United States Environmental
Environmental Protection Criteria and
Agency Assessment Office EPA-600/8-80- 014t/
SEPA Long-Term Effects
Of Air Pollutants: in Canine Species
Do not weed. This document
should be retained in the EPA
Region 5 Library Collection.
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EPA-600 / 8-80-014
July, 1980
V
v*.
Long-Term
Effects of
Air Pollutants:
in Canine Species
Editors:
U.S. Environmental Protection Agency J. R Stara
Region 5, Library (PL-12J) D. L. Dungworth*
77 West Jackson Boulevacd, 12th Floor J. G. Qrthoefer
Chicago, IL 60604-3590 w s Tyler*
Environmental Criteria and Assessment Office
U.S. Environmental Protection Agency
Cincinnati, Ohio
and
*Department of Pathology
School of Veterinary Medicine
University of California
Davis, California
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, Cincin-
nati, Ohio, and approved for publication. Mention of trade names or
commercial products does not constitute endorsement or recommen-
dation for use.
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FOREWORD
The Clean Air Act of 1970 as amended in 1977 requires that a comprehensive data base
be established to assess human health effects caused by air pollution from mobile
sources. The spectrum of potential toxic effects can be viewed from two perspectives:
The first is the identification of toxic effects from combined pollutants contained in
mobile emissions; the second is detailing the long-term, low-level effects of the individ-
ual major ambient air pollutants, which are combustion by-products of automotive
exhaust. Ideally, the major components of a data base used to develop health risk
assessments are well-designed epidemiological studies and long-term, low-level animal
studies. In epidemiological studies, the obvious limitation is the identification of the
concentrations to which the specified population was exposed. Acute animal studies,
although pertinent in some situations, cannot provide the information necessary for
evaluating the potential hazard of the continuous, low-level exposure which is often
more representative of existing environmental conditions. Chronic studies frequently
establish effect levels below those used in acute studies, and provide the opportunity to
assess irreversible tissue and organ changes which are often the major toxic manifesta-
tions of low-level insult.
The 9-year study presented in this monograph reviews the effects following exposure of
dogs for 68 months to automotive exhaust, simulated smog, oxides of nitrogen, oxides
of sulfur, and their combinations. Studies using canine species over extended periods of
time have proven useful in the evaluation of risk to humans, especially when combined
with epidemiological studies and human clinical investigations.
All of the data were reviewed by invited expert scientists at a conference held at
Asilomar, California. Their evaluations and judgments form a significant segment of
this monograph.
This research demonstrates a close alliance between government and university labora-
tories. For the first 6 years, the study was performed at the Environmental Toxicology
Research Laboratory, U.S. Environmental Protection Agency, in Cincinnati. The con-
cluding 3 years of the study were completed at the University of California, Davis. The
collaborative effort between scientists of both institutions has produced data which will
be a valuable contribution in the quest for a better understanding of the effects of
mobile source pollution.
-V&r^
-jr
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EXECUTIVE SUMMARY
Female beagle dogs comprising one control group (20) and seven treatment groups (12 each)
were exposed 16 hours daily for 68 months to filtered air, raw or photochemically reacted
auto exhaust, oxides of sulfur or nitrogen, or their combination. Mixtures of pollutants were
chosen to provide various combinations of components generated by automobile emissions.
The concentrations chosen were mostly in the approximate range of two to six times greater
than averaged daily maxima occurring in several large cities during the exposure period.
Specific exposures were to clean filtered air (CA), raw auto exhaust (R), irradiated auto
exhaust (I), sulfur oxides (SOX: 1100 ^g/m3 S02 and 90 Mg/m3 H2S04), raw auto exhaust with
added sulfur oxides (R + SOX), irradiated auto exhaust with sulfur oxides (I + SOX), and two
mixtures of oxides of nitrogen — a high nitrogen dioxide level (NOL+N02H: 1210 /^g/m3
N02 and 310 Mg/m3 NO) and a low nitrogen dioxide level (NOR + N02L: 270 Mg/m3 N02 and
2050 ng/m3 NO). Following the end of the 5-year exposure period, the dogs were kept for
approximately 3 years in a normal indoor atmosphere to determine whether their health
status would improve or worsen.
Clinical examinations were conducted regularly throughout the 8V6-year experimental period.
Hematologic studies were done at 6-month intervals. Special clinical examinations were:
pulmonary function studies after 18, 36, and 61 months of exposure, and again 24 months
after exposure ceased; cardiovascular studies after 48 and 54 months of exposure, and
immediately prior to necropsy 32 to 36 months after the end of exposure; and neurologic
assessment (visual evoked brain potentials) in the last month of exposure. Radiologic
examinations were done 18 and 24 months after the end of exposure. Morphologic studies
were based on necropsies performed 32 to 36 months after the end of exposure, though 16
pulmonary biopsy specimens obtained from control and two treatment groups were also
examined. Samples of lung and bone were obtained at necropsy for subsequent collagen and
lead determinations, and samples of brain, liver, heart, and lungs were obtained for lipid
analysis.
The statistically significant and important findings were exposure-related abnormalities in
pulmonary function and structure in the beagles. There was a good level of correlation
between functional and structural abnormalities using pooled data of grouped animals.
Current further analysis indicates good correlation on an individual dog basis. This correla-
tion between functional and structural findings strengthens the validity and implications of
the data.
Small abnormalities in pulmonary function were detected after 61 months of exposure, 7
months before it ended. When the same dogs were studied 2 years after the termination of
exposure, many of their pulmonary function values showed an even greater difference from
those of controls. The controls appeared to have stable pulmonary function values similar to
those of other healthy beagles. Since these dogs were handled the same except for exposure
and were of the same age and breed, it can be concluded that the differences measured were
due to the effects of the exposure regimens. Exposed beagles had abnormalities in ventilatory,
resistance, blood-gas exchange, and lung volume values. Groups exposed to R and R + SOX
had the greatest changes in indicators of airway disease — increases in pulmonary resistance,
physiological dead space, and pulmonary arterial carbon dioxide. This finding was in
accordance with subsecpient morphologic observations. Groups exposed to SOX and N02 had
IV
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the greatest changes indicating pulmonary parenchymal abnormality — increases in lung
volumes and dynamic compliance and decrease in pulmonary diffusing capacity. This finding
also correlated well with the fact that these groups were most affected by early
emphysematous-type lesions.
Two important structural abnormalities were present in lungs of exposed dogs. Morphologic
and morphometric analysis revealed enlargement and breakdown of air spaces centered on
respiratory bronchioles and alveolar ducts in all but the groups exposed to R and R + SO*.
It was most severe in the dogs exposed to high N02 or SOX. The abnormality in air spaces
can be considered to be analogous to early human centrilobular (centriacinar) emphysema.
Although mild compared to clinically significant emphysema in man, it was sufficient to cause
abnormalities in pulmonary function. The other major structural abnormality was hyperplasia
of bronchiolar epithelial cells. It occurred to some degrees in all exposure groups but was
worse in the dogs exposed to R and R + SO* — the two groups lacking emphysematous-type
changes.
Several important implications are raised by this study relative to assessment of the risks of
air pollution on human populations and the establishment of air quality criteria. Mild but
unequivocal functional and structural changes were produced in the lungs of beagles exposed
to several mixes of pollutant at concentrations in the range of two to six times average daily
maxima in selected cities. Taking high N02 as an example, the exposure concentration was
1210 Mg/m3 to which was added 310 ^g/m3 of the less toxic NO. In severe pollution episodes,
peaks of N02 can exceed 300 Mg/m3. The National Ambient Air Quality Standard for NOa is
an annual arithmetic mean 24-hour value of 100 Mg/m3. This is approximately one-tenth of
the level that proved damaging to beagle lungs in the 68-month, 16-hour-a-day exposure.
Similarly, with the SOX exposure the levels were 1100 Mg/m3 of S02 and 90 Mg/m3 of r^SCU.
Although the situation is compounded by the role of particulates and by difficulties in
determining the exact states of sulfur oxides, S02 concentrations as such can exceed 250
Mg/m3 in severe pollution episodes. The air quality standard is an annual arithmetic mean
24-hour value of 80 /^g/m3. This is slightly less than one-tenth of the level causing pulmonary
damage in the exposed beagles.
The structural abnormalities persisted for 3 years following the end of the 68-month exposure.
The indication from the continued loss of pulmonary function after exposure ceased is that
the damage worsened. The conclusion that the long-term, low-level exposures produced
persistent damage, which although slight appeared to be progressive, makes the findings even
more important in predicting possible effects in man over a life span. They provide strong
support for no relaxation in the air quality standards.
A further conclusion is that because of the complexity of biological interactions in the lungs
of an exposed man or animal, judiciously selected long-term studies will be necessary to fill
the gap between short-term in vitro or in vivo experimental studies and epidemiological
observations of human populations.
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ACKNOWLEDGEMENTS
This research encompassed a period of over 9 years; during this time it was con-
ducted uninterrupted at both the U.S. Environmental Protection Agency research
laboratories in Cincinnati and the University of California, Davis. The editors
acknowledge the assistance of all who were involved in the project: this includes
scientists who have contributed to the study design and collection of data, and
technical personnel who were involved in the daily operation of the exposure sys-
tem, the animal chamber maintenance, animal care, physical and biological mea-
surements and other research requirements. A special thanks goes to Dr. F.
Gordon Hueter, presently the Director of the Health Effects Research Laboratory-
RTF, Research Triangle Park, North Carolina, who initiated the study in 1965.
Furthermore, the editors would like to acknowledge the individuals who compiled
and reduced the enormous amount of data into an acceptable form.
In order to review and evaluate the data, a conference was held at Asilomar.
California. Many individuals contributed to its technical and organizational
success, and they have our appreciation. Special recognition is extended to:
Carol Haynes (U.S. EPA) and Arlene Kasmire (UC, Davis), who made arrange-
ments for the conference and speakers; and Dotty Morrissey (UC, Davis), Verna
Tilford (U.S. EPA), and Donna Consiglio (UC, Davis), who typed and trans-
cribed the proceedings. Their dedicated help made this monograph possible.
We also wish to express our thanks to Jan Connery of Energy Resources Co., Inc.,
who spent many long hours correcting and editing this monograph.
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TABLE OF CONTENTS
Page
FOREWORD iii'
EXECUTIVE SUMMARY iv
ACKNOWLEDGEMENTS vi
LIST OF FIGURES ix
LIST OF TABLES xiii
INTRODUCTION 1
J.E Stara
CHAPTER
1. Study Overview, Rationale, Experimental Design, Experimental Facilities
and Exposure Atmospheres 11
J.F. Stara, K. Busch, R.H. Hinners and J.K. Burkart
2. Exposure Chamber Atmospheres. Sampling and Analysis 41
Af. Malanchuk
3. Clinical Summary of Beagle Study. Physical Examinations, Ocukr
Examinations, Hematology Examination (Post-Exposure) and
Blood Chemistries 55
J.G. Orthoefer, J.E Stara, Y.Y. Yang and K.I. Campbell
4. Collagen and Prolyl Hydroxlyase Levels in Lungs in Dogs Exposed
to Automobile Exhaust and Other Noxious Gas Mixtures 71
R.S. Bhatnagar
5. The Effects of Air Pollutants on Membrane Lipids 87
G. Rouser and R. Aloia
6. Pulmonary and Cardiovascular Physiology Studies
During Exposure 97
T.R. Lewis and W.J. Moorman
1. Review of the Cardiovascular and Pulmonary Function Studies on Beagles
Exposed for 68 Months to Auto Exhaust and Other Air Pollutants 115
J.R. Gillespie
8. Review of Radiologic Studies 155
D. Dungworth
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9. Effects of Air Pollutants on Visual Evoked Brain Potentials in Chronically Exposed
Beagle Dogs ' 167
El,. Johnson, J.G. Orthoefer andJ.E Stara
10. Ultrastructure of Biopsied Tissue 179
RJ. Stephens and G. Freeman
11. Necropsy 183
J.G. Orthoefer
12. Morphometric and Morphologic Evaluation of Pulmonary Lesions in Beagle Dogs
Chronically Exposed to High Ambient Levels of Air Pollutants 195
D. Hyde, J.G. Orthoefer, D. Dungworth, W. Tyler,
R. Carter and H. Lum
13. Critical Review. Implications and Plans for the Future 263
W. Tyler, D. Dungworth and J.E Stara
vni
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LIST OF FIGURES
Page
FRONT COVER
Scanning electron micrograph of bronchiokr epithelial cell hyperplasia
partially occluding a distal terminal bronchiole.
