. (1977) Enhanced 

binding of autologous cells to the macrophage plasma membrane as a sensitive indicator of pollutant damage. In: Sanders, C. L.; Schneider, R. 

P.; Dagle G. E.; Ragan, H. A., eds. Pulmonary macrophage and epithelial 

cells: proceedings of the sixteenth annual Hanford biology symposium; 

September 1976; Richland, WA. Washington, DC: Energy Research and 

Development Administration; pp. 1-21. (ERDA symposium series: v. 43). 

Available from: NTIS, Springfield, VA; CONF-760927. 

Halliwell, B. (1982) Production of superoxide, hydrogen peroxide and hydroxyl 

radicals by phagocytic cells: a cause of chronic inflammatory disease? 

Cell Biol. Int. Rep. 6: 529-542. 

Hamburger, S. J.; Goldstein, B. D. (1979) Effect of ozone on the agglutination 

of erythrocytes by concanavalin A. I: . Studies in rats. Environ. 

Res. 19: 292-298. 

Hamburger, S. J.; Goldstein, B. D.; Buckley, R. D.; Hackney, J. D.; Amoruso, M. 

A. (1979) Effect of ozone on the agglutination of erythrocytes by concanavalin A. II: Study of human subjects receiving supplemental vitamin E. 

Environ. Res. 19: 299-305. 

Hamelin, C.; Chung, Y. S. (1974) Resistance a 1'ozone chez Escherichia coli. 

I. Relations avec certains mecanismes de reparation de 1'ADN [Ozone 

resistance in Escherichia coli. I. Relations with some DNA repair 

mechanisms]. Mol. Gen. Genet. 129: 177-184. 

Hamelin, C.; Chung, Y. S. (1975a) The effect of low concentrations of ozone on 

Escherichia coli chromosome. Mutat. Res. 28: 131-132. 

Hamelin, C.; Chung, Y. S. (1975b) Characterization of mucoid mutants of 

Escherichia coli K-12 isolated after exposure to ozone. J. Bacteriol. 

122: 19-24. 

Hamelin, C.; Chung, Y. S. (1978) Role of the pol, rec, and DNA gene products 

in the repair of lesions produced in Escherichia coli DNA by ozone. Stud. 

Biophys. 68: 229-335. 

Hamelin, C.; Sarhan, F.; Chung, Y. S. (1977a) Ozone-induced DNA degradation 

in different DNA polymerase I mutants of Escherichia coli K12. Biochem. 

Biophys. Res. Commun. 77: 220-224. 

Hamelin, C.; Sarhan, F.; Chung, Y. S. (1977b) DNA-degradation caused by ozone 

in mucoid mutants of Escherichia coli K12. FEMS Microbiol. Lett. 2: 

149-151. 

9-274 
Hammond, P. B.; Beliles, R. P. (1980) Metals: ch. 17. In: Doull, J.; 

Klaassen, C. D.; Amdur, M. 0., eds. Toxicology, the basic science of 

poisons. New York, NY: Macmillan Press Company, Inc.; pp. 409-467. 

Hathaway, J. A.; Terrill, R. E. (1962) Metabolic effects of chronic ozone 

exposure on rats. Am. Ind. Hyg. Assoc. J. 23: 392-395. 

Hattori, K.; Kato, N.; Kinoshita, M.; Kinoshita, S.; Sunada, T. (1963) Protective effect of ozone in mice against whole-body X-irradiation. Nature 

(London) 198: 1220. 

Hayes, W. J., Jr. (1975) Toxicology of pesticides. Baltimore, MD: William 

and Wilkins Company; pp. 118-127. 

Henschler, D. (1960) Schutzwirkung einer Vorbehandlung mit geringen Gaskonzentrationen gegen todliche Reizgas-Lungenodeme [Protective effect of 

previous treatment with low gas concentrations against lethal pulmonary 

edema from irritant gas]. Naunyn Schmiedeberg's Arch. Exper. Pathoi. 

Pharmakol. 238: 66-67. 

Hesterberg, T. W.; Last, J. A. (1981) Ozone-induced acute pulmonary fibrosis 

in rats: prevention of increased rates of collagen synthesis by methylprednisolone. Am. Rev. Respir. Dis. 123: 47-52. 

Hilding, D. A.; Hilding, A. C. (1966) Ultrastructure of tracheal ci1ia and 

cell during regeneration. Ann. Otol. Rhinol. Laryngol. 75: 281-294. 

Holtzman, M. J.; Fabbri, L. M.; Skoogh, B.-E.; 0'Byrne, P. M.; Walters, E. H.; 

'Aizawa, H.; Nadel, J. A. (1983a) Time course of airway hyperresponsiveness 

induced by ozone in dogs. J. Appl. Physiol.: Respir. Environ. Exercise 

Physiol. 55: 1232-1236. 

Holtzman, M. J. ; Fabbri , L. M. ; 0' Byrne, P. M. ; Gold, B. D. ; Aizawa, H. ; 

Walters, E. H.; Alpert, S. E.; Nadel; J. A. (1983b) Importance of airway 

inflammation for hyperresponsiveness induced by ozone. Am. Rev. Respir. 

Dis. 127: 686-690. 

Holzman, R. S.; Gardner, D. E.; Coffin, D. L. (1968) In vivo inactivation of 

lysozyme by ozone. J. Bacteriol. 96: 1562-1566. 

Hu, P. C.; Miller, F. J.; Daniels, M. J.; Hatch, G. E.; Graham, J. A.; Gardner, 

D. E.; Selgrade, M. K. (1982) Protein accumulation in lung lavage fluid 

following ozone exposure. Environ. Res. 29: 377-388. 

Huber, G. L. ; Mason, R. J. ; LaForce, M. ; Spencer, N. J. ; Gardner, D. E. ; 

Coffin, D. L. (1971) Alterations in the lung following the administration 

of ozone. Arch. Intern. Med. 128: 81-87. 

Hueter, F. G.; Contner, G. L.; Busch, K. A.; Hinners, R. G. (1966) Biological 

effects of atmospheres contaminated by auto exhaust. Arch. Environ. 

Health 12: 553-560. 

Hurst, D. J.; Coffin, D. L. (1971) Ozone effect on 'iysosomal hydrolases of 

alveoiar macrophages in vitro. Arch. Intern. Med. 127: 1059-1063. 

9-275 
Hurst, D. J. ; Gardner, D. E. ; Coffin, D. L. (1970) Effect of ozone on acid 

hydrolases of the pulmonary alveolar macrophage. J. Reticuloendothel. 

Soc. 8: 288-300. 

Hussain, M. Z.; Mustafa, M. G.; Chow, C. K.; Cross, C. E. (1976a) Ozone-induced 

increase of lung proline hydroxylase activity and hydroxyproline content. 

Chest 69(suppl. 2): 273-275. 

Hussain, M. Z.; Cross, C. E.; Mustafa, M. G.; Bhatnagar, R. S. (1976b) Hydroxyproline contents and prolyl hydroxylase activities in lungs of rats 

exposed to low levels of ozone. Life Sci. 18: 897-904. 

Hyde, D.; Orthoefer, J. G.; Dungworth, D.; Tyler, W.; Carter, R.; Lum, H. (1978) 

Morphometric and morphologic evaluation of pulmonary lesions in beagle 

dogs chronically exposed to high ambient levels of air pollutants. Lab. 

Invest. 38: 455-469. 

Ibrahim, A. L.; Zee, Y. C.; Osebold, J. W. (1976) The effects of ozone on the 

respiratory epithelium and alveolar macrophages of mice. I. Interferon 

production. Proc. Soc. Exp. Biol. Med. 152: 483-488. 

Ibrahim, A. L.; Zee, Y. C.; Osebold, J. W. (1980) The effects of ozone on the 

respiratory epithelium of mice. II. Ultrastructural alterations. J. 

Environ. Pathol. Toxicol. 3: 251-258. 

Ikematsu, T. (1978) Influence of ozone on rabbit's tonsils--observation of 

the surface of the tonsil with scanning electron microscope. Nippon Jibi 

Inkoka Gakkai Kaiho 81: 1447-1458. 

Illing, J. W.; Miller, F. J.; Gardner, D. E. (1980) Decreased resistance to 

infection in exercised mice exposed to NO 2 and 03 . J. Toxicol. Environ. 

Health 6: 843-851. 

Inoue, H.; Sato, S.; Hirose, T.; Kikuchi, Y.; Ubukata, T.; Nagashima, S.; 

Sasaki, T.; Takishima, T. (1979) A comparative study between functional 

and pathologic alterations in lungs of rabbits exposed to an ambient 

level of ozone: functional aspects. Nikkyo Shikkai-Shi 17: 288-296. 

Ishidate, M., Jr.,; Yoshikawa, K. (1980) Chromosome aberration tests with 

Chinese hamster cells in vitro with and without metabolic activation--a 

comparative study on mutagens and carcinogens. Arch. Toxicol. Suppl. 4: 

41-44. 

Jackson, R. M.; Frank, L. (1984)-Ozone-induced tolerance to hyperoxia in rats. 

Am. Rev. Respir. Dis. 129: 425-429. 

Jegier, Z. (1973) L'ozone en tant que polluant atmospherique [Ozone as an air 

pollutant]. Can. J. Public Health 64: 161-166. 

Johnson, D. A. (1980) Ozone inactivation of human a l -proteinase inhibitor. 

Am. Rev. Respir. Dis. 121: 1031-1038. 

9-276 
Johnson, B. L.; Orthoefer, J. G.; Lewis, T. R.; Xintaras, C. (1976) The effect 

of ozone on brain function. In: Finkel, A. J.; Duel, W. C., eds. 

Clinical implications of air pollution research: [proceedings of the] air 

pollution medical research conference; December 1974; San Francisco, 

CA. Acton, MA: Publishing Sciences Group, Inc.; pp. 233-245. 

Johnson, K. I.; Fantone, J. C., III; Kaplan, J.; Ward, P. A. (1981) In vivo 

damage of rat lungs by oxygen metabolites. Clin. Invest. 67: 983-993. 

Juhos, L. T.; Evans, M. J.; Mussenden-Harvey, R.; Furiosi, N. J.; Lapple, C. 

E.; Freeman, G. (1978) Limited exposure of rats to H 2 SO4 with and without 

03 . J. Environ. Sci. Health C13: 33-47. 

Kaplan, J. ; Smaldone, G. C. ; Menkes, H. A. ; Swift, D. L. ; Traystman, R. J. 

(1981) Response of collateral channels to histamine: lack of vagal 

effect. J. Appl. Physiol.: Respir. Environ. Exercise Physiol. 51: 1314- 

1319. 

Kavlock, R.; Daston, G.; Grabowski, C. T. (1979) Studies on the developmental 

toxicity of ozone. I. Prenatal effects. Toxicol. Appl. Pharmacol. 48: 

19-28. 

Kavlock, R. J.; Meyer, E.; Grabowski, C. T. (1980) Studies on the developmental 

toxicity of ozone: postnatal effects. Toxicol. Lett. 5: 3-9. 

Kawachi , T. ; Yahagi , T. ; Kada, T. ; Tazima, Y. ; Ishidate, M. ; Sasaki , M. ; 

Sugiyama, T. (1980) Cooperative programme on short-term assays for carcinogenicity in Japan. In: Montesano, R.; Bartsch, H.; Tomatis, L.; Davis, 

W., eds. Molecular and cellular aspects of carcinogen screening tests; 

June 1979; Hanover, West Germany. Lyon, France: International Agency 

for Research on Cancer; pp. 323-330. (IARC scientific publications: 

no. 27). 

Kenoyer, J. L.; Phalen, R. F.; Davis, J. R. (1981) Particle clearance from the 

respiratory tract as a test of toxicity: effect of ozone on short and 

long term clearance. Exp. Lung Respir. 2: 111-120. 

Kensler, C. J.; Battista, S. D. (1966) Chemical and physical factors affecting 

mammalian ciliary activity. In: Symposium on structure, function and measurement of respiratory cilia; February 1965; Durham, NC." Am. Rev. Respir. 

Dis. 93: 93-102. 

Kesner, I+ .; +indya, R. J.; Chan, P. C. (1979) Inhibition of erythrocyte membrane 

(Na + K)-activated ATPase by ozone-treated phospholipids. J. Biol. Chem. 

254: 2705-2709. 

Kimura, A.; Goldstein, E. (1981) Effect of ozone on concentrations of lysozyme 

in phagocytizing alveolar macrophages. J. Infect. Dis. 143: 247-251. 