INTRODUCTION
Figure 1. Schedule of biological testing from 1965 to 1974 5
CHAPTER
1. Study Overview, Rationale, Experimental Design, Experimental Facilities
and Exposure Atmospheres
Figure 1. Example of experimental animal — ISinch beagle, female 12
Figure 2. Study time line 15
Figure 3. Inhalation chamber with placement of experimental animals 16
Figure 4. Five-year summary of experimental atmospheric levels 19
Figure 5. Engine room with the dynamometer system 24
Figure 6. Chronic auto exhaust study diagram 25
Figure 7. Irradiation chamber 26
Figure 8. Exposure chamber supply and exhaust flow diagram 27
Figure 9. System schematic diagram 29
Figure 10. Animal exposure chambers 33
Figure 11. Animal exposure chamber — Schematic diagram 34
2. Exposure Chamber Atmospheres. Sampling and Analysis
Figure 1. Pattern of differences in mean CAE study lead concentrations 48
3. Clinical Summary of Beagle Study. Physical Examinations, Ocular
Examinations, Hematology Examination (Post-Exposure) and
Blood Chemistries
Figure 1. Prevalence of chronic dermatitis in experimental animals 56
Figure 2. Prevalence of epiphora in experimental animals 57
Figure 3. The effect of exposure to auto exhaust on blood indices
in the beagle dogs pre-exposure and exposure 59
Figure 4. The effect of exposure to auto exhaust on blood indices
in the beagle dogs post-exposure 60
7. Review of the Cardiovascular and Pulmonary Function Studies on Beagles
Exposed for 68 Months to Auto Exhaust and Other Air Pollutants
Figure 1. Mean values with standard errors for respiratory (rsX
pulmonary (p), and chest wall (cw) resistances for each group
of beagles 135
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Figure 2. Mean values with standard errors for total lung capacity,
functional residual volume, and residual volume (top to
bottom of each bar) for each group of beagles 137
Figure 3. Mean dynamic compliance values (Cjyj,) at different
breathing frequencies for each group of beagles identified on
the left of each Cjyn/f plot 137
Figure 4. Changes in each dog's residual volume (RV) between
termination of exposure (TE) and two years after termina-
tion of exposure (2YR) 140
Figure 5. Changes in each dog's inspiratory lung compliance (CL)
between termination of exposure (TE) and two years after
termination of exposure (2YR). 141
9. Effects of Air Pollutants on Visual Evoked Brain Potentials in Chronically Exposed
Beagle Dogs
Figure 1. Typical averaged visual brain potential (VER) from a
control treatment beagle dog 171
Figure 2. Means and standard errors for VER component N i latency
versus air pollution treatment 172
Figure 3. Means and standard errors for VER component Pj latency
versus air pollution treatment 173
Figure 4. Means and standard errors for VER component Nj
amplitude versus air pollution treatment 174
Figure 5. Means and standard errors for VER component PI
amplitude versus air pollution treatment 175
Figure 6. Means and standard errors for VER overall amplitude
(RMS) versus air pollution treatment 176
11. Necropsy
Figure 1. Fixation of the right lung was accomplished using a dilute
Karnovsky's fixture at 30 cm H20 pressure for 16 to 18 hours 183
12. Morphometric and Morphologic Evaluation of Pulmonary Lesions in Beagle Dogs
Chronically Exposed to High Ambient Levels of Air Pollutants
Figure 1. Cranial, middle, and caudal lobes of the canine right lung are
depicted from the lateral view; the accessory lobe is depicted from
the caudal view. Blocks of tissue for histologic evaluation were
selected from indicated sites in transverse plane for cranial and
middle lobes, sagittal plane for caudal lobe, and frontal plane for
accessory lobe 198
Figure 2. a. Pulmonary acinus from a control dog originates from a
distal bronchus (b) and consists of a terminal bronchiole (Tb)
with a nonuniform branching pattern of respiratory bronchioles
(Rb) and alveolar ducts, b. Lung from the N02-high group of
dogs at the same magnification illustrates distal air space enlarge-
ment and strands of tissue (arrow). 204
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Figure 3. Comparison between the control and all other exposed
groups by surface density (Sva, m2/cm3) and internal surface area of
the right lung (S^, nvfy The control group to the left and all other
exposed groups from left to right are plotted by decreases in Sva,
Each bar represents the group mean ± 1 standard error. Significant
differences from the control groups are marked by an asterisk 206
Figure 4. Comparison of distal airways and parenchyma in lungs
from control and exposed groups, a. Numerous respiratory bron-
chioles (Rb) and alveolar ducts (Ad) are interspersed among alveoli in
a lung from the control group, b. Slight enlargement of respiratory
bronchioles (Rb) and alveolar ducts (Ad) of a representative lung from
the NO-high group, c. Mild enlargement and loss of interalveolar
septa in alveolar ducts (Ad) in a representative lung from the SOX
group, d. Mild to severe enlargement and loss of interalveolar septa
characteristic of a lung from the N02 -high group. A respiratory bron-
chiole (Rb) is adjacent to a greatly enlarged alveolar duct (Ad) 207
Figure 5. Comparison of distal airways in lungs from control and
exposed dogs. a. Respiratory bronchiole (Rb) branches into
alveolar ducts (Ad). Numerous alveoli and a moderate number
of alveolar pores (arrow) are present in this lung of a control dog.
b. Lung from the SOX group of dogs at the same magnification
illustrates alveolar duct (Ad) enlargement and loss of interalveo-
lar septa. It is similar to the lesion in Figure 4c. c. Higher magni-
fication in a region of b shows short interalveolar septa (black
arrow) and shallow alveoli (a). Few interalveolar pores are seen
(white arrow) 208
Figure 6. Respiratory bronchiole (Rb), alveolar ducts (Ad), and alveoli
have the same conformation as controls despite widespread increase
in interalveolar pores (inset, arrow) in a lung from the R group of
dogs 209
Figure 7. Junction between a distal respiratory bronchiole (Rb) and
proximal alveolar duct (Ad) shows air space enlargement with
numerous alveolar pores and fenestrations in a lung from the
NC>2 -high group of dogs. Fenestrations and trabeculae (arrow)
are common in alveolar ducts (inset). 211
Figure 8. Irregular hyperplasia of nonciliated bronchiolar cells (inset)
partially occludes the lumen of a terminal bronchiole in a lung
from the R group of dogs. An aggregation of inflammatory cells
(arrow) is seen in the distal region of the terminal bronchiole 212
Figure 9. Comparison of terminal bronchiolar epithelium in lungs from
control and exposed dogs. a. Epithelium of a terminal bronchiole
(Tb) from the lung of a control dog has a mixture of ciliated
(arrows) and nonciliated cells, b. Hyperplastic nodules in a
terminal bronchiole (Tb) of a lung from the I group of dogs
show a mixture of hypertrophied ciliated and nonciliated cells.
Goblet cells (arrow) are also visible (1 pm Epon-embedded
section). 213
XI
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Figure 10. Hyperplastic nodules composed primarily of nonciliated
cells protrudes into the lumen of a terminal bronchiole (Tb) in
a lung from the I group of dogs. The clear areas in the nonciliated
cells (NC) represent areas of fixative-leached glycogen. A macrc-
phage (M) and segments of a connective tissue core (arrows) are
also present An alveolar macrophage (AM) is in the airway lumen 214
Figure 11. In a lung from the NC^-high group of dogs, a ciliated cell
(C) lies deep within a bronchiolar hyperplastic nodule, close to
the basal lamina (bl). The cytoplasm has dispersed basal bodies
(bb) and cilia are in the intercellular space (arrow). Serial sections
showed cilia exiting from this cell 216
Figure 12. In the dorsal membraneous portion of the trachea, the
ciliated cells with short cilia (C) and long microvilli and the
apical portion of goblet cells (G) are easily seen in a lung from
a control dog 217
Figure 13. Focus of ciliary loss without squamous metaplasia in a
primary bronchus from a lung in the NC^-high group of dogs.
Ciliated cells are reduced in number and have fewer cilia per
cell. Goblet cells do not have apical projections (G), but
ciliated cells have numerous microvilli (C). 218
Figure 14. Focus of ciliary loss accompanied by squamous metaplasia
is seen as a proliferative lesion (P) which protrudes into an airway
lumen of a bronchus of a lung from the R group of dogs. The sur-
face of the lesion has numerous microvilli and surface folds (inset). 219
Figure 15. Section of a proliferative lesion (P) that protrudes into the
lumen (L) of a distal bronchus in a lung from the NC^-high group
of dogs illustrating squamous metaplasia. At least five cells deep,
the lesion has a connective tissue core (C) containing numerous
mononuclear inflammatory cells. (1 /urn Epon-embedded section). 220
Figure 16. Thin section adjacent to the area seen in Figure 15. Colum-
nar- to spindle-shaped cells with intracellular fibrils (F) and
numerous intercellular processes connected by desmosomes (D)
(inset) were the distinguishing characteristics of squamous meta-
plasia. A ciliated cell (C) sends cilia into the bronchial lumen (L). 221
xn
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LIST OF TABLES
CHAPTER Page
1. Study Overview, Rationale, Experimental Design, Experimental Facilities
and Exposure Atmospheres
Table 1. Random Distribution and Number of Experimental
Animals Per Chamber 13
Table 2. Composition of Exposure Atmospheres and
Abbreviations Used 17
Table 3. Estimated Exposure Concentrations 17
Table 4. Comparison of Pollutant Levels in Chronic Dog Study
with Ambient Atmospheric Concentrations in Four Cities —
1966-1971 18
Table 5. Methods for Reducing Errors in Animal Exposure
Experiments 20
Table 6. Analysis of Variance of Red Blood Cell Counts (RBC)
on 96 Young Female Beagles 22
2. Exposure Chamber Atmospheres. Sampling and Analysis
Table 1. Major Atmosphere Components and Measurement Strategy 42
Table 2. Pollutants Measured Automatically and Continuously 42
Table 3. Pollutants Measured Periodically 43
Table 4. Pollutant Concentrations in the Exposure Chamber
Atmospheres During the Chronic Exposure Study 44
Table 5. Hydrocarbon Concentrations in the Exposure Atmospheres
at Equilibrium 45
Table 6. Phototape Sampler Transmission Values 46
Table 7. Anions Associated with Airborne Paniculate 46
Table 8. Acrolein 46
Table 9. Formaldehyde Concentrations 46
3. Clinical Summary of Beagle Study. Physical Examinations, Ocular
Examinations, Hematology Examination (Post-Exposure) and
Blood Chemistries
Table 1. Tabulation of Ocular Lesions, as Reported by Dr. Wyman 58
Table 2. Certain Hematological Abnormalities in Beagles in
Relation to Different Treatments 61
Table 3. Date and Cause of Pre-Euthanasia Deaths
in the Chronic Auto Exhaust Study ' 62
4. Collagen and Prolyl Hydroxylase Levels in Lungs in Dogs Exposed
to Automobile Exhaust and Other Noxious Gas Mixtures
Table 1. Analysis of Variance for Hydroxyproline/Ninhydrin 73
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Table 2. Mean Hydroxyproline/Ninhydrin (x 1(H) by Treatment and the
Results of Duncan's Multiple Range Test 73
Table 3. Analysis of Variance for Prolyl Hydroxykse 74
Table 4. Mean Prolyl Hydroxylase (dpm/mg) by Treatment and the
Result of Duncan's Multiple Range Test (a =0.05) 74
5. The Effects of Air Pollutants on Membrane Lipids
Table 1. Heart Lipids (Mean Values) 88
Table 2. Liver Lipids (Mean Values) 90
Table 3. Lung Lipids (Mean Values) 91
6. Pulmonary and Cardiovascular Physiology Studies
During Exposure
Table 1. Means and Standard Deviations of Body Weight and
Pulmonary Function Tests — 18 Months 99
Table 2. Pulmonary Function Means by Treatment and by
Variable at 36 Months of Exposure 100
Table 3. Pulmonary Function Means by Treatment and by
Variable at 61 Months of Exposure 102
Table 4. Summary of the Electrocardiographic Data 104
Table 5. Summary of Vectorcardiographic Data 104
Table 6. Mean Widened-QRS-Complex (W-QRS-C) Indices for
Air Pollutant-Exposed Groups of Dogs 105
Table 7. Mean Canine Blood Viscosity (Centipoise) 106
Table 8. Average Carboxyhemoglobin and
Methemoglobin Concentrations in Canine Blood 106
7. Review of the Cardiovascular and Pulmonary Function Studies on Beagles
Exposed for 68 Months to Auto Exhaust and Other Air Pollutants
Table 1. Grading System for Electrocardiograms 120
Table 2. Some EGG Interpretation Grading Criteria 120
Table 3a. EGG, VCG, and Other Diagnoses, by Sampling Period 121
Table 3b. EGG, VCG, and Other Diagnoses, by Sampling Period 122
Table 3c. EGG, VCG, and Other Diagnoses, by Sampling Period 123
Table 4. Selected Blood Pressures 124
Table 5. Phonocardiographic and Physical Abnormalities 125
Table 6. Dog Illnesses During Observation Period 126
Table 7. Dog Deaths During Observation Period 127
Table 8. Average Hematocrit, Carboxyhemoglobin, and
Methemoglobin Levels 128
Table 9. Cardiovascular Function of Each Group 3 Years
After Termination of Exposure 129
Table 10. Mean Intravascular Pressures of Each Group 3 Years
After Termination of Exposure 130
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Table 11. Mean Values and Standard Errors of Peripheral Vascular
Resistance (Rper), Pulmonary Vascular Resistance (Rpv) and
Left Ventricular Work (WLV) for Each Group 3 Years after
Termination of Exposure 131
Table 12. Body Weight (WT), Blood Gases (pHa) Breathing
Frequency (f), Tidal Volume (V>p), Minute Volume (V „,;„),
Percent Dead Spac^ (%VD), Dead Space (Vpj), and Dead
Space Ventilation (VD) of Beagles 133
Table 13. Total Dead Space of Control Group Compared with
Exposed Groups 2 Years After Termination of Exposure 134
Table 14. Pulmonary Diffusion Capacity (DLco) and DL^ to
Total Lung Capacity (TLC) Ratio (DLc0/TLC) for the
Beagles 136
Table 15. Inspiratory and Expiratory Lung and Chest Wall
Quasistatic Compliance Values for the Beagles 136
Table 16. Lung Volumes of the Beagles in the Control Group at
the Termination of Exposure (IE) and 2 Years After
Termination of Exposure (2YR) Compared with Values
from a Similar Group of Beagles Previously Studied at
UC-Davis (External Controls) 138
Table 17. Compliances of Lung (CLi&E) an^ °f Chest Wall
(C^) of the Beagles in the Control Group at the Termina-
tion of Exposure (TE) and 2 Years after Termination of
Exposure (2YR) Compared with Values from a Similar Group
of Beagles Previously Studied at UC-Davis (External Controls) 138
Table 18. Diffusing Capacity (D^o), DLc0/Total Lung Volume
Ratio (DLco/TLC) and Total Pulmonary Resistance (Rpui) of
the Beagles in the Control Group at the Termination of Expo-
sure (TE) and 2 Years After the Termination of Exposure
(2YR) Compared with Values from a Similar Group of
Beagles Previously Studied at UC-Davis (External Controls) 138
Table 19. The Mean Pulmonary Function Values of the Beagles
Immediately Following Termination of Exposure (TE) and
2 Years after Termination (2YR) 139
Table 20. Mean Total Lung Capacity (ml) of Each Group of
Beagles at End of Exposure (TE, column l\ 2 Years
After End of Exposure (2YR, column 2), Change in Volume
Between TE and 2YR (column 3), Predicted Value from
External Controls (column 4), and the Difference Between
Predicted and 2YR (column 5) 142
11. Necropsy
Table 1. Total Body Weights and Organ Weights and
Standard Deviations for 70 Beagle Dogs 184
Table 2. The Frequency Distribution of Mammary Tumors
for Beagles Exposed to Exhaust and Non-Exhaust
Atmospheres 185
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Table 3. The Frequency Distribution of Mammary Tumors
for the Beagle Exposed to Different Atmospheres 185
Table 4. Mucoidal Degeneration of A-V Valves 186
Table 5. Skin Lesions and Papillomas 186
Table 6. Lung Lesions 187
Table 7. Lead Analysis of Femurs from Dogs (Beagles) Exposed
to Chronic Auto Exhaust 187
12. Morphometric and Morphologic Evaluation of Pulmonary Lesions in Beagle Dogs
Chronically Exposed to High Ambient Levels of Air Pollutants
Table 1. Measured Pollutant Concentrations to Which Dogs
Were Exposed 16 Hours a Day for 68 Months 197
Table 2. Morphometric Parameters for Dog Lungs from
Control and Exposed Groups 200
Table 3. Grading System for Selected Pulmonary
Histologic Lesions 201
Table 4. Grading System for Selected Pulmonary Lesions
Evaluated by SEM 202
Table 5. Summary of Results for Morphometric Parameters 205
Table 6. Summary of Results of Pulmonary Lesions
Evaluated by SEM 210
xvi
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INTRODUCTION
/. F. Stara
As a result of the increasing concern about the hazards of chemical and physical agents which
pollute our environment, a vast amount of research has been initiated to assess their health
effects in exposed populations (1,2). Epidemiological studies have demonstrated that high air
pollution levels generated from stationary/industrial sources and associated with increased
levels of sulfur compounds and particulates have caused increased morbidity and mortality,
particularly in sensitive populations, e.g., among people with chronic pulmonary or cardiac
disease. At the same time the literature reveals that there is less convincing epidemiologic
evidence linking increased incidence of disease with increased photochemical smog levels in
which the principal compounds identified are oxidants. In support of the epidemiological
findings, a large number of animal toxicologic studies have been carried out to identify the
health effects of common ambient air pollutants. However, the great majority of these
investigations have been short-term and have used pollutant concentrations many times those
encountered in the urban or rural ambient atmosphere (3,4). Since the major objective of the
U.S. Environmental Protection Agency is to abate or control environmental pollution to levels
of acceptable risk, research investigations must provide a sufficient body of data so that
informed judgments concerning acceptable levels of pollution in the environment can be
made.
There is general agreement that, due to the extremely large number of potentially toxic
agents present in the environment, a satisfactory assessment program must utilize a carefully
conceived and comprehensively designed evaluation system. The best approach thus far is the
employment of bioassay screening techniques for ranking the toxic potential of the many
pollutants, and thus reducing the volume of compounds to a manageable number for further
in vivo toxicity testing. Data obtained from the bioassay screening programs, along with
appropriate analytical procedures and monitoring of the effluents and emissions from
pollution sources, are then used to select a greatly reduced number of compounds for detailed
biochemical, physiological and pathological toxicity studies of intermediate dosage and
duration. Finally, on the basis of this information, only a few of the most ubiquitous and
potentially harmful pollutants are selected for long-term mammalian studies at levels actually
found in the environment. In this step-wise fashion, a comprehensive and reliable toxicologic
assessment is obtained from which a sound administrative decision-making process and
abatement action program can be promulgated.
In this introduction, the contributions of long-term studies in large mammalian species are
reviewed in terms of their importance as valid predictors of similar clinical manifestations of
harmful effects in man (5). To demonstrate the dependability of results and to confirm that
such data probably could not have been obtained in shorter-lived species such as rodents
because of the latency period needed for development of harmful biological effects, dogs and
other higher mammals have been successfully used in a number of investigations, particularly
in the fields of radiation and pesticide effects and anti-cancer drug research. For example, in
a study of effects of anti-cancer drugs in dogs and monkeys, it was confirmed that the large
animal species, over the long run, "... served to alert the physician to a significant proportion
of the total spectrum of drug effects, which were encountered later during the clinical use of a
new anti-cancer compound."
-------
Dougherty et aL (6) have found that the important biological end-points observed in dogs
exposed to 226fta an^ 228fta are quite comparable to those observed in humans and that
deposition patterns of 239pu ;n dogs more closely approximate distribution in humans than
do deposition patterns in rats. Clarke and Bair (7) administered 239pu()2 to dogs as an
aerosol; death resulted from 55 to 855 days post-exposure. Pathological effects observed
included severe fibrosis followed by alveolar cell, bronchiolar and squamous types of
metaplasia. Goldman and Delia Rosa (8) have reported on the dynamics of 90§r metabolism
in dogs exposed from intra-uterine time to adulthood. Their results indicate that the long-
term dynamics of 90Sr metabolism are similar in some respects to that of 90Sr m man. Mays
et aL (9) investigated the comparative effectiveness of 226]^ 239pU) 228ga Q^ 90Sr
exposure in dogs in the induction of osteosarcomas in an attempt to elucidate the mechanism
of radiation-induced bone cancer. Dungworth et aL (10) have investigated a spectrum of
myeloproliferative disorders in response to 90Sr exposure in dogs in a series of experiments
which they hope will elucidate the relationship between granulocytic leukemia and myelofi-
brosis with myeloid metaplasia in man. Bustad et aL (11) have investigated hematopoietic
changes in dogs maintained on a 90Sr diet (six dose levels from 0.03 to 12 ^tCi/day) for the
first 1.5 years of their lives. A persistent dose-related leukocyte depression was noted at the
highest two dose levels. Eleven dogs 1.3 to 5 years old, fed the two highest dose levels,
exhibited a terminal anemia and myeloid leukemia. Leach et aL (12) subjected dogs, monkeys
and rats to the inhalation of natural U02 dust at a concentration of 5 mg/m3,6 hours per day,
5 days per week for periods up to 5 years. The lung and tracheobronchial lymphonode data
for dogs and monkeys in this study suggest that there may be a radiation hazard in these
tissues at or below the recommended TLV. These dogs were exposed for periods up to 5 years
with little evidence of injury; however, 2 to 6 years post-exposure a high percentage of the
dogs developed pulmonary neoplasia (13). Hobbs et aL (14), in comparing the toxicity of
inhaled 144Ce02 in dogs, mice and hamsters, found much lower doses to be effective in
reducing the lifespan of the dogs compared to that of rodents on a /^Ci/kg basis. The rationale
suggested is that rodents consistently have a much shorter lung retention of inhaled, relatively
insoluble particles than dogs. In another study, Hobbs et aL (15) were unable to demonstrate
pulmonary neoplasms in hamsters exposed to lung burdens of 239puQ2 equivalent to those
shown to produce neoplasms in dogs 3 years post-exposure. It is suggested that the latent
period for tumor development may not be proportional to lifespan, in which case hamsters
may not live long enough to develop pulmonary neoplasms.
Dogs have also proven useful in the evaluation of risk from insult imposed by a variety of
chemicals, particularly pesticides. One study which illustrates the potential value of long-term
dog studies of pesticide toxicity in terms of their value in the assessment of human health
effects is that of Deichmann et aL (16). In this study dogs were dosed for 14 months with DDT.
The pesticide concentration in the blood and fat during this time was comparable to levels in
healthy occupationally exposed workers. The only abnormality noted in the dogs was an
increase in alkaline phosphatase activity. Attempts were made to breed experimental males
with experimental females after the cessation of pesticide feeding. Among the effects noted
were: suppressed mammary development and milk production, stillbirths in one-half of the
deliveries and depression of weanling survival rate from the normal of 84% to 32%. Other
studies in which dogs have proven useful include: Yeary (17) (ferrocome); Worden et aL (18)
(lenacil>, Gallo (19) (phosalone>, Mollello et aL (20) (probucol); Wernick et aL (21) (hair dyes);
Woodard et aL (22) (xanthan gum>, Worden et aL (23) (gusathion); Smyth et aL (24) (1, 2, 6
hexanetriol); Smith and Case (25) (fluorocarbons); Worden et aL (26) (ronnel>, Earl et aL (27)
-------
(diazinon>, Hansen et aL (28) (2, 4 dichlorophenoxyacetic acid>, King et aL (29) (succistearin>,
Case et aL (30) (nefopam hydrochloride); Muacevic et al. (31) (bromophos); Jenkins et aL (32)
(benzene, toluene, xylene, and cumene); and Lyon et aL (33) (acrolein).
Another area in which dogs have proven useful test subjects concerns the quantification of
effects from cigarette smoking. Auerbach et aL (34) have reported elevated heart weight to
body weight ratios in smoking dogs, as well as pulmonary fibrosis and emphysema, which
were similar to conditions reported in humans. In addition, Auerbach et aL (35) have shown a
thickening of myocardial arteriole walls in smoking dogs. Thickening increased with the
duration of smoking and with the number of cigarettes smoked, and in dogs smoking
nonfilter as opposed to filter cigarettes. These results parallel effects noted in humans.
Hammond et aL (36) have found that the responses observed in dogs following smoking
closely parallel those observed in man, in that the types of histologic changes produced in the
lung parenchyma were the same in both species. In both species there was a dose-response
relationship and the degree of damage to the lung parenchyma increased with increasing
duration of cigarette smoking. Battista et al (37) have found that following exposure to five
cigarettes daily, dogs exhibited a 7% increase in central airway resistance when exposed to
whole smoke and less than a 1% increase when exposed to the gas phase alone; a 30%
increase in minute volume following whole smoke exposure and only a 1 % increase following
gas phase exposure; and a 5% decrease in peripheral airway resistance for both whole smoke
and gas phase exposure.
Another large mammal which has been used extensively in long-term toxicity testing is the
miniature swine. This animal has proven to be especially useful in the study of 90Sr-induced
leukemia. Clarke et al. (38), Clarke et al (39), Howard and Clarke (40) and Ragan et al (41)
have published a series of reports which describe various aspects of a study in which
miniature swine were dosed orally with 90Sr at levels from 1 to 3100 ^Ci/day for 7 to 10 years.