Kindya, R. J.; Chan, P. C. (1976) Effect of ozone on erythrocyte membrane 

adenosine triphosphatase. Biochim. Biophys. Acta 429: 608-615. 

Klebanoff, S. J.; Clark, R. A. (1975) Hemolysis and iodination of erythrocyte 

components by a myeloperoxidase-mediated system. Blood 45: 699-707. 

9-277 
Kliment, V. (1972) Geometry of guinea pig respiratory tract and application of 

Landahl's model of deposition or aerosol particles. J. Hyg. Epidemiol. 

Microbiol. Immunol. 16: 107-114. 

Kliment, V. (1973) Similarity and dimensional analysis, evaluation of aerosol 

deposition in the lungs of laboratory, animals and man. Folia Morphol. 

21: 59-64. 

Konigsberg, A. S.; Bachman, C. H. (1970) Ozonized atmosphere and gross motor 

activity of rats. Int. J. Biometeorol. 14: 261-266. 

Koontz, A. E.; ieatfh, R. L. (1979) Ozone alteration of transport of cations 

and the Na /K -ATPase in human erythrocytes. Arch. Biochem. Biophys. 

198: 493-500. 

Kotin, P.; Thomas, M. (1957) Effect of air contaminants on reproduction and 

offspring survival in mice. AMA Arch. Ind. Health 16: 411-413. 

Kotlikoff, M. I.; Jackson, A. C.; Watson, J. W. (1984) Oscillatory mechanics 

of the respiratory system in ozone-exposed rats. J. Appl. Physiol.: 

Respir. Environ. Exercise Physiol. 56: 182-186. 

Krupnick, A. J.; Frank, R. (1984) Synergistic effects on rat lungs of mixtures 

of oxidant air pollutants (ozone or nitrogen dioxide) and respirable 

aerosols [letter to the editor]. Am. Rev. Respir. Dis. 129: 885. 

Kulle, T. J.; Cooper, G. P. (1975) Effects . of formaldehyde and ozone on the 

trigeminal nasal sensory system. Arch. Environ. Health 30: 237-243. 

Kyei-Aboagye, K.; Hazucha, M.; Wyszogrodski, I.; Rubinstein, D.; Avery, M. E. 

(1973) The effect of ozone exposure in vivo on the appearance of lung 

tissue lipids in the endobronchial lavage of rabbits. Biochem. Biophys. 

Res. Commun. 54: 907-913. 

LaBelle, C. W.; Long, J. E.; Christofano, E. E. (1955) Synergistic effects of 

aerosols: particulates as carriers of toxic vapors. AMA Arch. Ind. Health 

11: 297-304. 

Laqueur, E.; Magnus, R. (1921) Experimental and theoretical bases for the 

therapy of phosgene sickness. Z. Gesamte Exp. Med. 13: 284-291. 

Larkin, E. C.; Rao, G. A. (1984) Reduced growth in rats exposed to ozone as 

compared to pair-fed controls breathing air. IRCS Med. Sci. 12: 741-742. 

Larkin, E. C.; Kimzey, S. L.; Siler, K. (1978) Response of the rat erythrocyte 

to ozone exposure. J. Appl. Physiol.: Respir. Environ. Exercise Physiol. 

45: 893-898. 

Larkin, E. C.; Goheen, S. C.; Rao, G. A. (1983) Morphology and fatty acid 

composition of erythrocytes from monkeys exposed to ozone for one year. 

Environ. Res. 32: 445-454. 

9-278 
Last, J. A. (1983) Health effects of indoor air pollution: synergistic effects 

of nitrogen dioxide and a respirable aerosol. Environ. Int. (U.S.A.) 9: 

319-322. 

Last, J. A.; Cross, C. E. (1978) A new model for health effects of air pollutants: evidence for synergistic effects of mixtures of ozone and sulfuric 

acid aerosols on rat lungs. J. Lab. Clin. Med. 91: 328-339. 

Last, J. A.; Greenberg, D. B. (1980) Ozone-induced alterations in collagen 

metabolism of rat lungs. II. Long-term exposure. Toxicol. Appl. Pharmacol. 

55: 108-114. 

Last, J. A.; Kaizu, T. (1980) Mucus glycoprotein secretion by tracheal explants: 

effects of pollutants. EHP Environ. Health Perspect. 35: 131-138. 

Last, J. A.; Jennings, M. D.; Schwartz, L. W.; Cross, C. E. (1977) Glycoprotein 

secretion by tracheal explants cultured from rats exposed to ozone. Am. 

Rev. Respir. Dis. 116: 695-703. 

Last, J. A.; Greenberg, D. B.; Castleman, W. L. (1979) Ozone- induced al terations in collagen metabolism of rat lungs. Toxicol. Appl. Pharmacol. 51: 

247-258. 

Last, J. A.; Dasgupta, P. K.; DeCesare, K.; Tarkington, B. K. (1982) Inhalation 

toxicology of ammonium persulfate, an oxidant aerosol, in rats. Toxicol. 

Appl. Pharmacol. 63: 257-263. 

Last, J. A.; Gerriets, J. E.; Hyde, D. M. (1983) Synergistic effects on rat 

lungs of mixtures of oxidant 'air pollutants (azone or nitrogen dioxide) 

and respirable aerosols. Am. Rev. Respir. Dis. 128: 539-544. 

Last, J. A.; Hyde, D. M.; Chang, D. P. Y. (1984a) A mechanism of synergistic 

lung damage by ozone and a respirable aerosol. Exp. Lung Res. 7: 223-235. 

Last, J. A.; Reiser, K. M.; Tyler, W. S.; Rucker, R. B. (1984b) Long-term 

consequences of exposure to ozone. I. Lung collagen content. Toxicol. 

Appl. Pharmacol. 72: 111-118. 

Lee, L.-Y.; Bleecker, E. R.; Nadel, J. A. (1977) Effect of ozone on bronchomotor 

response to inhaled histamine aerosol in dogs. J. Appl. Physiol.: Respir. 

Environ. Exercise Physiol. 43: 626-631. 

Lee, L.-Y.; Dumont, C.; Djokic, T. D.; Menzel, T. E.; Nadel, J. A. (1979) 

Mechanism of rapid, shallow breathing after ozone exposure in conscious 

dogs. J. Appl. Physiol.: Respir. Environ. Exercise Physiol. 46: 1108-1114. 

Lee, L.-Y.; Djokic, T. D.; Dumont, C.; Graf, P. D.; Nadel, J. A. (1980) Mechanism 

of ozone-induced tachypneic response to hypoxia and hypercapnia in conscious 

dogs. J. Appl. Physiol.: Respir. Environ. Exercise Physiol. 48: 163-168. 

Lewis, T. R.; Hueter, F. G.; Busch, K. A. (1967) Irradiated automobile exhaust: 

its effects on the reproduction of mice. Arch. Environ. Health 15: 26-35. 

9-279 
Lewis, T. R.; Moorman, W. J.; Yang, Y.; Stara, J. F. (1974) Long-term exposure 

to auto exhaust and other pollutant mixtures: effects on pulmonary function 

in the beagle. Arch. Environ. Health 29: 102-106. 

Ling, N. R.; Kay, J. E. (1975) Lymphocyte stimulation. Amsterdam, The Netherlands: North-Holland Publishing Co. 

Lum, H.; Schwartz, L. W.; Dungworth, D. L.; Tyler, W. S. (1978) A comparative 

study of cell renewal after exposure to ozone or oxygen: response of 

terminal bronchiolar epithelium in the rat. Am. Rev. Respir. Dis. 118: 

335-345. 

Lunan, K. D.; Short, P.; Negi, D.; Stephens, R. J. (1977) Glucose-6-phosphate 

dehydrogenase response of postnatal lungs to NO 2 and 03 . In: Sanders, 

C. L. ; Schneider, R. P. ; Dagle, G. E. ; Ragan, H. A. , eds. Pulmonary 

macrophage and epithelial cells: proceedings of the sixteenth annual 

Hanford biology symposium; September 1976; Richland, WA. Washington, DC: 

Energy Research and Development Administration; pp. 236-247. (ERDA 

symposium series: 43). Available from: NTIS, Springfield, VA; CONF-760927. 

MacLean, S. A.; Longwell, A. C.; Blogoslawski, W. J. (1973) Effects of ozonetreated seawater on the spawned, fertilized, meiotic, and cleaving eggs 

of the commercial American oyster. Mutat. Res. 21: 283-285. 

MacRae, W. D.; Stich, H. F. (1979) Induction of sister chromatid exchanges in 

Chinese hamster ovary cells by thiol and hydrazene compounds. Mutat. 

Res. 68: 351-365. 

Martin, C. J.; Boatman, E. S.; Ward, G. (1983) Mechanical properties of alveolar wall after pneumonectomy and ozone exposure. J. Appl. Physiol.: Respir. 

Environ. Exercise Physiol. 54: 785-788. 

Matsumura, Y. (1970) The effects of ozone, nitrogen dioxide, and sulfur dioxide 

on the experimentally induced allergic respiratory disorder in guinea 

pigs. III. The effect on the occurrence of dyspneic attacks. Am. Rev. 

Respir. Dis. 102: 444-447. 

Matsumura, Y., Mizuno, K.; Miyamoto, T.; Suzuki, T.; Oshima, Y. (1972) The 

effects of ozone, nitrogen dioxide, and sulfur dioxide on the experimentally induced allergic respiratory disorder in guinea pigs. IV. 

Effects on respiratory sensitivity to inhaled acetylcholine. Am. Rev. 

Respir. Dis. 105: 262-267. 

Matzen, R. N. (1957a) Development of tolerance to ozone in reference to pulmonary edema. Am. J. Physiol. 190: 84-88. 

Matzen, R. N. (1957b) Effect of vitamin C and hydrocortisone on the pulmonary 

edema produced by ozone in mice. J. Appl. Physiol. 11: 105-109. 

McAllen, S. J.; Chiu, S. P.; Phalen, R. F.; Rasmussen, R. E. (1981) Effect of 

in viyo ozone exposure on in vitro pulmonary alveolar macrophage mobility. 

J. Toxicol. Environ. Health 7: 373-381. 

• :rl 
McCord, J. M.; Fridovich, I. (1978) The biology and pathology of oxygen radicals. 

Ann. Intern. Med. 89: 122-127. 

McJilton, C.; Thielke, J.; Frank, R. (1972) Ozone uptake model for the respiratory system. In: Abstracts of technical papers: American industrial 

hygiene conference; May; San Francisco, CA. Am. Ind. Hyg. Assoc. J. 

33: paper no. 45. 

Meiners, B. A.; Peters, R. E.; Mudd, J. B. (1977) Effects of ozone on indole 

compounds and rat lung monoamine oxidase. Environ. Res. 14: 99-112. 

Mellick, P. W.; Mustafa, M. G.; Tyler, W. S.; Dungworth, D. L. (1975) Morphologic and biochemical changes in primate lung due to low-level ozone 

exposure. In: Proceedings of the 23rd international.tuberculosis conference; 

September; Mexico. Bull. Int. Union Tubercul. 51: 565-567. 

Mellick, P. W.; Dungworth, D. L.; Schwartz, L. W.; Tyler, W. S. (1977) Short 

term morphologic effects of high ambient levels of ozone on lungs of 

rhesus monkeys. Lab. Invest. 36: 82-90. 

Mendenhall, R. M.; Stokinger, H. E. (1959) Tolerance and cross-tolerance 

development to atmospheric pollutants ketene and ozone. J. Appl. Physiol. 

14: 923-926. 

Menzel, D. B. (1970) Toxicity of ozone, oxygen, and radiation. Annu. Rev. 

Pharmacol. 10: 379-394. 

Menzel, D. B. (1971) Oxidation of biologically active reducing substances by 

ozone. Arch. Environ. Health 23: 149-153. 

Menzel, D. B. (1976) The role of free radicals in the toxicity of air pollutants 

(nitrogen oxides and ozone). In: Pryor, W. A., ed. Free radicals in 

biology: v.. 2. New York, NY: Academic Press, Inc.; pp. 181-202. 

Menzel, D. B. (1983) Metabolic effects of ozone exposure. In: Lee, S. D.; 

Mustafa, M. G.; Mehiman, M. A., eds. International symposium on the 

biomedical effects of ozone and related photochemical oxidants; March 

1982; Pinehurst, NC. Princeton, NJ: Princeton Scientific Publishers, 

Inc.; pp. 47-55. (Advances in modern environmental toxicology: v. 5). 

Menzel, D. B.; Roehm, J. N.; Lee, S. D. (1972) Vitamin E: the biological and 

environmentai antioxidant. J. Agric. Food Chem. 20: 481-486. 

Menzel, D. B.; Slaughter, R.; Donovan, D.; Bryant, A. (1973) Fatty acid ozonides 

as the toxic agents on ozone inhalation. Pharmacologist 15: 239. 

Menzel, D. B.; Slaughter, R. J.; Bryant, A. M.; Jauregui, H. 0. (1975a) Heinz 

bodies formed in erythrocytes by fatty acid ozonides and ozone. Arch. 

Environ. Health 30: 296-301. 

Menzel, D. B.; Slaughter, R. J.; Bryant, A. M.; Jauregui, H. 0. (1975b) Prevention of ozonide-induced Heinz bodies in human erythrocytes by vitamin E. 

Arch. Environ. Health 30: 234-236. 

9-281 
Menzel, D. B.; Anderson, W. G.; Abou-Donia, M. B. (1976) Ozone exposure modifies prostaglandin biosynthesis in perfused rat lungs. Res. Commun. 

Chem. Pathol. Pharmacol. 15: 135-147. 

Menzel, D. B.; Donovan, D. H.; Sabransky, M. (1978) Dietary vitamin E and fat: 

effect on murine lung and red blood cell glutathione peroxidase activity. 

Presented at: 62nd annual meeting, Federation of American Societies for 

Experimental Biology; April; Atlantic City, NJ. Fed. Proc. Fed. Am. Soc. 

Exp. Biol. 37: 707. 

Menzel, D. B.; Smolko, E. D.; Gardner, D. E.; Graham, J. A. (1983) Frontiers 

in short-term testing of pneumotoxicants. In: Woodhead, ed. Short-term 

tests for environmentally-induced chronic health effects. Washington, 

DC: U.S. Environmental Protection Agency, Office of Research and 

Development; EPA report no. EPA-600/8-83-002. 

Miller, F. J. (1977) A mathematical model of transport and removal of ozone in 

mammalian lungs [Ph.D. thesis]. Raleigh, NC: North Carolina State 

University. Available from: University Microfilms, Ann Arbor, MI; publication no. 77-21,560. 

Miller, F. J. (1979) Biomathematical modeling applications in the evaluation 

of ozone-toxicity. In: Mudd, J.; Lee, S. D.; eds. Assessing toxic effects 

of environmental pollutants. Ann Arbor, MI: Ann Arbor Science Publishers, 

Inc.; pp. 263-286. 

Miller, S.; Ehrlich, R. (1958) Susceptibility to respiratory infections of 

animals exposed to ozone. I. Susceptibility to Klebsiella pneumoniae . J. 

Infect. Dis. 103: 145-149. 

Miller, F. J.; Illing, J. W.; Gardner, D. E. (1978a) Effect of urban ozone 

levels on laboratory-induced respiratory infections. Toxicol. Lett. 

2: 163-169. 

Miller, F. J.; Menzel, D. B.; Coffin, D. L. (1978b) Similarity between man and 

laboratory animals in regional deposition of ozone. Environ. Res. 17: 

84-101. 

Mi l ler, F. J. ; McNeal , C. A. ; Ki rtz, J. M. ; Gardner, D. E. ; Coffi n, D. L. ; 

Menzel, D. B. (1979) Nasopharyngeal removal of ozone in rabbits and 

guinea pigs. Toxicology 14: 273-281. 

Miller, F. J.; Overton, J. H., Jr.; Jaskot, R. H.; Menzel, D. B.; (1985) 

A model of the regional uptake of gaseous pollutants in the lung. I. The 

sensitivity of the uptake of ozone in the human lung to lower respiratory 

tract secretions and to exercise. Toxicol. Appl. Pharmacol. 79: 11-27. 

Mizoguchi, I.; Osawa, M.; Sato, Y.; Makino, K.; Yagyu, H. (1973) Studies on 

erythrocyte and photochemical smog. I. Effects of air pollutants on 

erythrocyte resistance. Taiki Osen Kenkyu 8: 414. 

Mole, M. L., Jr.; Stead, A. G.; Gardner, D. E. ; Miller, F. J.; Graham, J. A. 

(1985) Effect of ozone on serum lipids and lippoproteins in the rat. 

Toxicol. Appl. Pharmacol. 80: 367-376. 

9-282 
Montgomery, M. R.; Niewoehner, D. E. (1979) Oxidant-induced alterations in 

puimonary microsomal mixed-function oxidation: acute effects of paraquat 

and ozone. J. Environ. Sci. Health C13: 205-219. 

Moore, P. F.; Schwartz, L. W. (1981) Morphological effects of prolonged exposure 

to ozone and sulfuric acid aerosol on the rat lung. Exp. Mol. Pathol. 

35: 108-123. 

Moore, G. S.; Calabrese, E. J.; Grinberg-Funes, R. A. (1980) The C57L/J mouse 

strain as a model for extrapulmonary effects of ozone exposure. Bull. 

Environ. Contam. Toxicol. 25: 578-585. 

Moore, G. S.; Calabrese, E. J.; Labato, F. J. (1981a) Erythrocyte survival in 

sheep exposed to ozone. Bull. Environ. Contam.. Toxicol. 27: 126-138. 

Moore, G. S.; Calabrese, E. J.; Schulz, E. (1981b) Effect of in vivo ozone 

exposure to Dorset sheep, an animal model with low levels of erythrocyte 

glucose-6-phosphate dehydrogenase activity. Bull. Environ. Contam. Toxicol. 

26: 273-280. 

Moore, G. S. ; Calabrese, E. J.; Molteni, K. H. (1984) Plasmodium berghei 

infection in mice: effect of low-level ozone exposure. Bull. Environ. 

Contam. Toxicol. 33: 99-105. 

Moorman, W. J.; Chmiel, J. J.; Stara, J. F.; Lewis, T. R. (1973) Comparative 

decomposition of ozone in the nasopharynx of beagles: acute vs chronic 

exposure. Arch. Environ. Health 26: 153-155. 

Morgan, M. S.; Frank, R. (1977) Uptake of pollutant gases by the respiratory 

system. In: Brain, J. D.; Proctor, D. F., eds. Respiratory defense 

mechanisms. Part I. New York, NY: Marcel Dekker, Inc.; pp. 157-189. 

Mountain, J. T. (1963) Detecting hypersusceptibility to toxic substances: an 

appraisal of simple blood tests. Arch. Environ. Health 6: 357-365. 

Mudd, J. B.; Freeman, B. A. (1977) Reaction of ozone with biological membranes. 

In: Lee, S. D., ed. Biochemical effects of environmental pollutants. 

Ann Arbor, MI: Ann Arbor Science Publishers, Inc.; pp. 97-133. 

Mudd, J. B.; Leavitt, R.; Ongun, A.; McManus, T. T. (1969) Reaction of ozone 

with amino acids and proteins. Atmos. Environ. 3: 669-681. 

Murlas, C. G.; Roum, J. H. (1985) Sequence of pathologic changes in the airway 

mucosa of guinea pigs during ozone-induced bronchial hyperractivity. Am. 

Rev. Respir. Dis. 131: 314-320. 

Murphy, S. D. (1964) A review of effects on animals of exposure to auto exhaust 

and some of its components. J. Air Pollut. Control Assoc. 14: 303-308. 

Murphy, S. D. ; Leng, J. K. ; Ulrich, C. E. ; Davis, H. V. (1963) Effects on 

animals of exposure to auto exhaust. Arch. Environ. Health 7: 60-70. 

Murphy, S. D.; Ulrich, C. E.; Frankowitz, S. H.; Xintaras, C. (1964) Altered 

function in animals inhaling low concentrations of ozone and nitrogen 

dioxide. Am. Ind. Hyg. Assoc. J. 25: 246-253. 

9-283 
Mustafa, M. G. (1975) Influence of dietary vitamin E on lung cellular sensitivity to ozone in rats. Nutr. ,Rep. Int. 11: 473-476. 

Mustafa, M. G.; Cross, C. E. (1974) Effects of short-term ozone exposure on 

lung mitochondrial oxidative and energy metabolism. Arch. Biochem. 

Biophys. 162: 585-594. 

Mustafa, M. G.; Lee, S. D. (1976) Pulmonary biochemical alterations resulting 

from ozone exposure. Ann. Occup. Hyg. 19: 17-26. 

Mustafa, M. G.; Lee, S. D. (1979) Biological effects of environmental pollutants: methods for assessing biochemical changes. In: Lee, S. D.; 

Mudd, J. B., eds. Assessing toxic effects of environmental pollutants. 

Ann Arbor, MI: Ann Arbor Science Publishers, Inc.; pp. 105-120. 

Mustafa, M. G.; Tierney, D. F. (1978) Biochemical and metabolic changes in the 

lung with oxygen, ozone, and nitrogen dioxide toxicity. Am. Rev. Respir. 

Dis. 118: 1061-1090. 

Mustafa, M. G. ; DeLucia, A. J. ; York, G. K. ; Arth, C. ; Cross, C. E. (1973) 

Ozone interaction with rodent lung. II. Effects on oxygen consumption of 

mitochondria. J. Lab. Clin. Med. 82: 357-365. 

Mustafa, M. G.; Ospital, J. J.; Hacker, A. D.; Lee, S. D. (1976) Effects of ozone 

and nitrogen dioxide exposure on lung metabolism. EHP Environ. Health 

Perspect. 16: 184. 

Mustafa, M. G. ; Hacker, A. D. ; Ospital , J. J. ; Hussai n, M. Z. ; Lee, S. D. 

(1977) Biochemical effects of environmental oxidants pollutants in animal 

lungs. In: Lee, S. D., ed. Biochemical effects of environmental pollutants. Ann Arbor, MI: Ann Arbor Science Publishers, Inc.; pp. 59-96. 

Mustafa, M. G.; Faeder, E. J.; Lee, S. D. (1980) Biochemical basis of pulmonary 

response to ozone and nitrogen dioxide injury. In: Bhatnagar, R. S., 

ed. Molecular basis of environmental toxicity. Ann Arbor, MI: Ann Arbor 

Science Publishers, Inc.; pp. 151-172. 

Mustafa, M. G.; Elsayed, N. M.; Quinn, C. L.; Postlethwait, E. M.; Gardner, D. 

E.; Graham, J. A. (1982) Comparison of pulmonary biochemical effects of 

low level ozone exposure on mice and rats. J. Toxicol. Environ. Health 

9: 857-865. 

Mustafa, M. G.; Elsayed, N. M.; Graham, J. A.; Gardner, D. E. (1983) Effects 

of ozone exposure on lung metabolism: influence of animal age, species, 

and exposure conditions. In: Lee, S. D.; Mustafa, M.. G.; Mehlman, M. 

A., eds. International symposium on the biomedical effects of ozone and 

related photochemical oxidants; March 1982; Pinehurst, NC. Princeton, NJ: 

Princeton Scientific Publishers, Inc.; pp. 57-73. (Advances in modern 

environmental toxicology: v. 5). 

Mustafa, M. G.; Elsayed, N. M.; von Dohlen, F. M.; Hassett, C. M.; Postlethwait, 

E. M.; Quinn, C. L.; Graham, J. A.; Gardner, D. E. (1984) A comparison of 

biochemical effects of nitrogen dioxide, ozone, and their combination in 

mouse lung. I. Intermittent exposures. Toxicol. Appl. Pharmacol. 72: 82-90. 

9-284 
Myers, B. A.; Dubick, M. A.; Reiser, K. M.; Gerriets, J. E.; Last, J. A. 

Rucker, R. B. (1984) Ozone exposure, food restriction and protein 

deficiency: changes in collagen and elastin in rodent lung. Toxicol. 

Lett. 23: 43-49. 

Nadel, J. A. (1977) Autonomic control of airway smooth muscie and airway 

secretions. Am. Rev. Respir. Dis. 115: 117-126. 

Nakajima, T.; Kusumoto, S.; Tsubota, Y.; Yonekawa, E.; Yoshida, R.; Motomiya, 

K.; Ito, K.; .Ide, G.; Otsu, H. (1972). Histopathological studies on the 

respiratory organs of mice exposed to photochemical oxidants and automobile exhaust. Osaka-furitsu Koshu Eisei Kenkyusho Kenlcyu Hokoku Rodo 

Eisei Hen 10: 35-42. 