These reports indicate that bone marrow damage rather than osteosarcomas may be the
limiting factor in chronic oral toxicity of 90Sr and that the latent period for leukemia
induction is considerably shorter than that for osteosarcoma development In these studies
the parental generation, which was exposed to 90Sr at levels from 25 to 125 ^Ci/day from 9
months of age, exhibited primarily myeloproliferative disorders consisting of a progressive
decrease in circulating leukocytes and platelets and a precipitous terminal fall in erythrocytes.
A second syndrome was noted primarily in groups dosed at levels of 125 and 625 ^Ci/day.
This syndrome was characterized by broad spectrum myeloproliferation ranging from
myeloid metaplasia to frank blast cell leukemia. In contrast, the offspring demonstrated a
marked increase in the incidence of lymphoproliferative disorders in addition to the myelo-
proliferations. In addition, 9"Sr, up to levels where females did not survive long enough to
bear young, did got affect fetal or neonatal mortality.
Primates constitute another group of animals frequently chosen for long-term toxicity studies
when extrapolation to the human population is a goal. Examples of studies in which primates
have proven useful include a series of experiments reported by Spalding et al (42, 43, 44).
These studies involve the effects of radiation exposure in primates with respect to dose
protraction and residual injury and recovery rate expecially in relation to the concept of
equivalent residual dose.
-------
Another animal which has been used extensively and has proven to be quite useful in a
number of long-term studies is the cat. Several papers which discuss a number of parameters
relating to orally administered 89§r have been published by Berman and Stara (45), Nelson et
al (46), Stara and Wolfangel (47), Nelson et al (48), and Rcfsenstein and Nelson (49). Topics
discussed in these papers include: tissues uniquely sensitive to radiation-induced
lymphoproliferative or myeloproliferative disorders during growth and development of the
cat; assessment of the radiation dose rate delivered to the bone marrow and other organs in
cats of different ages; and the relationship between the effective dose of 89$r in cats and the
90Sr dose which is leukemogenic in dogs and miniature swine. In two more recent studies
with dogs, Benjamin et aL (50) have shown differences in the cause of death depending on
time following inhalation exposure to 144Ce and 90sr. In dogs dying within 2 years of
exposure, deaths were attributable to non-neoplastic radiation lesions, whereas in dogs dying
later than 2 years following exposure, neoplasms were the primary cause of death. Norris et
al (51) are in the process of developing dose-response curves for dogs exposed to 60Co (X&Y)
radiation. Preliminary data indicate a shift in the response curve at low dosages so that injury
is independent of dose rate and dependent only upon the total accumulated dose.
This monograph presents the results of a major study of the effects of air pollutants in
beagles, a high mammalian species. The work was performed at the EPA's Health Effects
Research Laboratory in Cincinnati, Ohio, from 1965 to 1971, after which the dogs were
moved to the University of California at Davis for further study during the next three years.
The primary objective of the study was to examine the effects of long-term exposure to
environmentally realistic levels of whole automobile exhaust and its individual components.
The irreversible chronic effects in dogs were assessed in order to provide valuable data on the
potential toxic effects in humans.
The schedule of biological tests conducted during the investigation is shown in Figure 1.
Results for the major parameters of interest (pulmonary, cardiovascular, hematological, and
pathological) are presented in the chapters of this monograph, together with neurophysiologic
(EEC) observations, clinical chemistry findings, assessment of immune competence, the
results of continuous clinical observations of the animals, and other significant findings.
To assess the implications of these data, a special conference was organized exclusively to
review and evaluate all aspects of the study. It was held at Asilomar, California, on August 25,
26 and 27, 1976, and attended by 21 scientists and academicians from government agencies,
research institutes, and universities. The attendees and their affiliations are listed below.
Portions of the discussions that took place at the Asilomar Conference are presented at the
end of each chapter. Chapter 13, which consists solely of conference discussions, provides a
detailed critical review of the study, and presents ideas for future studies of long-term,
low-level pollutant effects in higher mammals. The work presented in the following chapters
demonstrates the dire need for and the value of long-term animal toxicology data which
parallel more closely the. effects on man of chronic low-level exposures to environmental and
occupational contaminants.
-------
P
;
P
F*
CV
CV P
1
=" NP
EXPOSURE
P
CV
F MO
POST-EXPOSURE
1
and
3PH.
n
10
20
30
40
50
60
70
80
90
100 104
Figure 1
Schedule of biological testing from 1965 to 1974.
MONTHS
LEGEND PF - PULMONARY FUNCTION STUDIES
CV — CARDIOVASCULAR STUDIES
MORPH — QUANTITATIVE MORPHOLOGY STUDIES
NP — NEUROPHYSIOLOGIC (EEG) STUDIES
— DESIGNATES CINCINNATI STUDIES
NOTE HEMATOLOGY EVERY SIX MONTHS
CONT CLINICAL OBSERVATIONS AND TREATMENT
(ESP AFTER 9/69)
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Asilomar Conference Discussion Participants
Dr. Roy Albert
U.S. Environmental Protection Agency
Dr. Joseph Brain
Harvard School of Public Health
Dr. Rajendra Bhatnagar
University of California
Mr. Kenneth Busch
National Institute of Occupational Safety
and Health
Dr. Donald Dungworth
University of California
Dr. Jerry Gillespie
University of California
Dr. F. Gordon Hueter
U.S. Environmental Protection Agency
Dr. Dallas Hyde
University of Florida
Dr. Jerome Kleinerman
St. Luke's Hospital
Dr. Si Duk Lee
U.S. Environmental Protection Agency
Dr. Trent Lewis
National Institute of Occupational Safety
and Health
Dr. Neil Littlefield
National Center for lexicological Research
Dr. James MacEwen
University of California
Mr. Myron Malanchuk
U.S. Environmental Protection Agency
Dr. Bryon McLees
National Institutes of Health
Dr. Paul Nettesheim
Oak Ridge National Laboratory
Dr. John Orthoefer
U.S. Environmental Protection Agency
Dr. George Rouser
City of Hope National Medical Center
Dr. Jerry Stara
U.S. Environmental Protection Agency
Dr. Robert Stephens
Stanford Research Institute
Dr. Whitney Thurlbeck
University of Manitoba
Dr. Walter Tyler
University of California
References
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2. Brain, J.D. 1977. The respiratory tract and the environment Environ. Health Perspec. 20:
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3. MacFarland, H.N. 1975. Inhalation toxicology. J. Assoc. Off. AnaL Chem. 58: 689-91.
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-------
6. Dougherty, T.F., B J. Stover, J.H. Dougherty, W.S.S. Jee, C.W. Mays, C.E. Rehfeld, W.R.
Christensen, and H.C. Goldthorpe. 1962. Studies of the biological effects of 226Ra>
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7. Clarke, WJ., and WJ. Bair. 1964. Plutonium inhalation studies — VI. Pathologic effects
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8. Goldman, M., and RJ. Delia Rosa. 1967. Studies on the dynamics of strontium metabo-
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9. Mays, C.W., T.F. Dougherty, G.N. Taylor, R.D. Lloyd, BJ. Stover, W.S.S. Jee, W.R.
Christensen, J.H. Dougherty, and D.R. Atherton. 1969. Radiation-induced bone cancer in
beagles. Pp. 387408 in C.W. Mays, et aL, eds. Delayed effects of bone-seeking radionu-
clides. University of Utah Press, Salt Lake City.
10. Dungworth, D.L., M. Goldman, J.W. Switzer, and D.H. McKelvie. 1969. Development of
a myeloproliferative disorder in beagles continuously exposed to 90Sr. Blood 34: 610.
11. Bustad, L.K., M. Goldman, L.S. Rosenblatt, D.H. McKelvie, and I.I. Hertzendorf. 1969.
Hematopoietic changes in beagles fed 90Sr. /ra C.W. Mays, et aL, eds. Dekyed effects of
bone-seeking radionuclides. University of Utah Press, Salt Lake City.
12. Leach, LJ., E.A. Maynard, H.G. Hodge, J.K. Scott, C.L. Yule, G.E. Sylvester, and H.B.
Wilson. 1970. A five-year inhalation study with natural uranium dioxide dust (UOs). I.
Retention and biologic effect in the monkey, dog and rat Health Physics 13: 559-612.
13. Leach, LJ., C.L. Yulie, H.C. Hodge, G.E. Sylvester, and H.B. Wilsoa 1973. A five-year
inhalation study with natural uranium oxide dust II. Post-exposure retention and
biological effects in the dog and rat Health Physics 25: 239-58.
14. Hobbs, C.H., R.O. McCleUen, and SA Benjamin. 1974. Toxicity of inhaled 144Ce02 in
immature, young adult and aged Syrian hamsters. II. Inhalation Toxicol. Res. Instit Ann.
Report, 1973-1974, Lovelace Fn., Albuquerque, N.M.
15. Hobbs, C.H., J.A. Mewhinney, D.A. Slauson, R.O. McClellen, and JJ. Meglio. 1974.
Toxicity of inhaled 239puC<2 in immature, young adult and aged Syrian hamsters. II.
Inhalation Toxicol. Res. Inst Ana Rep. 1973-1974, Lovelace Fn., Albuquerque, N.M.
16. Deichmann, W.B., W.E. MacDonald, A.G. Beasley, and D. Cubit 1971. Subnormal
reproduction in beagle dogs induced by DDT and aldrin. Ind. Med. Surg. 40: 10-20.
17. Yeary, R.A. 1969. Chronic toxicity of dicyclopentadienyliron (ferrocene) in dogs. ToxicoL
Appl. Pharmacol. 15: 667-76.
18. Worden, A.M., ER.B. Noel, L.E. Mawdesley-Thomas, A.K. Palmer, and MA. Fletcher.
1974. Feeding studies on lenacil in the rat and dog. Toxicol. Appl. Pharmacol. 27:215-24.
19. Gallo, M.A. 1976. Chronic toxicologic investigations of phosalone in the dog. Toxicol.
AppL Pharmacol. 36: 561-68.
20. Mollello, J.A., C.G. Gerbig, and V.B. Robinson. 1973. Toxicity of (4,4'-(isopropylindene-
dithio) bis (2,6-di-t-butylphenol)), probucol, in mice, rats, dogs and monkeys: demonstra-
tion of a species specific phenomenon. Toxicol. AppL Pharmacol. 24: 59-93.
21. Wernick, T., B.M. Lanman, and J.L. Fraux. 1975. Chronic toxicity, teratologic and
production studies with hair dyes. Toxicol. Appl. Pharmacol. 32: 450-60.
22. Woodard, G., M.W. Woodard, W.H. McNeely, P Kovacs, and M.T.I. Cronia 1973.
Xanthan gum: safety evaluation by 2-year feeding studies in rats and dogs and a
3-generation reproduction study in rats. Toxicol. Appl. Pharmacol. 24: 30-36.
23. Worden, A.M., G.H. Wheldon, ER.B. Noel, and L.E. Mawdesley-Thomas. 1973. Toxicity
of gusathion for the rat and dog. ToxicoL Appl. Pharmacol. 24: 405-12
-------
24. Smyth, H.F. Jr., U.C. Pozzani, C.S. Weil, MJ. Tallant, and C.P. Carpenter. 1969.
Experimental toxicity and metabolism of 1, 2, 6 hexanetriol. Toxicol. Appl. Pharmacol.
15: 282-86.
25. Smith, J.K., and M.T. Case. 1973. Subacute chronic toxicity studies of fluorocarbon
propellants in mice, rats and dogs. Toxicol. Appl. Pharmacol. 26: 438443.
26. Worden, A.N., RR.B. Noel, and L.E. Mawdesley-Thomas. 1972. Effect of ronnel after
chronic feeding to dogs. Toxicol. Appl. Pharmacol. 23: 1-9.
27. Earl, EL, B.E. Melveger, J.E. Reinwall, G.W. Bierbower, and J.M. Curtis. 1971. Diazinon
toxicity-comparative studies in dogs and miniature swine. Toxicol. Appl. Pharmacol. 18:
285-95.
28. Hansen, W.H., M.L. Quaife, R.T. Habermann, and O.G. Fitzhugh. 1971. Chronic toxicity
of 2, 4-dichlorophenoxyacetic acid in rats. Toxicol. Appl. Pharmacol. 20: 122-29.
29. King, W.R., W.R. Michael, and R.H. Coots. 1971. Feeding of succistearin to rats. Toxicol.
Appl. Pharmacol. 18: 26-34.
30. Case, M.T., J.K. Smith, and R.A. Nelson. 1976. Chronic oral toxicity studies of nefopam
hydrochloride in rats and dogs. Toxicol. Appl. Pharmacol. 36: 301-306.