Nambu, Z.; Yokoyama, E. (1983) Antitoxidant system and ozone tolerance. Environ. 

Res. 32: 111-117. 

Nasr, A. N. M. (1967) Biochemical aspects of ozone intoxication: a review. J. 

Occup. Med. 9: 589-597. 

National Air Poliution Control Administration. (1970) Air quality criteria for 

photochemical oxidants. Washington, DC: U.S. Department of Health, 

Education and Welfare, Public Health Service; NAPCA publication no. AP-63. 

Available from: NTIS, Springfield, VA; PB-190262. 

National Institutes of Health. (1983) Supplement: comparative biology of the 

lung: workshop; June 1982. Am. Rev. Respir. Dis. Suppl. 128: (2, pt. 2): 

S1-S91. 

National Institutes of Health (1985) The definition of emphysema: report of a 

National Heart, Lung, and Blood Institute, Division of Lung Diseases 

workshop. Am. Rev. Respir. Dis. 132: 182-185. 

National Research Council. (1977) Ozone and other photochemical 

oxidants. Washington, DC: National Academy of Sciences, Committee on 

Medical and Biologic Effects of Environmental Pollutants. 

Nayak, D. P.; Keiley, G. W.; Young, G. A.; Underdahl, N.R. (1964). Progressive 

descending influenza infection in mice determined by immunofluorescence. 

Proc. Soc. Exp. Biol. Med. 116: 200-206. 

Niewoehner, D. E.; Kleinerman, J.; Rice, D. B. (1974) Pathologic changes in 

the peripheral airways of young cigarette smokers. N. Engl. J. Med. 

291: 755-758. 

North Atlantic Treaty Organization. (1974) Air quality criteria for photochemical 

oxidants and related hydrocarbons: a report by the expert panei on air 

quality criteria. Brussels, Belgium: NATO, Committee on the Challenges of 

Modern Society; NATOICCMS n. 29. 

0' Byrne, P. M. ; Walters, E. H. ; Gold, B. D. ; Aizawa, H. A. ; Fabbri , L. M. ; 

Alpert, S. E.; Nadel, J. A.; Holtzman, M. J. (1984a) Neutrophil depletion 

inhibits airway hyperresponsiveness iiiduced by ozone exposure. Am. Rev. 

Respir. Dis. 130: 214-219. 

9-285 
0`Byrne, P. M.; Walters, E. H.; Aizawa, H.; Fabbri, L. M.; Holtzman, M. J.; 

Nadel, J. A. (1984b) Indomethacin inhibits the airway hypperresponsiveness 

but not the neutrophil influx induced by ozone in dogs. Am. Rev. Respir. 

Dis. 130: 220-224. 

0'Byrne, P. M.; Aizawa, H.; Bethel, R. A.; Chung, K. F.; Nadel, J. A.; Holtzman, 

M. J. (1984c) Prostaglandin F 2a increases responsiveness of pulmonary 

airways in dogs. Prostaglandins 28: 537-543. 

Oberst, F. W.; Comstock, C. C.; Hackley, E. B. (1954) Inhalation toxicity of 

ninety percent hydrogen peroxide vapor. AMA Arch. Ind. Hyg. Occup. Med. 

10: 319-327. 

Oomichi, S.; Kita, H. (1974) Effect of air pollutants on ciliary activity of 

respiratory tract. Bull. Tokyo Med. Dent. Univ. 21: 327-343. 

Orthoefer, J. G.; Bhatnagar, R. S.; Rahman, A.; Yang, Y. Y.; Lee, S. D.; Stara, 

J. F. (1976) Collagen and prolyl hydroxylase levels in lungs of beagles 

exposed to air pollutants. Environ. Res. 12: 299-305. 

Osebold, J. W.; Owens, S. L.; Zee, Y. C.; Dotson, W. M.; LaBarre, D. D. (1979) 

Immunological alterations in the lungs of mice following ozone exposure: 

changes in immunoglobulin levels and anti body-contai ni ng cells. Arch. 

Environ. Health 34: 258-265. 

Osebold, J. W.; Gershwin, L. J.; Zee, Y. C. (1980) Studies on the enhancement 

of allergic lung sensitization by inhalation of ozone and sulfuric acid 

aerosol. J. Environ. Pathol. Toxicol. 3: 221-234. 

Pack, A.; Hooper, M. B.; Nixon, W.; Taylor, J. C. (1977) A computational 

model of pulmonary gas transport incorporating effective diffusion. 

Respir. Physiol. 29: 101-124. 

Paiva, M. (1973) Gas transport in the human lung. J. Appl. Physiol. 35: 401- 

410. 

Palmer, M. S.; Swanson, D. H.; Coffin, D. L. (1971) Effect of ozone on benzpyrene 

hydroxylase activity in the Syrian golden hamster. Cancer Res. 31: 

730-733. 

Palmer, M. S.; Exley, R. W.; Coffin, D. L. (1972) Influence of pollutant gases 

on benzpyrene hydroxylase activity. Arch. Environ. Health 25: 439-442. 

P'an, A. Y. S.; Jegier, Z. (1971) The serum trypsin inhibitor capacity during 

ozone exposure. Arch. Environ. Health 23: 215-219. 

P'an, A. Y. S.; Jegier, Z. (1972) Trypsin protein esterase in relation to 

ozone-induced vascular damage. Arch. Environ. Health 24: 233-236. 

P`an, A. Y. S.; Jegier, Z. (1976) Serum protein changes during 'exposure to ozone. 

Am. Ind. Hyg. Assoc. J. 37: 329-334. 

P'an, A. Y. S.; Beland, J.; Jegier, Z. (1972) Ozone-induced arterial lesions. 

Arch. Environ. Health 24: 229-232. 

• :. 
Pemsingh, R. S.; Atwal, 0. S. (1983) Occurrence of APUD-type cells in the 

ciliated cyst of the parathyroid gland of ozone-exposed dogs. Acta Anat. 

116: 97-105. 

Penha, P. D.; Werthamer, S. (1974) Pulmonary lesions induced by long-term 

exposure to ozone. II. Ultrastructure observations of proliferative and 

regressive iesions. Arch. Envi. ron. Health 29: 282-289. 

Pepelko, W. E.; Mattox, J. K.; Yang, Y. Y. (1980) Arterial blood gases, pulmonary 

function and pathology in rats exposed to 0.75 or 1.0 ppm ozone. J. 

Environ. Pathol. Toxicol. 3: 247-259. 

Peterson, D. C.; Andrews, H. L. (1963) The role of ozone in radiation avoidance 

in the mouse. Radiat. Res. 19: 331-336. 

Peterson, M. L.; Rummo, N.; House, D.; Harder, S. (1978a) The effect of ozone 

on human immunity: in vitro responsiveness of lymphocytes to phytohemmagglutinin. Arch. Environ. Health 33: 59-63. 

Peterson, M. L.; Harder, S.; Rummo, N.; House, D. (1978b). Effect of ozone on 

leukocyte function in exposed human subjects. Environ. Res. 15: 485-493: 

Phalen, R. F.; Kenoyer, J. L.; Crocker, T. T.; McClure, T. R. (1980) Effects 

of sulfate aerosols in combination with ozone on elimination of tracer 

particles inhaled by rats. J. Toxicol. Environ. Heaith 6: 797-810. 

Pirquet, C.; Schittemhelm, A., eds. (1921) Uber Kampfgasvergiftungen [War gas 

poisoning]. Z. Gesamte Exp. Med. 13: 284-296. 

Plopper; C. G. ; Chow, C. K. ; Dungworth, D. L. ; Brummer, M. ; Nemeth, T. J. 

(1978) Effect of low level of ozone on rat lungs. II. Morphological 

responses during recovery and re-exposure. Exp. Mol. Pathol. 29: 400-411. 

Plopper, C. G.; Chow, C. K.; Dungworth, D. L.; Tyler, W. S. (1979) Pulmonary 

alterations in rats exposed to 0.2 and 0.1 ppm ozone: a correlated morphological and biochemical study. Arch. Environ. Health 34: 390-395. 

Prat, R.; Nofre, C.; Cier, A. (1968) Effets de l'hypochlorite de sodium de 

1'ozone et des radiations ionisantes sur les constituants pyrimidiques d' 

Escherichia coli [Effects of sodium hypochlorite, ozone, and ionizing 

radiations on pyrimidine constituents of Escherichia coli]. Ann. Inst. 

Pasteur Paris 114: 595-607. ~ 

Previero, A.; Signor, A.; Bezzi, S. (1964) Tryptophan modification in polypeptide chains. Nature (London) 204: 687-688. 

Pryor, W. A. (1976) Free radical reactions in biology: initiation of lipid 

autoxidation by ozone and nitrogen dioxide. EHP Environ. Health Perspect. 

16: 180-181. 

Pryor, W. A.; Stanley, J. P.; Blair, E; Cullen, G. B. (1976) Autoxidation of 

polyunsaturated fatty acids. Part I. Effect of ozone on the autoxidation 

of neat methyl linoleate and methyl linolenate. Arch. Environ. Health 31: 

201-210. 

9-287 
Pryor, W. A.; Dooley, M. M.; Church, D. F. (1983) Mechanisms for the reaction 

of ozone with biological molecules, the source of the toxic effects of 

ozone. In: Lee, S. D.; Mustafa, M. G.; Mehlman, M. A., eds. International 

symposium on the biomedical effects of ozone and photochemical oxidants; 

March 1982; Pinehurst, NC. Princeton, NJ: Princeton Scientific Publishers, 

Inc.; pp. 7-19. (Advances in modern environmental toxicology: v. 5). 

Raub, J. A.; Miller, F. J.; Graham, 

exposure on pulmonary function 

S. D.; Mustafa, M. G.; Mehlman, 

the biomedical effects of ozone 

1982; Pinehurst, NC Princetgn, 

Inc.; pp. 363-367. (Advances in 

J. A. (1983a) Effects of low-level ozone 

in adult and neonatal rats. In: Lee, 

M. A., eds. International symposium on 

and related photochemical oxidants; March 

NJ: Princeton Scientific Publishers, 

modern environmental toxicology: v. 5). 

Raub, J. A. ; Mi 11 er, F. J. ; Graham, J. A. ; Gardner, D. E. ; 0' Nei 1, J. J. 

(1983b) Pulmonary function in normal and elastase-treated hamsters exposed 

to a complex mixture of olefin-ozone-sulfur dioxide reaction products. 

Environ. Res. 31: 302-310. 

Raut, S. ; Hurd, J. ; Cureton, R. J. ; Blandford, G. ; Heath, R. B. (1975) The 

pathogenesis of infections of the mouse caused by virulent and avirulent 

variants of an influenza virus. J. Med. Microbiol. 8: 127-136. 

Razumovskii, S. D.; Zaikov, G. E. (1972) Effect of structure of an unsaturated 

compound on rate of its reaction with ozone. J. Org . Chem. USSR (Engl. 

Transl.) 8: 468-472. 

Reasor, M. J.; Adams, G. K., III; Brooks, J. K.; Rubin, R. J. (1979) Enrichment 

of albumin and IgG in the airway secretions of dogs breathing ozone. J. 

Environ. Sci. Health C13: 335-346. 

Reddy, C. C.; Thomas, C. E.; Scholz, 

(1983) Effects of ozone exposure 

nutrition on enzymes associated 

D. ; Mustafa, M. G. ; Mehlman, M. 

biomedical effects of ozone and 

1982, Pinehurst, NC. Princeton, 

Inc., pp. 395-410. (Advances in 

R. W.; Labosh, T. J.; Massaro, E. J. 

under inadequate vitamin E and/or selenium 

with xenobiotic metabolism. In: Lee, S. 

A., eds. International symposium on the 

related photochemical oxidants; March 

NJ: Princeton Scientific Publishers, 

modern environmental toxicology: v. 5). 

Revis, N. W.; Major, T.; Dalbey, W. E. (1981) Cardiovascular effects of ozone 

and cadmium inhalation in the rat. In: Northrop Services, Inc., ed. 

Proceedings of the research planning workshop on health effects of oxidants; 

January 1980; Raleigh, NC. Research Triangle Park, NC: U.S. Environmental 

Protection Agency, Health Effects Research Laboratory; EPA-600/9-81-001; 

pp. 171-179. Available from: NTIS, Springfield, VA; PB81-178832. 

Reynolds, R. W.; Chaffee, R. R. J. (1970) Studies on the combined effects of 

ozone and a hot envi ronment on reaction time in subhuman primates. In: 

Project clean air: v. 2. Santa Barbara, CA: University of California; 

research praject S-6. 