31. Muacevic, G., E. Dirks, HJ. Kinkel, and E Leuschner. 1970. Anticholinesterase activity of
bromophos in rats and dogs. Toxicol. Appl. Pharmacol. 16: 585-96.
32. Jenkins, LJ., Jr., R.A. Jones, and J. Siegel. 1970. Long-term inhalation screening studies
of benzene, toluene, 0-xylene and cumene on experimental animals. Toxicol. Appl.
Pharmacol. 16: 818-23.
33. Lyon, J.P., J. Jenkins, Jr., R.A. Jones, RA. Coon, and J. Siegel. 1970. Repeated and
continuous exposure of laboratory animals to acrolein. Toxicol. Appl. Pharmacol. 17:
726-732.
34. Auerbach, 0., E.G. Hammond, D. Kirman, and L. 1967. Garfinkel. Emphysema produced
in dogs by cigarette smoking. J.A.M.A. 199: 241-46.
35. Auerbach, O., E.G. Hammond, and L. Garfinkel. 1971. Thickness of walls of myocardial
arterioles in relation to smoking and age. Arch. Environ. Health 22: 20-27.
36. Hammond, E.G., D. Kirman, and L. GarfinkeL 1970. Effects of cigarette smoking on
dogs. Arch. Environ. Health 21: 740-53.
37. Battista, S.P., W.B. Steber, and CJ. Kensler. 1974. Pulmonary function in spontaneously
breathing dogs with lower airway catheters. Arch. Environ. Health 28: 164.
38. Clarke, WJ., R.F. Palmer, E.B. Howard, and PL. Hackett. 1970. Strontium-90: Effects of
chronic ingestion on farrowing performance of miniature swine. Science 169: 598-600.
39. Clarke, W.J., E.B. Howard, and PL. Hackett. 1969. Strontium-90 induced neoplasia in
swine. Pp. 263-77 in C.W. Mays et aL, eds. Delayed effects of bone-seeking radionuclides.
University of Utah Press, Salt Lake City.
40. Howard, E.B., and WJ. Clarke. 1970. Induction of hematopoietic neoplasms in miniature
swine by chronic feeding of 90Sr. J. Nad Cancer Instit. 44: 21-38.
41. Ragan, H.A., PL. Hackett, BJ. McClanhan, and WJ. Clarke. 1973. Pathologic effects of
chronic 90Sr ingestion in miniature swine. Pp. 919-929 in L.T. Harmison, ed. Research
animals in medicine. DHEW Pub. No. (NIH) 72-333 GPO, Washington, D.C.
42. Spalding, J.E, LM. Holland, O.S. Johnson and J.R. Prine. 1975. Recovery rate and
residual injury in monkeys exposed to large doses of radiation by fractionation. Radiation
Res. 62: 70-78.
43. Spalding, J.F., L.M. Holland, J.R. Prine, RM. LaBauve, and J.E. London. 1973. Compara-
tive effects of dose protraction and residual injury in dogs and monkeys. Radiation Res.
54: 453-62.
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44. Spalding, J.F., L.M. Holland, and J.R. Prine. 1972. The effects of dose protraction on
hematopoiesis in the primate and dog. Life Sciences and Space Research. X-Akademie-
Verlag, Berlin.
45. Berman, E., and J.F. Stara. 1968. Qualitative evaluation of the embryonic skeleton in cats.
Pp. 73-78 in Radiation Bioeffects Summary Report, Jan.-Dec. 1968, U.S. HEW.
46. Nelson, N.S., J.E Stara, and R.C. WolfangeL 1968. 85Sr distribution and weights of organ
systems in chronically-labelled cats. Pp. 70-72 in Radiation Bioeffects Summary Report,
Jan.-Dec. 1968, U.S. HEW.
47. Stara, J.E, and R.G. Wolfangel. 1968. Metabolism of 85sr in cats. Pp. 6669 in Radiation
Bioeffects Summary Report, Jan.-Dec. 1968, U.S. HEW.
48. Nelson, N.W., R.G. Wolfangel, and J.E Stara. 1968. Placental transport of strontium and
calcium in the Felis domestica. Pp. 63-65 in Radiation Bioeffects Summary Report,
Jan.-Dec. 1968, U.S. HEW.
49. Rosenstein, L.S., and N.S. Nelson. 1968. Dosimetry of radio-strontium in skeletal tissues
of cats. Pp. 73-78 in Radiation Bioeffects Summary Report, Jan.-Dec. 1968, U.S. HEW.
50. Benjamin, S.A., FE Hahn, T.L. Chiffelle, B.B. Boecker, C.H. Hobbs, R.K. Jones, and M.B.
Snipes. 1975. Occurrence of hemangiosarcomas in beagles with internally deposited
radionuclides. Cancer Res. 35: 1745-55.
51. Norris, W.P., SA. Tyler, and G.A. Sacher. 1975. An interspecies comparison of the
response of mice and dogs to continuous "OCoY-irradiation. IAEA Int. Symp. on
Biological Effects of Low Level Radiation Pertinent to the Protection of Man and His
Environment. Chicago, Illinois, Nov. 3-7, 1975.
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1. STUDY OVERVIEW, RATIONALE, EXPERIMENTAL DESIGN,
EXPERIMENTAL FACILITIES AND EXPOSURE ATMOSPHERES
J.E Stara, K. Busch, R. G. Hinners and J.K. Burkart
Introduction
A long-term research study was conducted in order to obtain information necessary to
elucidate the public health consequences of emissions from mobile and stationary sources and
to provide an appropriate data base for air quality criteria development. Automotive exhaust
and stationary source emissions are composed of a complex mixture of interacting chemical
substances of varying toxicity. A number of the components which pose a potential hazard to
human health have not yet been fully investigated, nor have their respective contributions to
human illness been fully determined. While brief animal exposures provide useful informa-
tion on acute toxicity of emission components, extended chronic exposures at realistic
environmental levels are necessary to evaluate the long-term effects of air contaminants on
biological systems. This introductory paper is intended to familiarize the reader with the
study rationale and experimental design, including study duration, the complicated inhala-
tion facility, the original pollutant mixtures used, and the various biological parameters
measured.
The experimental facilities designed for inhalation toxicologic studies of complex air pollu-
tion mixtures were unique in 1965 and continue to be so. Currently our laboratory staff is
involved in other inhalation studies investigating the health effects of emissions from diesel
engines and from automotive pollution control systems such as the catalytic converter. The
major individual pollutants implicated in the exhaust, e.g., sulfuric acids, sulfates, platinum,
palladium and ruthenium, are currently under study. The system is unique because the auto
exhaust, simulated smog and associated compounds are ducted to animal chambers for
determination of toxic effects at carefully controlled levels.
The major objective of the study reported in this monograph was the identification and, if
possible, the quantitation of the biological effects in dogs exposed to low-level automotive
emissions and individual ubiquitous ambient air pollutants during the most active portion of
their life spans. Of particular interest was the determination of possible irreversible effects
caused by the tested pollutants. Young female beagles were exposed for 16 hours daily to raw
and photochemically reacted auto exhaust, oxides of nitrogen, oxides of sulfur, and combina-
tions thereof, for approximately 6 years. Thereafter, they were allowed to live for 3 years in a
normal atmosphere in order to permit a recovery from transient effects.
Experimental Animals
Beagle dogs were selected as the experimental animals primarily because of their large size,
which is well suited for various physiological measurements and repeated sampling, and
because of their long life span, which permits a detailed observation of the incidence and
progress of developing disease patterns (Figure 1). From the previous long-term studies in
dogs reported in the introduction, it was felt that the progressive degenerative changes
observed in dogs would closely parallel the slowly developing harmful effects of low-level
pollutants in man. The use of smaller animals such as rodents, with their much shorter life
span, would not adequately satisfy these requirements.
11
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One hundred and four (104) female beagles, approximately 4 months of age, were obtained
from a similar genetic pool, and assembled into an experimental colony. After an environmen-
tal acclimatization period, the beagles were divided into seven groups of 12 animals and one
group of 20, and placed into 26 inhalation chambers, with 4 animals per chamber. Animal
group selections and chamber assignments were performed at random, i.e., a complete
randomization was performed in the allocation of animals to specific exposure atmospheres
and to the triplicate chambers for each atmosphere (See Table 1).
Table 1. Random Distribution and Number of Experimental Animals Per Chambers
Atmosphere"
(1)CA
(2)R
(3) I
(4) SOX
(5)R + SOX
(6)I + SOX
(7) NOL+NO2H
(8)NOH + NO2L
Total no. of a
Beginning
of exposure
X
Y
Z
X
Y
Z
X
Y
Z
X
Y
Z
X
Y
Z
X
Y
Z
X
Y
Z
X
Y
Z
#2
4
4
#5
4
3
#14
4
4
#4
4
3
#6
4
4
#7
4
4
#8
4
2
#3
4
4
#9
4
2
#12
4
4
#16
4
4
#10
4
4
#18
4
4
#15
4
3
#19
4
4
#13
4
3
#11 #21 #25
444
434
#23
4
3
#22
4
2
#20
4
1
#24
4
2
#17
4
4
#26
4
4
#27
4
3
20
12
12
12
12
12
12
12
End of
exposure
17
10
10
Totals
104
10
11
10
10
86
a«ey: X = Chamber number (selection was randomized).
Y = Number of dogs at outset of experiment.
2 = Number of surviving animals at the termination of exposure.
bSee Table 2 for precise composition of each exposure atmosphere.
13
-------
Study Duration and Exposure Atmospheres
The study was initiated in the fall of 1965 (Figure 2). At the outset of the experiment three
mammalian species, mice, guinea pigs and female beagle dogs, were placed into inhalation
exposure chambers. The rodents were placed on the top shelf and the beagles on the lower
shelf (Figure 3). The rodents were removed after a short period of exposure in order to study
acute and subacute effects. The exposure of dogs was discontinued after a 68-month period,
i.e., at the time when the animals were almost exactly 6 years of age. The post-exposure period
of observations and testing lasted approximately 3 years, at which time the animals were
euthanized and pathology studies were performed.
Two major reviews of this long-term investigation were performed by expert scientific
committees. After 4 years of exposure, when the dogs were 50 months old, a scientific
committee reviewed the data and recommended that additional tests be performed especially
addressing the cardiovascular system. The committee felt that the study should be conducted
over the life span of the animals in the animal inhalation chambers under a continuous
exposure regimen (3). However, the study was ended in 1970 because the chamber space was
needed for other research. Fortunately, an agreement was subsequently made to allow
continuation of the animal observation and testing outside of the exposure chambers, i.e., in
ambient air, for an additional period of time. An in-house committee decided in 1971
to contract for 3 years the maintenance, observation, and continuation of in vivo testing and
final sacrifice of all animals with the Department of Pathology, University of California at
Davis (5).
The exposure atmospheres consisted of a clean air control (CA) and seven experimental
exposures as follow: nonirradiated auto exhaust (R), irradiated auto exhaust (I), oxides of
sulfur (S02+H2S04), two concentrations of oxides of nitrogen (NO-low + NCh-high and
NO-high + NO 2-low), and two mixtures of auto exhaust and oxides of sulfur (R + SOX and
I + SOx) (Table 2). The initial exposure levels of the various atmospheres are presented in
Table 3. The intent in the study protocol (6) was to expose the dogs for a long period of time
to realistic urban pollutant levels. Table 4 presents maximum 24-hour means, maximum
annual point values and annual arithmetic means for ambient air pollutants in four highly
industrialized urban communities during the 6 years of this study (1966-1971) (7, 8).
Comparison to pollutant levels in animal chamber exposure atmospheres indicates that the
levels to which the dogs were exposed were generally from two to six times as great as the
reported maximum 24-hour means. In the case of SC"2 in Chicago during the years 1966,
1967, 1968 and 1969, exposure levels were directly comparable to the maximum 24-hour
means. In addition, in certain years maximum yearly values for NO, N02 and oxidants were
within the same range as experimental dog exposure levels. Although maximum 24-hour
means and maximum annual values are not precisely representative of ambient levels over
long periods, nevertheless these comparisons show that for various time intervals certain
segments of the population have been exposed to levels of pollutants which approximate the
levels used in this study for long-term exposure of beagles. For this reason, the authors feel
that the study can be appropriately termed a "long-term exposure of dogs to elevated urban
air pollution levels."
The 5-year summary of average measured chamber concentrations is presented in Figure 4.
All exposure levels of the various pollutants show a rather steady trend, with the exception of
nitrous oxide. The averages shown are over triplicate chambers for 4- to 6-month periods.