Roehm, J. N.; Hadley, J. G.; Menzel, D. B. (1971a) Antioxidants vs lung disease. 

Arch. Intern. Med. 128: 88-93. 

• :: 
Roehm, J. N.; Hadiey, J. G.; Menzel, D. B. (1971b) Oxidation of unsaturated 

fatty acids by ozone and nitrogen dioxide: a common mechanism of action. 

Arch. Environ. Health 23: 142-148. 

Roehm, J. N.; Hadley, J. G.; Menzel, D. B. (1972) The influence of vitamin E 

on the lung fatty acids of rats exposed to ozone. Arch. Environ. Health 

24: 237-242. 

Ross, B. K.; Hlastala, M. P.; Frank, R. (1979) Lack of ozone effects on oxygen 

hemoglobin affinity. Arch. Environ. Health 34: 161-163. 

Roth, R. P.; Tansy, M. F. (1972) Effects of gaseous air pollutants on gastric 

secreto-motor activities in the rat. J. Air Pollut. Control Assoc. 22: 

706-709. 

Roum, J. H.; Murlas, C. (1984) Ozone-induced changes in muscaranic bronchial 

reactivity by different testing methods. J. Appl. Physiol.: Respir. 

Environ. Exercise Physiol. 57: 1783-1789. 

Roycroft, J. H.; Gunter, W. B.; Menzel, D. B. (1977) Ozone toxicity: hormonelike oxidation products from arachidonic acid by ozone-catalyzed autoxidation. Toxicol. Lett. 1: 75-82. 

Sachsenmaier, W.; Siebs, W.; Tan, T.-A. (1965) Wirkung von Ozon auf 

Mauseacitestumorzellen und auf Huhnerfibroblasten in der Gewebekultur 

[Effect of ozone on mouse ascites on tumor cells and on chick fibroblasts 

in tissue culture]. Z. Krebsforsch. 67: 113-126. 

Saltzman, B. E.; Gilbert, N. (1959) Iodometric microdetermination of organic 

oxidants and ozone: resolution of mixtures by kinetic colorimetry. Anal. 

Chem. 31: 1914-1920. 

Sato, S.; Kawakami, M.; Maeda, S.; Takishima, T. (1976a) Scanning electron 

microscopy of the lungs of vitamin E-deficient rats exposed to a low 

co.ncentration of ozone. Am. Rev. Respir. Dis. 113: 809-821. 

Sato, S.; Kawakami, M.; Maeda, S.; Takishima, T. (1976b) Electron microscopic 

studies on the effects of ozone on the lungs of vitamin E-deficient rats. 

Nippon Kyobu Shikkan Gakkai Zasshi 14: 355-365. 

Sato, S.; Shimura, S.; Kawakami, M.; Hirose, T.; Maeda, S.; Takishima, T.; 

Kimura, S.; Yashiro, M.; Okazaki, S.; Ito, M. (1978) Biochemical and 

ultrastructural studies on the effects of long-term exposure of ozone on 

vitamin E=depleted rats. Nippon Kyobu Shikkan Gakkai Zasshi 16: 260-268. 

Sato, S. ; Shimura, S. ; Hirose, T. ; Maeda, S. ; Kawakami , M. ; Takishima, T. ; 

Kimura, S. (1980) Effects of long-term ozone exposure arsd dietary vitamin 

E in rats. Tohoku J. Exp. Med. 130: 117-128. 

Savino, A.; Peterson, M. L.; House, D.; Turner, A. G.; Jeffries, H. E.; Baker, 

R. (1978) The effects of ozone on human cellular and humoral immunity: 

Characterization of T and B lymphocytes by rosette formation. Environ. 

Res. 15: 65-69. 

s ~ • 
Savolainen, H.; Zitting, A. (1980) Glial cell effects of subacute formic acid 

vapour exposure [letter to the editor]. Acta Pharmacol. Toxicol. 47: 

239-240. 

Scheel, L. D.; Dobrogorski, 0. J.; Mountain, J. T.; Svirbely, J. L.; Stokinger, 

H. E. (1959) Physiologic, biochemical, immunologic and pathologic changes 

following ozone exposure. J. Appl. Physiol. 14: 67-80. . 

Scherer, P. W.; Shendalman, L. H.; Greene, N. M. (1972) Simultaneous diffusion 

and convection in single breath lung washout. Bull. Math. Biophys. 34: 

393-412. 

Scherer, P. W.; Shendalman, L. H.; Greene, N. M.; Bouhuys, A. (1975) Measurement of axial diffusivities in a model of the bronchial airways. J. 

Appl. Physiol. 38: 719-723. 

Schlipkoter, H.-W.; Bruch, J. (1973) Funktionelle und morphologische Veranderung 

bei Ozonexposition [Functional and morphological alterations caused by 

exposure to ozone]. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 

Abt. 1: Orig. Reihe B 156: 486-499. 

Schreider, J. P.; Raabe, 0. G. (1981) Anatomy of the nasal-pharyngeal airway 

of experimental animals. Anat. Rec. 200: 195-205. 

Schwartz, L. W.; Christman, C. A. (1979) Alveolar macrophage migration: influence of lung lining material and acute lung insult. Am. Rev. Respir. 

Dis. 120: 429-439. 

Schwartz, L. W.; Dungworth, D. L.; Mustafa, M. G.; Tarkington, B. K.; Tyler, 

W. S. (1976) Pulmonary responses of rats to ambient levels of ozone: 

effects of 7-day intermittent or continuous exposure. Lab. Invest. 34: 

565-578. 

Scott, M. L. (1970) Studies on vitamin E and related factors in nutrition and 

metabolism. In: DeLuch, H. F.; Suttie, J. W., eds.' The fat-soluble vitamins. Madison, WI: University of Wisconsin Press; pp. 355-374. 

Scott, D. B. M.; Lesher, E. C. (1963) Effect of ozone on survival and permeability of Escherichia coli. J. Bacteriol. 85: 567-576. 

Selgrade, M. J. K.; Mole, M. L.; Miller, F. J.; Hatch, G. E.; Gardner, D. E. 

Hu, P. C. (1981) Effect of NO 2 inhalation and vitamin C deficiency on 

protein and lipid accumulation in the lung. Environ. Res. 26: 422-437. 

Shakman, R. A. (1974) Nutritional influences on the toxicity of environmental 

pollutants: a review. Arch. Environ. Health 28: 105-113. 

Sherwin, R. P.; Richters, V.; Okimoto, D. (1983) Type 2 pneumocyte hyperplasia 

in the lungs of mice exposed to an ambient level (0.3 ppm) of ozone. In: 

Lee, S. D.; Mustafa, M. G.; Mehlman, M. A., eds. International symposium 

on the biomedical effects of ozone and related photochemical oxidants; 

March 1982; Pinehurst, NC. Princeton, NJ: Princeton Scientific Publishers, 

Inc.; pp. 289-297. (Advances in modern environmental toxicology: v. 5). 

9-290 
Sherwood, R. L.; Kimura, A.; Donovan, R.; Goldstein, E. (1984) Effect of 0.64 

ppm ozone on rats with chronic pulmonary bacterial infection. J. Toxicol. 

Environ. Health 13: 893-904. 

Shimura, S.; Maeda, S.; Takismima, T. (1984) Giant lamellar bodies in alveolar 

type II cells of rats exposed to a low concentration of ozone. Respiration 

46: 303-309. 

Shingu, H.; Sugiyama, M.; Watanabe, M.; Nakajima, T. (1980) Effects of ozone 

and photochemical oxidants on interferon production by rabbit alveolar 

macrophages. Bull. Environ. Contam. Toxicol. 24: 433-438. 

Shinriki, N.; Ishizaki, K.; Miura, K.; Ueda, T.; Harada, F. (1983) Degradation 

of nucleic acids with ozone. III. Mode of ozone-degradation of mouse proline transfer ribonucleic acid (tRNA) and isoleucine tRNA. Chem. Pharm. 

Bull. (Tokyo) 31: 3601-3608. 

Sielczak, M. W.; Denas, S. M.; Abraham, W. M. (1983) Airway cell changes in 

tracheal iavage of sheep after ozone exposure. J. Toxicol. Environ. Health 

11: 545-553. 

Sparrow, A. H.; Schairer, L. A. (1974) Mutagenic response of Tradescantia to 

treatment with X-rays, EMS, DBE, ozone, S0 2 , N2 0 and several insecticides. 

Am. Environ. Mutagen Soc. 445: 265-276. 

Speit, G.; Vogel, W-. (1982) The effect of sulfhydryl compounds on s i sterchromatid exchanges. II. The question of cell specificity and the role 

of H2O2 . Mutat. Res. 93: 175-183. 

Speit, G.; Vogel, W.; Wolf, M. (1982) Characterization of sister chromatid 

exchange induction by hydrogen peroxide. Environ. Mutagen. 4: 135-142. 

Stara, J. F.; Dungworth, D. L.; Orthoefer, J. G.; Tyler, W. S., eds. (1980) 

Long-term effects of air pollutants: in canine species. Cincinnati, OH: 

U.S. Environmental Protection Agency, Environmental Criteria and Assessment 

Office; EPA report no. EPA-600/8-80-014. Available from: NTIS, Springfield, 

VA; PB81-144875. 

Stephens, R. J.; Freeman, G.; Stara, J. F.; Coffin, D. L. (1973) Cytologic 

changes in dog lungs inducgd by chronic exposure to ozone. Am. J. Pathol. 

73: 711-726. 

Stephens, - R. - J:; Sloan, M. F:; Evans, M: J:; Freeman -, G. - (1974a) Early response 

of lung to low levels of ozone. Am. J. Pathol. 74: 31-58. 

Stephens, R. J.; Sloan, M. F.; Evans, M. J.; Freeman, G. (1974b) Alveolar type 

1 cell response to exposure to 0.5 ppm 03 for short periods. Exp. Mol. 

Pathol. 20: 11-23. 

Stephens, R. J.; Sloan, M. F.; Groth, D. G. (1976) Effects of long-term, low 

lev.el exposure of NO 2 or 03 on rat lungs. EHP Environ. Health Perspect. 

16: 178-179. 

9-291 
Stephens, R. J.; Sloan, M. F.; Groth, D. G.; Negi, D. S.; Lunan, K. D. (1978) 

Cytologic response of postnatal rat lungs to 0 3 or NO2 exposure. Am. J. 

Pathol. 93: 183-200. 

Stephens, R. J.; Buntman, D. J.; Negi, D. S.; Parkhurst, R. M.; Thomas, D. W. 

(1983) Tissue levels' of vitamin E in the lung and the cellular response 

to injury resulting from oxidant gas exposure. Chest 83: 37S-39S. 

Stewart, R. M.; Weir, E. K.; Montgomery, M. R.; Niewoehner, D. E. (1981) Hydrogen peroxide contracts airway smooth muscle: a possible endogenous mechanism. Respir. Physiol. 45: 333-342. 

Stich, H. F.; Wei, L.; Lam, P. (1978) The need for a mammalian test for mutagens: 

action of some reducing agents. Cancer Lett. 5: 199-204. 

Stinson, S. F.; Loosli, C. G. (1979) The effect of synthetic smog on voluntary 

activity of CD-1 mice. In: Animals as monitors of environmental pollutants: symposium on pathobiology of environmental pollutants, animal 

models and wildlife as monitors; 1977; Storrs, CT. Washington, DC: National 

Academy of Sciences; pp. 233-240. 

Stokinger, H. E.; Coffin, D. L. (1968) Biological effects of air pollutants. 

In: Stern, A. C., ed. Air pollution v. 1. pp. 446-546. 

Stokinger, H. E.; Wagner, W. D.; Wright, P•. G. (1956) Studies on ozone toxicity. 

I. Potentiating effects of exercise and tolerance development. AMA Arch. 

Ind. Health 14: 158-162. 

Stokinger, H. E.; Wagner, W. D.; Dobrogorski, 0. J. (1957) Ozone toxicity 

studies. III. Chronic injury to lungs of animals following exposure at 

low level. AMA Arch. Ind. Health 16: 514-522. 

Strawbridge, H. T. G. (1960) Chronic pulmonary emphysema (an experimental 

study). II. Spontaneous pulmonary emphysema in rabbits. Am. J. Pathol. 

37: 309-331. 