14
-------
SEPARATE EXPOSURES
START EXPOSURE & TERMINATED BEGINNING OF
(9-7-65) EXERCISE (5-7-71) SACRIFICE
i
EXPOSU
MICE, RATS, GUINEA
PIGS AND DOGS
3E PERIOD
DOGS ONLY
POST-EXPOSURE
m
-4 0
BIRTH
-------
Figure 3
Inhalation chamber with placement of experimental animals. (Two other dogs are located in
the rear of the chamber.)
16
-------
Standard deviations and fractiles of frequency distributions were also calculated but are not
shown. (For details see Chapter 2.)
Experimental Design
The investigation was designed in a spirit of complete cooperation between the statisticians
and the other senior scientists who were conducting the biological studies. The statistics staff
was asked to participate in the design of the study, and even though some compromises had
to be made based on engineering design realities, the study was based on a sound statistical
experimental design. The aspect of the experimental design given the greatest attention was
the control of different types of experimental error. The first among the basic types of errors
considered was inter-animal biological variability, i.e., the failure of different animals to
exhibit exactly the same response under the same conditions. We know, for example, that this
error is large for white blood cell counts when compared to red blood cell counts. The
coefficient of variation for inter-animal variation was as high as 35% for white blood cell
counts, which means that there could be 70% variation about the mean (at the 95%
probability level). The variation was only about one-half as great for red blood cell counts.
The second type of error considered was intra-animal sampling variation. For many types of
Table 2. Composition of Exposure Atmospheres and Abbreviations Used
Abbreviation Atmosphere
CA Control air
R Non-irradiated auto exhaust
I Irradiated auto exhaust
SOX 0.5 ppm SO2 + 100 ^g/ms H2SO4 mist in control air
R + SOx Non-irradiated auto exhaust + 0.5 ppm SC>2 + 100/jg/m3
H2SO4
I + SOX Irradiated auto exhaust + 0.5 ppm SO2 + 100jjg/rn3 H2SO4
NOu+ NC>2H 0.2 ppm nitric oxide + 0.5-1.0 ppm nitrogen dioxide
NOH + NO2L 1.5-2.0 ppm nitric oxide + 0.2 ppm nitrogen dioxide
Table 3. Estimated Exposure Concentrations (0800 to 2400 hr daily for 68 months)3
Component
CO (ppm)
HC as CH4
(ppm)
NO2 (ppm)
NO (ppm)
Ox as O3 (ppm)
SOX (ppm)
H2S04 fcg/m3)
R
100
24-30
0.1
1.5-2.0
—
—
—
I
100
24-30
0.5-1.0
0.1
0.2-0.4
—
—
SOX NOLH
—
—
- 0.5
- 0
—
0.5
100
NOH + N02L
0.2
0.2 1.5-2.0
The actual measured concentrations are given in Table 4 of Chapter 2.
17
-------
Table 4. Comparison of Pollutant Levels in Chronic Dog Study with Ambient Atmospheric Concentrations in Four Cities — 1966-1971
CO
City
Chicago
Cincinnati
Washington,
DC
Los Angeles
Aim.
(ppm)
CO
NO
S02
NO2
HC
Oxid
CO
NO
SO2
NO2
HC
Oxid
CO
NO
SO2
NO2
HC
Oxid
CO
NO
S02
NO2
HC
Oxid
19663
Max.
24-hr.
Means
32.5
034
055
015
57
008
93
037
013
009
(47)d
06
154
041
025
007
64
010
Max.
24-hr.
Means
22.9
0 189
0645
0 114
518
0077
130
0358
0330
0058
461
0089
146
0359
0 132
0088
—
0080
1967b
Ann.
Arith.
Means
—
0072
0125
0050
300
0029
56
0032
0021
0028
251
0031
49
0047
0048
0043
—
0025
Max. Max.
Ann. 24-hr.
Value Means
— 163
— 0213
— 0437
— 0096
— 516
— 0 113
— 324
— 0339
— 0097
- 0100
—
— 0054
— 138
- 0305
— 0 180
- 0082
- 572
— 0095
1968b
Ann.
Arilh.
Means
62
0072
0 117
0048
29
0024
56
0064
0017
0031
0022
33
0035
0036
0047
223
0027
Max.
Ann.
Value
400
061
—
0 17
123
0 18
420
1 02
—
0056
0 14
_
—
—
—
—
Max.
24-hr.
Means
235
—
1 61
0 13
63
—
109
—
007
007
53
008
109
_
0 10
009
35
005
202
—
005
024
70
009
19693
Ann.
Arilh.
Means
—
008
007
005
27
-
—
—
—
27
—
_
—
—
20
—
65
0098
002
0065
29
0032
Max.
Ann
Value
41 0
1 6
1 6
017
81
007
240
071
029
022
112
016
250
1 2
017
024
94
0 10
390
1 2
009
080
120
030
Max.
24-hr.
Means
140
—
023
0 13
37
—
72
_
009
0 10
38
003
138
—
008
015
52
006
172
—
006
022
68
008
1970b
Ann.
Arifh.
Means
64
009
004
0058
20
—
_
—
—
—
—
35
005
002
0052
1 8
—
60
011
002
0074
32
003
Max.
Ann
Value
350
1 8
050
020
74
016
21.0
080
050
024
7.4
008
250
1 2
021
030
116
0 16
450
1 2
020
030
116
016
Max.
24-hr.
Means
157
—
023
042
45
006
97
—
005
008
5.3
007
99
—
008
012
42
007
136
—
004
028
84
008
1971 a
Ann.
Arith.
Means
—
—
—
006
24
002
_
—
—
—
—
35
004
003
0038
19
0025
54
011
002
0092
36
0025
Max.
Ann.
Value
290
1 8
068
057
81
0 17
240
062
024
0 17
86
016
460
063
020
074
120
024
360
1 1
011
074
120
024
Experimental
Exposure
Levels
In Dogs
100
1 5-20
050
05-1 0
24-30
02-04
100
1 5-20
050
0 5-1 0
24-30
02-04
100
1 5-20
050
05-1 0
24-30
02-04
100
1 5-20
050
05-1 0
24-30
02-04
3NADB (AEROS. US EPA, Office of Air Quality Planning and Standards)
bAir Quality Data for 1967 & 1968 From NASN and contributing state and local networks APTD-0741, 1971
cOnlydata available
dlncomplete data
-------
CHAMBER ATMOSPHERE — I
CO
HC
19661 1967 I 1968 I 1969 < 1970 1971
YEAR
CHAMBER ATMOSPHERE — I + SO,,
~CO
HC
°3
'1966' 1967 I 1968 I 1969 I 1970 1971
YEAR
CHAMBER ATMOSPHERES — R, R + SO>, SO», NOL + NO2H
IDENTICAL FOR R AND R + SOx
NOH+N02L
19661 1967 I 1968 I 1969 I 1970 1971
Figure 4
Five-year summary of experimental atmospheric levels.
-------
biological measurements, intra-animal sampling variation within the same animal can be
considerable. For example, one respiration may not yield exactly the same respiratory
function measurement as another respiration. Such data reflect intra-animal sampling varia-
bility. An assay error is a failure to measure reproducibility constituents present in a
particular sample in repeated analyses using the same assay technique. For example, several
assays of the number of cells in a single blood sample may yield several different numerical
values. The differences observed are due to assay error. Another possible error in this study
was the intra-chamber induced error, which can be due to the movement of the animal about
the chamber, to variations in the quality of the atmospheres that were ducted, into the
chamber over time, and to the possibly unequal pollutant distributions within the chamber
space. There are degrees of variation realized between chambers receiving the same type and
level of experimental atmospheres, the so-called inter-chamber induced variation, which may
be an even greater source of error. These possible errors were controlled by physical means if
at all possible, or mathematically through such devices as covariance analysis, e.g., relating an
animal's post-exposure response to its pre-exposure response, or by arranging adequate
controls and test extra space groups within homogeneous sets of conditions, i.e., within
"blocks." In Table 5 the different methods for reducing the five basic types of errors are
indicated. In this experiment, we decided blocking was unnecessary because we were able to
eliminate most of the sources of variation that would relate to blocking (e.g., noise and light)
in the exposure rooms. In addition, the animals represented a very homogeneous population.
The physical steps that were taken to create a statistically valid experimental design included
the purchase of dogs in two shipments from the same supplier, and assembly of the dogs as a
stock colony before the study began so that groups could be chosen at random from the total
pool of available animals for the different exposures. With the exceptions mentioned below,
allocation of animals to specific exposures and assignments of animals to the triplicate
exposure chambers for each atmosphere were made at random. Some judicious compromises
of complete randomization were needed to prevent bias. In one instance there were two
sisters which were separated. In another case, the random selection would have paired the
largest dog with a very small animal. They were not placed together so that the small animal
would not be subjugated or dominated by the larger dog. Aside from these few instances, a
complete randomization was performed. One of the farsighted statistical features for the
Table 5. Methods for Reducing Errors in Animal Exposure Experiments
Methods for reducing errors
Basic error type
1) Inter-animal biological
variability
2) Intra-animal sampling
d)'
Physical
(2)
Mathematical
X
(3)
Blocking
X
(4&5)
Replication
(with randomization)
X
X
(sample of blood)
3) Assay error X — X X
4) Intra-chamber induced — — X X
variations
5) Inter-chamber induced — — X X
variation
20
-------
design was the use of a random order of sampling. All blood sampling, the analyses of the
samples, the lung function measurements, etc., were performed in random order over the
entire period of the study. The statistician furnished typewritten randomized schedules to the
biologists for selecting the animals for sampling. Independent randomizations were done for
each sampling period. Theoretically and practically, this was a very good system. In summary,
even though an attempt was made to control the known sources of errors by other techniques
mentioned earlier (e.g., blocking, mathematical corrections and differencing), still the ran-
domization method was a useful statistical device because it controlled or prevented other
unknown errors or bias and allowed them to be dealt with by probabilistic methods in
statistical analyses.
As mentioned earlier, the dogs were assigned randomly to the chambers, and the treatments
were applied to the chambers randomly with respect to chamber locations so that factors such
as human activity, noise, light, air from the halls when the doors were opened, etc., would not
confound the experiment. The triplicate exposure chambers were distributed randomly
throughout the array of 28 chambers. The only restriction was that the SOX chambers were
located closer to the source of the gas, since SOX atmosphere deteriorates rapidly in long
ducts. The location of the SOX chambers was not systematic with respect to any other factors.
Statistical analyses of the biological test data were performed using appropriate methods.
Analysis of variance was used to analyze biological measurement data. "Treatment" and
"chambers by treatments" were considered as sources of variation in the analysis. The
sources of variation and a sample analysis of variance of red blood cell counts on 96 dogs are
shown in Table 6. Expected mean squares indicate that the "chambers" mean square is the
proper denominator for the F-ratio, which tests for "treatment" effects. However, in the
absence of significant "chambers" effects, alternative tests were performed utilizing animal
variability as the error variance.
It was believed that the results obtained on different sampling days in a given period would
be statistically homogeneous, therefore day of sampling was omitted from the analysis of data.
The design of the experiment included three embedded 2x2 factorial treatment arrange-
ments as follows:
i) R x SOX Raw exhaust (2 levels)
CA R
SOx
(2 levels) SOX R+SOX
ii) I x SOX Irradiated exhaust (2 levels)
CA I
Exhaust vs. NOx
(2 levels) SOX I+SOX
iii) (Auto exhaust vs. NOX)
x Irradiation Irradiation (2 levels)
R I
SOX
(2 levels) NOH+N02L NOi+N02H
These factorial arrangements were exploited to improve the sensitivity of the data analysis.
21
-------
Table 6. Analysis of Variance of Red Blood Cell Counts (RBC) on 96 Young Female Beagles8
Source of variation
Atmospheres (treatments, T)
Chambers within atmospheres
(C)
Animals within chambers
(biological variation, B)
Duplicate assays (A)
Degrees of
freedom
7
16
72
96
Mean square
(X 1012)
4.02
0.98
0.91
0.03
F
4.12
1.07
25.06
—
Probability
0.009
0.40
0.000
—
EMS
(Expected mean square)
2 ,2 2
di + 2d! + 8dc
d^ + 2d|
«*A
aExperimental Design:
8 atmospheres: 7 contaminated, 1 clean air control
3 chambers per atmosphere
4 female dogs per chamber
-------
The 7 degrees of freedom for "treatments" were partitioned into single degree-of-freedom
contrasts corresponding to main effects and interactions for the above-listed 2x2 factorial
arrangements. The details of the statistical approaches used are referenced (9,10).
Exposure Facilities
The exhaust gases were generated by an automobile engine coupled to a dynamometer unit
designed to produce exhaust according to an established driving pattern (Figure 5). Clean
(CBR-filtered), conditioned air was mixed with the gases in a dilution system to produce the
desired concentration. From the dilution system, the exhaust gas mixture was divided with
one part flowing directly to animal exposure chambers, and the remainder flowing through
irradiation chambers to other animal chambers (Figure 6). The irradiation chambers (Figure
7) were lighted by a composite of three types of fluorescent tubes designed to simulate
sunlight. The facility included 28 animal exposure chambers and six irradiation chambers
(see Figure 8).