Suttorp, N.; Simon, L. M.; (1982) Lung cell oxidant injury: enhancement of 

polymorphonuclear leukocyte-mediated cytotoxicity in lung cells exposed to 

sustained in vitro hyperoxia. J. Clin. Invest. 70: 342-350. 

Suzuki, T. (1976) Studies on the acute toxicity of 5-hydroxytryptamine in the 

rats pre-exposed to ozone. Bull. Tokyo Med. Dent. Univ. 23: 217-226. 

Swann, H. E., Jr.; Balchum, 0. J. (1966) Biological effects of urban air 

pollution. IV. Effects of acute smog episodes on respiration of guinea pigs. 

Arch. Environ. Health 12: 698=704. 

Sweet, F. ; Kao, M. -S. ; iee, S. D. ; Hagar, W. L. ; Sweet, W. E. (1980) Ozone 

selectively inhibits growth of human cancer cells. Science (Washington, 

DC) 209: 931-933. 

Teige, B.; McManus, T. T.; Mucld, J. B. (1974) Reaction of ozone with phosphatidylcholine liposomes and the lytic effect of products on red blood cells. 

Chem. Phys. Lipids 12: 153-171. 

9-292 
Tepper, J. L.; Weiss, B.; Cox, C. (1982),Microanalysis of ozone depression of 

motor activity..Toxicol. Appl. Pharmacol. 64: 317-326. 

Tepper, J. L.; Weiss, B.; Wood, R. W. (1983) Behavioral indices of ozone 

exposure. In: Lee, S. D.; Mustafa, M. G.; Mehlman, M. A., eds. International symposium on the biomedical effects of ozone and related photochemical oxidants; March 1982; Pinehurst, NC. Princeton, NJ: Princeton 

Scientific Publishers, Inc.; pp. 515-526. (Advances in modern environmental toxicology: v. 5). 

Thienes, C. H.; Skillen, R. G.; Hoyt, A.; Bogen, E. (1965) Effects of ozone on 

experimental tuberculosis and on natural pulmonary infections in mice. 

Am. Ind. Hyg. Assoc. J. 26: 255-260. 

Thomas, G.; Fenters, J. D.; Ehrlich, R. (1979) Effect of exposure to PAN and 

ozone on susceptibility to chronic bacterial infection. Research Triangle 

Park, NC: U.S. Environmental Protection Agency, Health Effects Research 

Laboratory; EPA report no. EPA-600/1-79-001. Available from: NTIS, 

Springfield, VA; PB-292267. 

Thomas, G. B.; Fenters, J. D.; Ehrlich, R.; Gardner, D. E. (1981a) Effects of 

exposure to peroxyacetyl nitrate on susceptibility to acute and chronic 

bacterial infection. J. Toxicol. Environ. Health 8: 559-574. 

Thomas, G. B.; Fenters, J. D.; Ehrlich, R.; Gardner, D. E. (1981b) Effects of 

exposure to ozone on susceptibility to experimental tuberculosis. Toxicol. 

Lett. 9: 11-17. 

Thompson, D. J.; Lebowitz, M.; Casseil E. J. (1970) Health and the urban 

environment, air pollution, weather and the common cold. Am. J. Public 

Health 60: 731-739. 

Tice, R. R. ; Bender, M. A. ; Ivett, J. L. ; Drew, R. T. (1978) Cytogenetic 

effects of inhaled ozone. Mutat. Res. 58: 293-304. 

Trams, E. G.; Lauter, C. J.; Brown, E. A. B.; Young, 0. (1972) Cerebral cortical metabolism after chronic exposure to ozone. Arch. Environ. Health 24: 

153-159. 

Tremer, H. M.; Falk. H. L.; Kotan, P. (1959) Effect of air pollutants on ciliated mucus-secreting epithelium. JNCI J. Natl. Cancer Inst. 23: 979-994. 

Tyson,. C., . A..; _.Lunan., _ K.. a . D. ;_ Stephens,__ R.... J.._ (1982)..Age-rel.ated_. di ffer.ences. i n 

GSH-shuttle enzymes in NO 2 - or 03-exposed rat lungs. Arch. Environ. 

Health 37: 167-176. 

U.S. Department of Health, Education and Welfare. (1967) The health consequences 

of smoking. Washington, DC: Public Health Service; publication no. 1696. 

U.S. Department of Health, Education and Weifare. (1969) The health consequences 

of smoking: 1969 supplement to the 1967 Public Health Service review. 

Washington, DC: Public Health Service. 

9-293 
U.S. Environmental Protection Agency. (1978) Air quality criteria for ozone 

and photochemical oxidants. Research Triangle Park, NC: U.S. Environmental Protection Agency, Environmental Criteria and Assessment Office; 

EPA-600/8-78-004. Available --from: NTIS, Springfield, VA; PB80-124753. 

U.S. Environmental Protection Agency. (1982) Air quality criteria for particulate matter and sulfur oxides. Research Triangle Park, NC: U.S. Environmental Protection Agency, Environmental Criteria and Assessment Office; 

EPA report no. EPA-600/8-82-029a,b,c. Available from: NTIS, Springfield, 

VA; PB84-156777. 

Vaughan, T. R., Jr.; Jennelle, L. F.; Lewis, T. R. (1969) Long-term exposure 

to low levels of air pollutants: effects on pulmonary function in the 

beagle. Arch. Environ. Health 19: 45-50. 

Vaughan, W. J.; Adamson, G. L.; Lindgren, F. T.; Schooley, J. C. (1984) Serum 

lipid and lipoprotein concentrations following exposure to ozone. J. 

Environ. Pathol. Toxicol. Oncol. 5: 165-173. 

Veninga, T. S. (1967) Toxicity of ozone in comparison with ionizing radiation. 

Strahlentherapie 134: 469-477. 

Veninga, T. S. (1970) Ozone-induced alterations in murine blood and liver. 

Presented at: Second international clean air congress; December; 

Washington, DC. Washington, DC: International Union of Air .Pollution 

Prevention Associations; paper no. MB-15E. 

Veninga, T. S.; Lemstra, W. (1975) Extrapulmonary effects of ozone whether in 

the presence of nitrogen dioxide or not. Int. Arch. Arbeitsmed. 34: 

209-220. 

Veninga, T. S.; Wagenaar, J.; Lemstra, W. (1981) Distinct enzymatic responses 

in mice exposed to a range of low doses of ozone. EHP Environ. Health 

Perspect. 39: 153-157. 

Verweij, H.; Van Steveninck, J. (1980) Effects of semicarbazide on oxidative 

processes in human red blood cell membranes. Biochim. Biophys. Acta 602: 

591-599. 

Verweij, H., Van Steveninck, J. (1981) Protective effects of semicarbazide and 

p-aminobenzoic acid against ozone toxicity. Biochem. Pharmacol. 30: 1033- 

1037. 

Warshauer, D.; Goldstein, E.; Hoeprich, P. D.; Lippert, W. (1974) Effect of 

vitamin E and ozone on the pulmonary antibacterial defense mechanisms. 

J. Clin. Med. 83: 228-240. 

Watanabe, S.; Frank, R:; Yokoyama, E. (1973) Acute effects of ozone on lungs 

of cats. I. Functional. Am. Rev. Respir. Dis. 108: 1141-1151. 

Wayne, L. G.; Chambers, L. A. (1968) Biological effects of urban air pollution. V. A study of effects of Los Angeles atmosphere on laboratory 

rodents. Arch. Environ. Health 16: 871-885. 

9-294 
Wegner, C. D. (1982) Characterization of dynamic respiratory mechanics by measuring pulmonary and respiratory system impedances in adult bonnet 

monkeys (Macaca radiata): including the effects of long-term exposure to 

low-levei ozone [dissertation]. Davis, CA: University of California. 

Available from: University Micr.ofil-ms,.Ann Arbor, MI; publication 

no. 82-27900. 

Weibel, E. R. (1963) Morphometry of the human lung. New York, NY: Academic 

Press, Inc. 

Weiss, B.; Ferin, J.; Merigan, W.; Stern, S.; Cox, C. (1981) Modification of 

rat operant behavior by ozone exposure. Toxicol. Appl. Pharmacol. 

58: 244-251. 

Wilkins, E. S.; Fettissoff, P. (1981) The effect of air pollutants on lung 

surfactant surface tension. J. Envi•ron. Sci. Health A16: 477-491. 

Williams, P. S.; Caiabrese, E., J.; Moore, G. S. (1983a) An evaluation of the 

Dorset sheep as a predictive animal model for the response of G-6-PD 

deficient human erythrocytes to a proposed systemic toxic ozone intermediate. J. Environ. Sci. Health A18: 1-17. 

Williams, P. S.; Calabrese, E. J.; Moore, G. S. (1983b) The effect of inethyl 

linoleate hydroperoxide (MLHP), a possible toxic intermediate of ozone, 

on human normal and glucose-6-phosphate dehydrogenase (G-6-PD) deficient 

erythrocytes. J. Environ. Sci. Health A18: 37-49. 

Williams, P.; Calabrese, E. J.; Moore, G. S. (1983c) The effect of inethyl 

oleate hydroperoxide, a possible toxic ozone intermediate, on human 

normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes. 

Ecotoxicol. Environ. Saf. 7: 242-248. 

Williams, S. J.; Charles, J. M.; Menzel, D. B. (1980) Ozone induced alterations in phenol red absorption from the rat lung. Toxicol. Lett. 6: 

213-219. 

Wilmer, J. W. G. M.; Natarajan, A. T. (1981) Induction of sister-chromatid 

exchanges and chromosome aberrations by y-irradiated nucleic acid constituents in CH0 cells. Mutat. Res. 88: 99-107. 

Wilson, D. W.; Plopper, 

,C. G.; Dungworth, D. L. (1984) The response of the 

macaque tracheobronchial epithelium to acute ozone injury. A quantitative 

ultrastructural and autoradiographic stUdy. Am. J. Pathol. 116: 193-206. 

Winterbourn, C. C. (1979) Protection by ascorbate against acetylphenylhydrazineinduced Heinz body formation in glucose-6-phosphate dehydrogenase deficient 

erythrocytes. Br. J. Haematol. 41: 245-252. 

Witz, G.; Amoruso, M. A.; Goldstein, B. D. (1983) Effect of ozone on alveolar 

macrophage function: membrane dynamic properties. In: Lee, S. D.; 

Mustafa, M. G.; Mehlman, M. A., eds. International symposium on the 

biomedical effects of ozone and related photochemical oxidants; March 

1982; Pinehurst, NC. Princeton, NJ: Princeton Scientific Publishers, 

Inc.; pp. 263-272. (Advances in modern environmental toxicology: v. 5). 

9-295 
Woicott, J. A.; Zee, Y. C.; Osebold, J. W. (1982) Exposure to ozone reduces 

influenza disease severity and alters distribution of influenza viral 

antigens in murine lungs. Appl. Environ: Microbiol. 44: 723-731. 

Wong, D. S.; Hochstein, P. (1981) The potentiation of ozone toxicity by thyroxine. Res. Commun. Chem. Pathol. Pharmacol. 31: 483-492. 

Wood, R. W. (1979) Behavioral evaluation of sensory irritation evoked by 

ammonia. Toxicol. Appl. Pharmacol. 50: 157-162. 

World Health Organization. (1961) Chronic cor pulmonale: report of an expert' 

committee. Geneva, Switzerland: World Health Organization. (Technical 

report series: no. 213). 

Wright, J. L.; Lawson, L. M.; Pare, P. D.; Wiggs, B. J.; Kennedy, S.; Hogg, J. C. 

(1983) Morphology of peripheral airways in current smokers and ex-smokers. 

Am. Rev. Respir. Dis. 127: 474-477. 

Xi ntaras, C. ; Johnson, B. L. ; U1 ri ch, C. E. ; Terri 11 , R. E. ; Sobecki , M. F. 

(1966) Application of the evoked response technique in air pollution 

toxicology. Toxicol.•Appl. Pharmacol. 8: 77-87. 

Yilma, T.; Zee, Y. C.; Osebold, J. W. (1979) Immunofluorescence determination 

of the pathogenesis of infection with influenza virus in mice following 

exposure to aerosolized virus. J. Infect: Dis. 139: 458-464. 

Yokoyama, E. (1969) A comparison of the effects of S0 2 , NO2 and 0 3 on the 

pulmonary ventilation: guinea pig exposure experiments. Sangyo Igaku 

11: 563-568. 

Yokoyama, E. (1972) The effects of 0 3 on lung functions. In: Proceedings of 

the science lecture meeting on air pollution caused by photochemical 

reaction; March; Tokyo, Japan. Tokyo, Japan: Japan Society of Air 

Pollution; pp. 123-143. 