To approximate the exhaust generated by an automobile in city traffic, the engine dynamo-
meter unit was accelerated and decelerated as an automobile would be in moving traffic. In a
5-minute period, the engine cycle was the following: acceleration from a 10- to 30-mph cruise,
deceleration to 15 mph, acceleration to a 30-mph cruise, and deceleration back to 10 mph.
The diluted exhaust generated during a 5-minute engine cycle had a variation in the CO
content of 50 to 180 parts per million (ppm) with an integrated average of 100 ppm.
Dilution System
The engine-dynamometer unit generated an average CO concentration of approximately
57,000 ppm or 5.7% by volume over the 5-minute engine cycle. After cooling in a heat
exchanger and passing through a surge tank, the raw exhaust entered the dilution system
where it was mixed with clean air at the throat of a venturi. The venturi throat was
maintained at a negative pressure of 1 inch of water. The dilution ratio of clean air to auto
exhaust gas for an atmosphere of 100 ppm carbon monoxide was 570 to 1. This required a
raw-exhaust flow rate of 0.114 cubic foot per minute (cfm) to the throat of the mixing venturi.
To provide a means for adjusting the dilution ratio, a clean air line controlled by a vernier
needle valve was connected to a 'T' connection in the raw-exhaust line before the mixing
venturi. This needle valve was rotated by the correction servo motor on the output of the
automatic control system. To measure the CO content, a sample cylinder was used to
integrate the fluctuations over a 5-minute engine cycle. During the 2V6-minute measurement
and correction time, the average diluted gas sample was pumped by the sample cylinder
piston to an infrared CO analyzer. A direct current proportional to the CO content of the gas
sample from the output of the analyzer was used to energize the moving coil of a meter relay.
The contacts of the meter relay were positioned at the high and low limits of the required CO
concentration.
The direction of rotation of the needle valve correction servo motor was determined by
whether the high or low limit contacts of the meter relay were closed. If the limits imposed on
the CO concentration were not exceeded, the correction servo motor did not operate and the
amount of clean air added to the raw exhaust remained constant
23
-------
Figure 5
Engine room with the dynamometer system
-------
-\
AIR CONDITIONING
AND PURIFICATION
GAS
GENERATION
GAS
IRRADIATION
ANIMAL EXPOSURE
CHAMBER
MONITORING AND
RECORDING
Figure 6
Chronic auto exhaust study diagram.
-------
Figure 7
Irradiation chamber.
-------
EXHAUST STACK
i 1 — T 1 1 1 r"
1
E
2
CA
3
NOX
4
SOX
5
R
6
R+SOX
7
I+SOX
» f
/ / / 7
\ \\
--[ r~
8
NOX
9
CA
~T~
10
so*
~T~
11
CA
~r~
12
R
~T~
13
NOX
1
14
l
28
E
27
NO,
26
NOX
25
CA
24
R'SOX
23
R
22
I I L I I 1 1 >_^
E EPISODE
I IRRADIATED AUTO EXHAUST
NOXNOANDNO2
CA CONTROL AIR
R NON-IRRADIATED AUTO EXHAUST
* AUTO EXHAUST INJECTION POINT
Figure 8
Exposure chamber supply and exhaust flow diagram.
-------
The system schematic diagram in Figure 9 shows only one secondary system, since all four
were similar. The primary dilution occurred at point "A" where the air pressure was throttled
by a vernier needle valve described above. Secondary dilution took place at the venturi, where
negative throat pressure pulled the primary mixture into the duct system. Dampers affecting
the system are symbolized in Figure 9. Adjustment of any dampers resulted in a change in
CO concentration. The heat exchanger or surge tank "dump" dampers forced more raw
exhaust into the secondary when closed. The secondary and bleed damper were operated
simultaneously to maintain 33 cfm flowing in the main to the chambers. Each of the four
secondary mains had three branches so that the system of 12 chambers receiving auto
exhaust was balanced.
Instrumentation
The instrumentation was designed to analyze 29 gas samples (9 SO2, 6 Os, 6 HC, 6 CO, 1
NO, and 1 N02) by time-sharing the sample gases into a Flame Emission S02 Analyzer, a
Hydrogen Flame HC Analyzer, an Infrared CO Analyzer, and a Ratio Photometer NO-N02
Analyzer.
Sample lines of Vi-inch Teflon from the animal exposure chambers terminated in stainless-
steel solenoid-valve-operated manifolds located on the instrument table. The sample gases
were routed to the appropriate gas analyzer through the manifolds. The solenoid valves were
operated by selector switches on the multipoint strip chart recorders so that the gas-sample
line corresponding to the point to be printed on the recorder was open to the gas
measurement instrument.
The output of the SO 2 analyzer was connected to the input of a 12-print multipoint strip chart
recorder. The S02 concentrations in nine exposure chambers were sequentially recorded on
the first nine prints of the recorder. The tenth print of the recorder was used to indicate the
S02 concentration in the exposure-chamber room and was connected to an alarm system to
protect the operating personnel in case of a gas leak The eleventh and twelfth prints were not
used. The multipoint selector switch on the recorder sequentially operated the solenoid valves
on the manifold connected to the input of the S02 analyzer. Consequently, the S02 analyzer
received the gas sample represented by a point on the recorder at the time when that point
was to be printed. The S02 manifold was supplied with sample gases from three exposure
chambers fed with irradiated auto exhaust plus bottled SOa gas, three chambers fed with
nonirradiated auto exhaust plus S02, and three chambers supplied with clean air and bottled
S02 gas.
Eighteen points of a 24-print multipoint strip chart recorder were used to record the
concentrations of Os, CO, and HC in six animal exposure chambers fed with irradiated auto
exhaust. A 19th point was used to monitor the CO concentration in the exposure-chamber
room and sound the alarm if a leak developed. The multipoint selector switch on this recorder
controlled the solenoid valves on the CO-HC input manifold allowing only the gas sample,
represented by the corresponding point on the recorder, to flow into the CO and HC gas
analyzers. Another section of the multipoint switch connected the input of the recorder to the
signal output of the six Os meters, the CO analyzer, and the HC analyzer in the sequential
order of 0 3-CO-HC for each of the six exposure chambers. The solenoid valve at the
termination of the exposure-chamber sample line and the input of the CO-HC manifold was
opened when the selector switch reached the Os position, and remained open until the HC
28
-------
RAW EXHAUST HFAT HEA1 tXCHANUb DUMP
FROM EN
PURE AIR _
SUPPLY ~
RINF EXCHANGER V
PRESSURE (T\-,
GAUGE VL/
/
SERVO L— J s
lf"\l V
» _r~^ *""i
VERNIER
NEEDLE VALVE
i
» o r — f
PROPORTIONAL
' ' SAMPLE
€)— MBV
SURGE \SUHGblANKDUMP
TANK / (p "
)— BALL VALVE
/T^ NEGATIVE THROAT
^__^---\_y PRESSURE
I M .. 100ppm BL^,ED
I * 1 — ^~— J ' ~~~v. V
TO ATMOSPHERE
VENTURI
TO
INFRARED
ANALYZER
TO
EXPOSURE^
CHAMBERS
Figure 9
System schematic diagram.
SAMPLE CYLINDER
-------
concentration for the chamber had been recorded. As a result, the solenoid valves on the
CO-HC manifold were switched every three points on the strip chart recorder. This
arrangement allowed enough time for the CO and HC analyzers to settle at the concentration
in the sample line.
The NO and N02 concentrations in six animal exposure chambers fed with irradiated auto
exhaust were measured by a ratio photometer analyzer connected to the input of a two-print
multipoint recorder. One point recorded NO and the second point recorded N02. Because of
the 30-minute settling time required by this instrument, the inputs to the analyzer were
manually switched from one exposure chamber sample line to the other by a toggle-valve-
operated manifold.
A calibration manifold was added to the system to provide a means of putting conditioned air,
CO standard, HC standard, nitrogen or a bag sample into the CO and HC gas analyzers. This
was accomplished with toggle-switch-operated solenoid valves for each calibration gas, and
three-way solenoid valves that interrupted the output of the automatic sampling manifold.
The standards used were 90 ppm CO in nitrogen and 40 ppm carbon in nitrogen.
Sample gases from four animal exposure chambers supplied with non-irradiated auto exhaust
could be put through the HC and CO gas analyzers by manually operating a toggle-valve-
operated manifold and the toggle-switch-operated three-way solenoid valves. The toggle valve
was used to select the sample gas to be routed through the gas analyzers. The three-way
solenoid valves interrupted the flow of irradiated gas samples from the manifold controlled by
the multipoint strip chart selector switch.
Data Acquisition and Safety Alarm Systems
The data acquisition system provided a periodic record of 29 gas samples registered
sequentially on punched paper tape. The main function of the data system was to eliminate
the necessity of transferring the information on gas concentrations from the multipoint strip
chart recorders for computer analysis. The data system contained a digital clock, an input
scanner, an integrating digital voltmeter and a paper tape punch with parallel-to-serial
coupler. A sequence control unit synchronized the data system with the three independent
multipoint strip chart recorders controlling the input of the gas analyzers.
The safety alarm system was designed to indicate unsafe conditions both for personnel
working in the exposure-chamber room and for the experimental animals. Because some of
the pollutants fed into the animal exposure chambers were bottled gases, a warning system
was necessary to alert operating personnel if a gas leak developed. The instruments used to
measure the CO and S02 concentrations in the animal exposure chambers were also used to
measure the room concentrations of these gases. This was accomplished by adding solenoid
valves to the CO and S02 manifolds, which allowed room air to flow through the CO analyzer
when the Os-HC-CO recorder was on its nineteenth or last point, and through the S02
analyzer when the S02 recorder was on its tenth or last point.
Engine — Dynamometer Unit
The source of auto exhaust emissions was one or the other of two identical six-cylinder,
144-cubic-inch gasoline engines (see Figure 5). Each was equipped with an automatic
transmission and could be coupled to opposite ends of the dynamometer unit. Both engines
30
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had the necessary fuel, ignition, and cooling systems for separate operation with a minimum
of time required for switching from one engine to the other.
The dynamometer power absorption unit was an air gap, eddy current, double ended, dual
rotational type that used water cooling and had a power absorption rating of 50 horsepower
in a speed range of 1600 to 6000 rpm. A 300-pound flywheel was directly coupled to the
dynamometer unit to provide the rotational energy to simulate the inertial mass of a moving
2500-pound vehicle. The dynamometer field current could be set at constant value or made
dependent on the flywheel speed. Road speed and load conditions versus cycle time were
programmed and recorded to maintain reproducibility of operation. Drive shaft speed was
monitored by a built-in "Tach-Generator" and a load cell on the torque arm of the
dynamometer provided load readout.
Air Purification Unit
The cleanliness, temperature, and humidity of the dilution air used in the irradiation
chambers duplicated that encountered in clean urban atmospheres prior to contamination.
The air purifying system consisted of a pressure blower, filters, heat transfer coils, and a
humidifier combined into a single unit, together with a separate refrigeration compressor and
cooling tower. This portion of the facility supplied approximately 500 cfm of air at a pressure
of an 8-inch water column above atmospheric, measured at the outlet of the humidifier
section. The pneumatic controls were completely automatic once outlet temperature and
relative humidity had been set on the appropriate controllers.
The 7.5-ton capacity refrigeration compressor was a water-cooled type with an unloading
control. A forced-draft indoor cooling tower supplied recirculating cooling water to the
condenser coils. The sensing element for the unloader, located in the exit side of the cooling
section, actuated a thermostat-operated switch that controlled the compressor unloader and
maintained the cooling coil at the required operating temperature. Saturated air at 40° dry
bulb leaving the coil was then brought to the desired final conditions by the temperature and
humidity controls.
Hydrocarbons, organic and inorganic gases, and solid particulates, were reduced to accept-
able levels by a filtering system. A prefilter 2 inches thick on the intake of the blower was
followed by four absolute filter elements to remove particles down to 0.3 micron diameter,
and four activated charcoal filters to remove odors and gases. The entire air distribution
system ahead of the exposure chambers was constructed of stainless steel wherever the air was
in contact with a metal surface.
Irradiation Chambers
The general dimensions of each of the six irradiation chambers were 23.5 feet long by 47
inches wide by 8 feet high and each had a volume of approximately 683 cubic feet. No metals
other than aluminum and stainless steel were employed in any location that came into contact
with the atmosphere of the chamber. The sides were of a framework of aluminum structural
members. The plastic films that formed windows to pass the irradiating energy were clamped
and sealed by means of pressure screws and gasketed channels. The top, bottom, and ends of
the chamber were formed of V4-inch thick aluminum plate welded inside and outside at all
seams to prevent leakage. A sliding manhole door secured in place by screw clamps was
provided for access to the interior of the chamber (see Figure 7).