Yokoyama, E. (1973) The effects of low-concentration ozone on the lung pressure 

of rabbits and rats. Presented at: 46th annual meeting of the Japan 

Society of Industrial Hygiene; April; paper 223. 

Yokoyama, E. (1974) On the maximal expiratory flow volume curve of rabbits' 

exposed to ozone. Nipon Kyobu Shikkan Gakkai Zasshi 12: 556-561. 

Yokoyama, E.; Frank, R. (1972)__Respiratory uptake of ozone in . dogs. Arch 

Environ. Health 25: 132-138. 

Yokoyama, E.; Ichikawa, I. (1974) Study on the biological effects of atmospheric pollutants (FY 1972-1975). In: Research report for funds of the 

Environmental Agency in 1974. Tokyo, Japan: Institute of Public Health, 

Department of Industrial Health; pp. 16-1 - 16-6. 

Yokoyama, E.; Ichikawa, I.; Nambu, Z.; Kawai, K.; Kyono, Y. J. (1984) 

Respiratory effects of intermittent exposure to ozone of rats. Environ. 

Res. 33: 271-283. 

9-296 
Younes, M.; Siegers, C.-P. (1980) Lipid peroxidation as a consequence of 

glutathione depletion in rat and mouse liver. Res. Commun. Chem. Pathol. 

Pharm. 27: 119-128. 

Yu (19 

_ ......_.._..__.. 

, C. P. 75) On equations of gas transport in the lung. Respir. Physiol. 

23: 257-266. 

Zelac, R. E.; Cromroy, H. L.; Bolch, W. E., Jr.; Dunavant, B. G.; Bevis, H. A. 

(1971a) Inhaled ozone as a mutagen. I. Chromosome aberrations induced in 

Chinese hamster lymphocytes. Environ. Res. 4: 262-282. 

Zelac, R. E.; Cromroy, H. L.; Bolch, W. E., Jr.; Dunavant, B. G.; Bevis, H. A. 

(1971b) Inhaled ozone as a mutagen. II. Effect on the frequency of chromosome aberrations observed in irradiated Chinese hamsters. Environ. Res. 

4: 325-342. 

Zitnik, L. A.; Schwartz, L. W.; McQuillen, N. K.; Zee, Y. C.; Osebold, J. W. 

(1978) Pulmonary changes induced by low-level ozone: morphological observations. J. Environ. Pathol. Toxicol. 1: 365-376. 

Zitting, A.; Savolainen, H. (1980) Biochemical effects of subacuteformic acid 

vapor exposure. Res. Commun. Chem. Pathol. Pharmacol. 27: 157-162. 

9-297 

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 driplets Pat are dispersed or suspended 

in a gas, ranging in size from 10 to 10 micrometers ({am). 

Air spaces: A11 alveolar ducts, alveolar sacs, and alveoli. To be contrasted 

with AIRWAYS. 

Airway conductance (Gaw): Reciprocal of airway resistance. Gaw =(1/Raw). 

Airway resistance (Raw): The (frictional) resistance to airflow afforded by 

the airways betwe.en the airway opening at the mouth and the alveoli. 

Airways: All passageways of the respiratory tract from mouth or nares down to 

and including respiratory bronchioles. To be contrasted with AIR SPACES. 

Allergen: A material that, as a result of coming into contact with appropriate 

tissues of an animal body, induces a state of allergy or hypersensitivity; 

generally associated with idiosyncratic hypersensitivities. 

Alveolar-arterial oxygen pressure difference [P(A-a)0 2 ]: The difference in 

partial pressure of 0 in the alveolar gas spaces and that in the systemic 

arterial blood, measu ~ ed in torr. 

Alveolar-capillary membrane: A fine membrane (0.2 to 0.4 pm) separating 

alveolus from capillary; composed of epithelial cells lining the alveolus, 

a thin layer of connective tissue, and a layer of capillary endothelial 

cells. 

Alveolar carbon dioxide pressure (p CO ): 'Partial pressure of carbon dioxide 

in the air contained in the lung alv eoli. 

Alveolar oxygen partial pressure (P 0): Partial pressure of oxygen in the 

air contained in the aiveoli ofA the lungs. 

Alveolar septum (pl. 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. Physiol. 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. 

A-1 
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 1 ung. 

Alveolus: Hexagonal or spherical air cells of the lungs. The majority of 

alveoli arise from the alveolar ducts which are lined with the alveoli. 

An alveolus is an ultimate respiratory unit where the gas exchange takes 

place. 

Anatomical dead space (V )• Volume of the conducting airways down to the 

level where, durinT a~tbreathing, gas exchange with blood can occur, a 

region probably situated at the entrance of the alveolar ducts. 

Arterial oxygen saturation (Sa0 2 ): Percent saturation of dissolved oxygen in 

arterial blood. 

Arterial partial pressure of carbon dioxide (PaCO 2 ): Partial pressure of 

dissolved carbon dioxide in arterial blood. 

Arterial partial pressure of oxygen (Pa0 2 ): Partial pressure of dissolved 

oxygen in arterial blood. 

Asthma: A disease characterized by an increased responsiveness of the airways 

to various stimuli and manifested by slowing of forced expiration which 

changes in severity either•spontaneously or as a result of therapy. The 

term asthma may be modified by words or phrases indicating its etiology, 

factors provoking attacks, or its duration. 

Atelectasis: State of collapse of air spaces with elimination of the gas 

phase. 

ATPS condition (ATPS): Ambient temperature and pressure, saturated with'water 

vapor. These are the conditions existing in a water spirometer. 

Atropine: A poisonous white crystalline alkaloid, C H NO , from belladonna 

and related plants, used to relieve spasms of s~oo~ r~uscles. 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 originating in the lungs. The reflex ares are formed by the pulmonary mechanoreceptors, the vagal afferent fibers, the respiratory centers, the medullospinal pathway, the motor neurons, and the respiratory muscles. The afferent 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 reflexes 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 disregarded by Breuer and Hering, in which sudden inflation may stimulate 

inspiratory muscles. 

A-2 
Bronchiole: One of the finer subdivisions of the airways, less than 1 mm in 

diameter, and having no cartilage in its wall. 

Bronchioiitis: 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 etiology, 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 ciassic 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 (diameter) 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 rigtat and left main bronchi 

which in turn form lobar, segmental, and subsegmental bronchi. 

BTPS conditions (BTPS): Body temperature, barometric pressure, and.saturated 

with water vapor. These are the conditions existing in the gas phase of 

the lungs. For man the normal temperature is taken as 37°C, the pressure 

as the barometric pressure, and the partial pressure of water vapor as 47 

torr. 

Carbachol: A parasympathetic stimulant (carbamoy-lchoiine chloride, C 6H15C1N20 2 ) 

that produces constriction of the bronchial smooth muscles. 

Carbon dioxide production (VCO 2 ): Rate of carbon dioxide production by organisms, tissues, or cells. Common units: ml CO 2 (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 2 . 

A-3 
Chronic obstructive lung disease (COLD): This term refers to diseases of 

uncertain etiology characterized by persistent slowing of airflow during 

forced expiration. It is recommended that a more specific term, such as 

chronic obstructive bronchitis or chronic obstructive emphysema, be used 

whenever possible. Synonymous with chronic obstructive pulmonary disease 

(COPD). 

Closing capacity (CC): Closing volume plus residual volume, often expressed 

as a ratio of TLC, i.e. (CC/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 VENTfL.oAHION and RESISTANCE. 

Collateral ventilation: Ventilation of air spaces via indirect pathways, 

e.g., through pores in alveolar septa, or anastomosing respiratory bronchioles. 

Compliance (C L ,C t): A measure of distensibility. Pulmonary compliance is 

given by th~ slope of a static volume-pressure curve at a point, or the 

linear approximation of a nearly straight portion of such a curve, expressed in liters/cm H 0 or ml/cm H 0. Since the static volume-pressure 

characteristics of lun6s are nonlin~ear (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 ineasurement 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 0, D CO , D CO): Amount of gas (0 

CO 3CO ) commonly expressed is m~ 6as ~'STF~D) cH ffusi ng between al veol?a

,

r 

gas ang pulmonary capillary blood pei^ torr mean gas pressure difference 

per min, i.e., ml 0 2/(min-torr). Synonymous with transfer factor and 

diffusion factor. 

Dynamic compliance (C n )• The ratio of the tidal volume to the change in 

intrapleural pres~~ure between the points of zero flow at the extremes of 

tidal volume in liters/cm H 0 or ml/cm H 2 O. Since at the points of zero 

airflow at the extremes of 2tidal 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. 

A-4 
Elastance (E): The reciprocal of COMPLIANCE; expressed in cm H 20/liter or cm 

H 20/ml. 

Electrocardiogram (ECG,-EKG): The graphic record of the electrical currents 

that are associated with the heart's contraction and relaxation. 

Emphysema: A condition of the lung characterized by abnorinal, permanent 

enlargement of airspaces distal to the terminal bronchiole, accompanied 

by the destruction of their walls, and without obvious fibrosis. 

Expiratory reserve volume (ERV): The maximal volume of air exhaled from the 

end-expiratory level. 

FEVt/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 

perXt 100 x FEVt/FVC. It is an index of airway obstruction. 

Flow volume curve: Graph of instantaneous forced expiratory flow recorded at 

the mouth, against corresponding lung volume. When recorded over the 

full vital capacity, the curve includes maximum expiratory flow rates at 

ai 1 1 ung vol umes i n the VC range and i s cal l ed a maxi inum expi ratory 

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 measurement is made. For example: 

FEF75~ = instantaneous forced expiratory flow after 75% 

of the FVC has been exhaled. 

FEF200-1200 = mean forced expiratory flow between 200 ml 

and 1200 ml of the FVC (formerly cailed 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)]. 

FEFinax = the maximal forced expiratory flow achieved during 

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 FEVO 5' 

FEV 5 , FEVl These values are often expressed as a percent of the 

for~e~ vital c4acity, e.g. (FEV1 0/VC) X 100. 

Forced inspiratory vital capacity (FIVC): The maximal voiume 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. 

A-5 

I 
Functional residual capacity (FRC): The sum of RV and ERV (the volume of air 

remaining in the lungs at the end-expiratory position). The method of 

measurement should be indicated as with RV. 

Gas exchange: Movement of oxygen from the alveoli into the pulmonary capillary 

blood as carbon dioxide enters the alveoli from the blood. In broader 

terms, the exchange of gases between alveoli and lung capillaries. 

Gas exchange ratio (R): See RESPIRATORY QUOTIENT. 

Gas trapping: Trapping of gas behind small airways that were opened during 

inspiration but closed during forceful expiration. It is a volume difference 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 hemg+ group. The heme is made of four pyrrole rings 

and a divalent iron (Fe -protoporphyrin) which combines reversibly wi'th 

molecular oxygen. 

Histamine: A depressor amine derived from the amino acid histidine and found 

in all body tissues, with the highest concentration in the lung; a powerful 

stimulant of gastric secretion, a constrictor of bronchial smooth muscle, 

and a vasodilator that causes a fall in blood pressure. 

Hypoxemia: A state in which the oxygen pressure and/or concentration in 

arterial and/or venous blood is lower than its normal value at sea level. 

Normal oxygen pressures at sea level are 85-100 torr in arterial blood 

and 37-44 torr in mixed venous blood. In adult humans the normal oxygen 

concentration is 17-23 ml 0/100 ml arterial blood; in mixed venous blood 

at rest it is 13-18 ml 0 2/160 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 1 evel . If the P Oi s 1 ow i n the envi ronment, whether because of 

decreased barometric 1~^essure or decreased fractional concentration of 

0, the condition is termed environmental hypoxia. Hypoxia when referring 

tg 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 

hypoxiaQ2 which occurs when the local circulation is low compared to the 

local metabolism. 

Inspiratory capacity (IC): 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. 

A-6 
Kilogram-meter/min (kg-m/min): The work performed each min to move a mass of 1 

kg through a vertical distance of 1 m against the force of gravity. 

Synonymous with kiiopond-meter/min. 

Lung volume (V ): Actual volume of the lung, including the volume of the 

conductin~ airways. 

Maximal aerobic capacity (max VO): The rate of oxygen uptake by the body 

during repetitive maximal r3spiratory effort. Synonymous with maximal 

oxygen consumption. 

Maximum breathing capacity (MBC): Maximal volume of air which can be breathed 

per minute by a subject breathing as quickly and as deeply as pos . sible. 

This tiring lung function test is usually limited to 12-20 sec, but given 

in liters (BTPS)/min. Synonymous with maximum voluntary ventiiation (MVV). 

Maximum expiratory flow (V ): Forced expiratory flow, related to the 

total lung capacity orm&xactual volume of the lung at which the measurement is made. Modifiers refer to the amount of iung volume remaining 

when the measurement is made. For example: 

V

max 75% - instantaneous forced expiratory flow when the 

lung is at 75% of its TLC. 

V

max 3.0 - instantaneous forced expiratory flow'when the 

lung voiume is 3.0 liters 

Maximum expiratory flow rate (MEFR): Synonymous with FEF200-1200- 

Maximum mid-expiratory flow rate (MMFR or MMEF): Synonymous with FEF25-75%. 

Maximum ventilation (max V): The volume of air breathed in one minute during 

repetitive maximal re ~piratory effort. Synonymous with maximum ventilatory 

minute volume. 

Maximum voluntary ventilation (MVV): The volume of air breathed by a subject 

during voluntary maximum hyperventilation iasting 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 transport. Methemoglobins are formed by various drugs and occur under pathological conditions. Many methods for hemoglobin measurements utiiize 

methemoglobin (chlorhemiglobin, cyanhemiglobin). 

Minute ventilation (V ): Volume of air breathed in one minute. It is a 

product of tidal olume (VT ) and breathing frequency (f B ). See VENTILATION. 

Minute volume: Synonymous with minute ventilation. 

Mucociliary transport: The process by which mucus is transported, by ciliary 

action, from the lungs. 

A-7 
Mucus: The clear, viscid secretion of mucous membranes, consisting of mucin, 

epithelial cells, leukocytes, and various inorganic salts suspended in 

water. 

Nasopharyngeal: Relating to the nose or the nasal cavity and the pharynx 

(throat). 

Nitrogen oxides: Compounds of N and 0 in ambient air; i.e., nitric oxide (NO) 

and others with a higher oxidation state of N, of which NO 2 is the most 

important toxicologically. 

Nitrogen washout (ON, dN ): The curve obtained by plotting the fractional 

concentration oi N ~in expired alveolar gas vs. time, for a subject 

switched from breat~ ing ambient air to an inspired mixture of pure 0. A 

progressive decrease of N concentration ensues which may be analy ~ed 

into two or more exponent?al components. Normally, after 4 min of pure 

0 breathing the fractional N2 concentration in expired alveolar gas is 

d26wn to less than 2%. 

Normoxia: A state in which the ambient oxygen pressure is approximately 150 ± 

10 torr (i.e., the partial pressure of oxygen in air at sea level). 

Oxidant: A chemical compound that has the ability to remove, accept, or share 

electrons from another chemical species, thereby oxidizing it. 

Oxygen consumption (V0 2 , QO ): Rate of oxygen uptake of organisms, tissues, 

or cells. Common units: ml 02 (STPD)/(kg•min) or ml 0(STPD)/(kg•hr). 

For whole organisms the oxygen consumption is commonly ~cpressed per unit 

surface area or some power of the body weight. For tissue samples or 

isolated cells Q 02 =Pl 02/hr per mg dry weight. 

Oxygen saturation (SO): The amount of oxygen combined with hemoglobin, 

expressed as a p~centage of the oxygen capacity of that hemoglobin. In 

arterial blood, Sa0 2 . 

Oxygen uptake (V0 2 ): Amount of oxygen taken up by the body from the environment, 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 0 2 stores 

are constant. 

Particulates: Fine solid particles such as dust, smoke, fumes, or smog, found 

in the air or in emissions. 

Pathogen: Any virus, microorganism, or etiologic agent causing disease. 

Peak expiratory flow (PEF): The highest forced expiratory flow measured with 

a peak fl ow meter. 

Peroxyacetyl nitrate (PAN): Pollutant created by action of UV component of 

sunlight on hydrocarbons and NO x in the air; an ingredient of photochemical smog. 

m 
Physiological dead space (V ): Calculated volume which accounts for the 

difference between the % ressures of CO in expired and alveolar gas (or 

arterial blood). Physiological dead s ~ace 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 

ventilationlperfusion ratio in the lung. 

Plethysmograph: A rigid chamber placed around a living structure for the 

purpose of ineasuring changes in the volume of the structure. In respiratory measurements, the entire body is ordinarily enclosed ("body plethysmograph") 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 paraliel capillary tubes (Fleisch type) or of 

fine-meshed screen (Silverman-Lilly type). 

Pulmonary alveolar proteinosis: A chronic or recurrent disease characterized 

by the filling of alveoli with an insoluble exudate, usually poor in 

cells, rich in lipids and proteins, and accompanied by minimal histologic 

alteration of the alveolar walls. 

Pulmonary edema: An accumulation of excessive amounts of fluid in the lung 

extravascular tissue and air spaces. 

Pulmonary emphysema: An abnormal, permanent enlargement of the air spaces 

distal to the terminal nonrespiratory bronchiole, accompanied by destructive 

changes of the alveolar walls and without obvious fibrosis. The term 

emphysema may be modified by words or phrases to indicate its etiology, 

its anatomic subtype, or any associated airways dysfunction. 

Residual volume (RV): That volume of air remaining in the lungs after maximal 

exhalation. The method of ineasurement should be indicated in the text 

or, when necessary, by appropriate qualifying symbols. 

A-9 
Resistance flow (R): The ratio of the flow-resistive components of pressure 

to simultaneous flow, in cm H 0/liter per sec. Flow-resistive components 

of pressure are obtained by Abtracting 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 ineasurement is necessary; see AIRWAY RESISTANCE, 

TISSUE RESISTANCE, TOTAL PULMONARY RESISTANCE, COLLATERAL RESISTANCE. 

Respiratory cycle: A respiratory cycle is constituted by the inspiration 

followed by the expiration of a given volume of gas, called tidal volume. 

The duration of the respiratory cycle is the respiratory or ventilatory 

period, whose reciprocal is the ventilatory frequency. 

Respiratory exchange ratio: See RESPIRATORY QUOTIENT. 

Respiratory frequency (f ): The number of breathing cycles per unit of time. 

Synonymous with bre ~thing frequency (fB ). 

Respiratory quotient (RQ, R): Quotient of the volume of CO 2 produced divided 

by the volume of 0 consumed by an organism, an.organ, or a tissue during 

a given period of 2time. 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 CO output to the 0 uptake by the lungs, "respiratory 

quoti ent" bei rig 2restri cted to the2actual metabol i c CO output and 0 

uptake by the tissues. With this definition, respira&y quotient agd 

respiratory exchange ratio are identical in the steady state, a condition 

which implies constancy of the 02 and CO 2 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-toleft shunt within the lung, heart, or large vessels due to malformations 

are more important in respiratory physiology. Flow from left to right 

through a shunt should be marked with a negative sign. 

Specific airway conductance (SGaw): Airway conductance divided by the lung 

volume at which it was measured, i.e., normalized airway conductance. 

SGaw = Gaw/TGV. 

Specific airway resistance (SRaw): Airway resistance multiplied by the volume at 

which it was measured. SRaw = Raw x TGV. 

Spirograph: Mechanical device, including bellows or other scaled, moving 

part, which collects and stores gases and provides a graphical record of 

volume changes. See BREATHING PATTERN, RESPIRATORY CYCLE. 

Spirometer: An apparatus similar to a spirograph but without recording facility. 

A-10 
Static lung compliance (C L): Lung compliance measured at zero f.low '(breathholding) over linear ~~ rtion of the volume-pressure curve above FRC. See 

COMPLIANCE. 

Static transpulmonary pressure (P t): Transpulmonary pressure measured at a 

specified lung volume; e.g., ~ TLC is static recoil pressure measured at 

TLC (maximum recoil pressure). st 

Sulfur dioxide (SO ): Colorless gas with pungent odor, released primarily from 

burning of fo3sil fuels, such as coal, containing sulfur. 

STPD conditions (STPD): Standard temperature and pressure, dry. These are 

the condi ti ons of a vol ume 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 VTshould be used. 

Tissue resistance (Rti ): Frictional resistance of the pulmonary and thoracic 

tissues. 

Torr: A unit of pressure equal to 1,333.22 dynes/cm 2or 1.33322 millibars. 

The torr is equal to the pressure required to support a column of inercury 

1 mm high when the mercury is of standar,d density and subjected to standard 

acceleration. These standard conditions are met ~t 0° C and 45°latitude, 

where the acceleration of gravity is 980.6 cm/sec . In reading a mercury 

aarometer 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 ineasurement 

should be indicated, as with RV. 

A-11 
Total pulmonary resistance (R ): Resistance measured by relating fiow-dependent 

transpulmonary pressure ~to airflow at the mouth. Represents the total 

(frictional) resistance of the lung tissue (Rti ) and the airways (Raw). 

RL = Raw + Rti . 

Trachea: Commonly known as the windpipe; a cartilaginous air tube extending 

from the larynx (voice box) into the thorax (chest) where it divides into 

left and right branches. 

Transpulmonary pressure (P ): Pressure difference between airway opening 

(mouth, nares, or cankula opening) and the visceral pleural surface, in 

cm H 0. Transpulmonary in the sense used includes extrapulmonary structureE, e.g., trachea and extrathoracic airways. This usage has come 

about for want of an anatomic term which includes all of the airways and 

the lungs together. 

Ventilation: Physiological process by which gas is renewed in the lungs. The 

word ventilation sometimes designates ventilatory flow rate (or ventilatory minute volume) which is the product of the tidal volume by the 

ventilatory frequency. Conditions are usualiy indicated as modifiers; 

i.e., 

VE= Expired volume per minute (BTPS), 

and 

VI=.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 A): Physiological process by which alveolar gas is 

completely removed and replaced with fresh gas. Alveolar ventilation is 

less than total ventilation because when a tidal volume of gas leaves the 

alveolar spaces,'the last part does not get expelled from the body but 

occupies the dead space, to be reinspired with the next inspiration. 

Thus the volume of alveolar gas actually expelled completely is equal to 

the tidal volume minus the volume of the dead space. This truly complete 

expiration volume times the ventilatory frequency constitutes the alveolar 

ventilation. 

Ventilation, dead-space (V ): Ventilation per minute of the physiologic dead 

space (wasted ventilaUon), BTPS, defined by the following equation: 

VD = VE (PaCO2 - P ECO2 )/(PaCO2 - P I CO2 ) 

Ventilation/perfusion ratio (V /Q): Ratio of the alveolar ventilation to the 

blood perfusion volume fl through the pulmonary parenchyma. This ratio 

is a fundamental determinant of the 0 2and CO pressure of the alveolar 

gas and of the end-capillary blood. Througgut the lungs the local 

ventilation/perfusion ratios vary, and consequently the local alveolar 

gas and end-capillary blood compositions also vary. 

Vital capacity (VC): The maximum volume of air exhaled from the point of 

maximum inspiration. 

•4.5. OQ'<t72*= MtC1t[Y06 OillCit 1966®646-116140607 

A-12 

Ca= av -_^® m 

~ A C =r a 

7' 0 m m = f8 

fe.o 0 °.°m 

m 

oC H 

m — 

o S 1 w I 

y O G n 7 

EB p ty' W 

M N 

fD l8 C y 

a~ o ~ 

m 

®m mm' 

~ 

CD 

~ a y 

~ 

o 0 a 'm 

~ 

fB n w 2 

y. IB 

7 ri m 

o.n 

. Q 

o 

m~ c'~ y = C 

m D d 

~ m ~m

Cr 

 cb 

m~ — 

m 

v D \ 

C31 O O 

\ 

W 

80 -La 

\ 

C) 

N O f~. ~1 

H 

~ O Dm m ~ m < o ~ . ~• 

o W 3 

-~ y fD 

~ 

m

' m 

G CD ~ 

~ (A 

-o 

O O 

C m 

b) l7 

O 

° N 7 

W 

O O 

n ~ 

n O 

:3 d 

d .+ _.• a• 

O~ 

x 

.~ 

cn 

N 

0) 

0 

CD 

i 

k 

o • 

~ 

0 

n 'U 

'O f0~ n 

o ~ D 

W C ~ ~ C 

o ~ ° ^ 

~ 

r =~m~~~ . 

~ ~ 

d 

n c ~ 

D m~ ~ m 

d 

~ 

cn I 

D 

v 

r,w