31
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Sources external to the chamber supplied intense ultraviolet radiation, which passed through
windows of "Teflon" FEP, type "c" fluorocarbon film, of 5 ml thickness (500 gauge). Each
irradiation chamber had four banks of 74 flourescent tubes mounted in four external cavity
reflectors. The 96-inch tubes were operated from ballasts remotely mounted at the ends of the
chamber room and both high and low voltage leads were brought into the panels. The
lighting panels were supported on trolley tracks for rolling back from the chamber side to
facilitate servicing light tubes and changing window films. Three types of tubes, 16 blue light,
46 black light, and 12 sunlamps comprised the 74 tubes in each lighting panel.
The blue fluorescent lamps radiated energy in a band extending from 3000 to 6000
angstroms with approximately half the total energy radiated in a 4000- to 5000-angstrom
band. A spectral peak was found at 4400 angstroms. The black fluorescent lamps radiated
most of their energy in a 3000-to-5000 band, peaking sharply in the 3500-angstrom region.
The sunlamps radiated ultraviolet energy in the 2800- to 3500-angstrom band. One-half of this
energy, however, was radiated in a band from 3000 to 3250 angstroms, with a sharp energy
peak at 3100 angstroms.
The switching was so arranged that circuit breakers on the control panel operated one-fourth
of the lamps on each of the four lighting panels to provide even distribution of reduced
intensities.
To maintain the lamps at their most efficient operating temperature, a system to remove the
generated heat from the lighting panels was provided. This consisted of an exhaust fan taking
air from the shallow plenum chambers mounted on the outside of the panel reflectors and
connected to the exhauster through duct-work on top of the chamber. Room air was drawn
through the space around the four sides of the panel, through a row of 5/32-inch holes in the
reflector immediately behind each lamp, and into the exhaust system. Quick-disconnect slip
joints were used to connect the duct to the mating duct on the panel, with a canvas
connection provided to facilitate alignment. The reflectors were painted with an indoor flat
white paint containing a high magnesium oxide content as an extender in combination with
titanium oxide for good reflection of ultraviolet radiation.
Animal Exposure Chamber Construction
The exposure chambers (Figures 10 and 11), constructed of 14-gauge type 304 stainless steel,
were 9 feet 10 inches high, 3 feet wide and 3 feet deep. Formed funnels, top to bottom, had an
angular slope of approximately 53°. A volume of 44 cubic feet, or 1,245 liters, between the
supply and exhaust funnels was available for animal exposure. At the bottom of the chamber
were four uncaged beagles. The access door, 54 inches high and 32 inches wide, was made of
3/8-inch plate glass set in a neoprene gasket and framed with stainless steel. The door had a
full-length piano hinge and five pressure clamps, permitting pressure adjustment to seal the
door tight against its neoprene gasket. The clamps were positioned at each corner opposite
the hinged side and midway between the corners, both horizontally and vertically. In the rear
of the chamber, a window of 3/8-inch plate glass 18 inches wide permitted observation from
both sides. The lower formed-funnel section was supported in a structural-angle-iron frame.
Adjusting bolts in the floor plates of the legs were provided for leveling the chamber.
Cage rack support pins were welded in the four corners of the chamber at six levels about 10
inches apart. The removable cage racks were formed of ^4-inch diameter stainless steel rods
32
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Figure 10
Animal exposure chambers.
-------
CLEAN AIR SUPPLY
EXHAUST
©
?5=*
/
/
WOOD PLATFORM
/
WASTE
1 SUPPLY DAMPER
2. ORIFICE PLATE
3 INJECTION PORT
4 PEGBOARD GAGE PANEL
5 SAMPLE PORTS
6. 2" GAS OUTLET VALVE
7 3" DRAIN VALVE
8 FLASHING
9 GRATING FLOOR
10 CAGE SUPPORTS
Figure 11
Animal exposure chamber — Schematic diagram.
34
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with eyelets in the corners to engage the support pins. Cages for rats or guinea pigs hung
from the racks and could slide in and out of the chamber on light-gauge stainless-steel angle
spot-welded to the sides of the cages. The floor of the chamber was 12-gauge stainless steel
11/16 x 1-25/32 inches expanded metal supported on a cage rack. Each chamber had six cage
racks and two expanded metal gratings. This construction allowed flexibility in chamber
loading and permitted exchange of racks when the animals were serviced.
Four V^-inch-diameter iron pipe size (ips) stainless steel couplings were welded through the
wall at the rear of each exposure chamber. Through one of these ports, atmosphere samples
at the breathing zone of the animals were taken continuously with a stainless steel tube
extending 18 inches inside the chamber wall. The gas was drawn through Teflon tubing to the
centrally located gas analyzing instruments.
The negative static pressure of 0.3 inch of water in the chamber was measured with a
magnehelic gauge connected by a tee fitting to the second coupling port. The third port
contained a well-type dial-face thermometer for observing the temperature inside the cham-
ber. The fourth opening was used to provide fresh drinking water to the dogs on the bottom
level of the chamber by means of an automatic watering device (see Figure 11).
Airflow, Cooling System and Miscellaneous Provisions
Airflow to each exposure chamber receiving irradiated or raw auto exhaust was metered by a
Venturi type flowmeter in the supply duct. In the clean air supply duct to the other chambers,
orifice-type flowmeters were used. The pressure taps of each flowmeter were connected to a
magnehelic pressure gauge on the panel board above each chamber. An airflow calibration
curve was drawn for each flowmeter, and the gauge was marked with a red arrow for the
proper flow. The flow through these meters could be varied but was set for 11 cubic feet per
minute of 15 air changes per hour through each chamber. Rates of airflow and static pressure
in the chamber were controlled by means of a butterfly damper located in the supply duct and
a globe valve in the exhaust duct. The damper in the supply duct was manually controlled at
each chamber through mechanical linkages that permitted fine adjustment. A negative static
pressure of 0.3 inch of water was maintained in the operating chambers by a globe valve in
the exhaust pipe. Air was exhausted through a 2-inch elbow turned downward and located
beneath the floor in the center of the chamber. The elbow was connected to the exhaust
header by a stainless steel pipe welded through the side of the lower funnel.
In addition to controlling the cleanliness, temperature, and humidity of the dilution air supply
described earlier, the temperature rise due to the irradiation chamber lights had to be
overcome. The 2-inch diameter auto exhaust lines from the six irradiation chambers to their
respective six animal exposure chambers were wrapped with 1/4-inch refrigeration copper
tubing for a distance of 5 feet, just before the entrance to the exposure chamber. Freon from
the refrigeration compressor for the dilution air supply was circulated through a main header
to each of the six locations requiring additional cooling. Solenoid valves were provided at the
branches, and there was a main switch to cut off the cooling when desired. The insulated lines
could lower the temperature inside the animal exposure chamber from 80 to 75 °F when in
operation. A seven-day circular-chart recording instrument for continuously monitoring
temperature and humidity of the air used for dilution was installed to check on the
performance of the air conditioning equipment.
35
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For each irradiation chamber, there was a magnehelic gauge with a differential pressure
switch preset to actuate on a 1-inch water column decreasing positive pressure. A central
alarm bell was used to indicate failure of any of the Teflon fluorethylene propylene co-polymer
windows in the six irradiation chambers or any other irregularity in pressure. A series of pilot
lights indicated if a chamber was operating below normal, and individual switches were
provided to deactivate the alarm for low pressure in each chamber. When the irradiation
chamber pressure returned to normal, the alarm bell would ring again until the switch had
been returned to the normal position.
One leg of the tee-fitting connected to an exposure chamber access port was provided with a
magnehelic gauge. The other leg was provided with a differential pressure switch preset to
actuate a red pilot light on the instrument panel above the chamber door on a decreasing
negative pressure of 0.1-inch water column. The light would indicate to the operator a
malfunction, such as an improperly sealed chamber door or a clogged globe valve in the
chamber exhaust line. For the latter, a 2-inch tee with a plug in one leg served as a cleanout
between the chamber and the common exhaust header for a row of seven chambers.
Precautions to insure a safe working environment for laboratory personnel included a 3,500
cfm exhaust fan in a room window. An abnormal concentration of CO, S02, or N02 in the
exposure room from a leaking bottle gas cylinder or any ruptured lines would automatically
start the fan. A pilot light on the instrument panel would light, and an alarm would ring by
means of a wiring circuit connected with the chart recorders of the instrumentation that
monitored the room atmosphere as well as the exposure chambers. The window exhaust fan
also served as a backup system in case of failure of the exposure-room air conditioning
system, which was a separate unit. Corridor air or outdoor air could be drawn through the
room to provide ventilation until the air conditioning unit was repaired.
A major consideration from the operator's point of view was keeping the chambers clean.
Around the perimeter of the chamber and directly beneath the stainless steel expanded metal
rack, the bottom level for animal exposure, was a 1/2-inch ips spray ring. Water from four
wide-angle nozzles flushed the excreta on the bottom cone to the drain. The water was
regulated to 102 °F to prevent steaming inside and to the rear of the individual chambers. A
3-inch quick-opening lever-operated gate valve located at the bottom of the chamber was
opened when the water spray ring was turned on. The drain valves from a row of chambers
were connected to a common line leading to the city sewer. Another water line discharged
into the sewer where the excreta entered to provide a flushing action and keep the sewer line
clean.
When the entire chamber, from top to bottom, required cleaning, the dogs were removed to a
mobile cart and the interior of the chamber was hosed down. A portable unit with a hose
attached to the hot water line was used where detergent chemical action was required to cut
any deposit buildup. The unit had a IVfe-quart refillable plastic container in which a mild acid
could be used. An injector containing an adjustable valve regulated the volume of cleaning
solution for best efficiency. Once the surface was completely clean, regular washing with the
high-pressure water hose nozzle would keep it clean for about one week.
36
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References
1. Stara, J.E et al 1974. Toxicology of atmospheric pollutants resulting from fuel additives
and emissions associated with the use of automotive catalytic converters. Pp. 751-779 in
Vol. II, Recent advances in the assessment of the health effects of pollution, International
Symposium Proceedings (Paris, June 1974).
2. Hueter, EG. et aL 1966. Biological effects of atmospheres contaminated by auto exhaust
Arch. Environ. Health 12: 553-560.
3. Stara, J.E 1970. Technical review committee on the chronic dog study. Report to NAPCA,
DREW.
4. Stara, J.E 1970. Chronic toxicity of auto exhaust and other air pollutants in beagles.
Report to the Director, DHER, NAPCA, DREW (September 1, 1970).
5. Stara, J.E 1970. Further comments related to the termination of the chronic auto exhaust
study in beagles. Memorandum to Director, DHER, NAPCA, DHEW (September 11,
1970).
6. Stara, J.E 1969. Chronic toxicity of auto exhaust and other air pollutants in rodents and
beagle dogs. Revised Study Protocol (November 7, 1969).
7. NABD (AEROS, U.S. EPA, Office of Air Quality, Planning and Standards).
8. Air Quality Data for 1967 and 1968. 1971. NASN and contributing state and local
networks, APTD-0741.
9. Cochran, W.G., and G.M. Cox. 1957. Experimental designs, 2nd ed. John Wiley and Sons,
Inc., New York.
10. Snedecor, G.W., and W.G. Cochran. 1967. Statistical methods, 6th ed. The Iowa State
University Press, Ames, Iowa.
11. Korth, M.W. 1963. Dynamic irradiation chamber tests of automotive exhaust. Environ-
mental Health Series, Air Pollution. U.S. DHEW, PHS.
12. Hinners, R.G. et aL 1968. Animal inhalation exposure chambers. Arch. Environ. Health
16: 194-206.
13. Stara, J.E 1972. Summary of the data from the chronic auto exhaust study on the beagle.
Internal Report No. 2 to Division of Health Effects, NAPCA, DHEW.
14. Stara, J.E 1973. Chronic effects of auto exhaust, simulated smog and other pollutants in
beagles. Internal Report No. 3 to Division of Processes and Effects, Office of Research
and Monitoring, EPA.
15. Stara, J.E et al 1972. Chronic effects of auto exhaust and other atmospheric pollutants in
female beagles. ETRL Annual Report for 1972, NERC, EPA.
16. Stara, J.E et aL 1976. Long-term effects of auto exhaust and related pollutants in beagles.
I. A study overview and pulmonary function effects. Proceedings of 172nd ACS Sympo-
sium, San Francisco, California, Section on Environment, ABS. No. 38.
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Asilomar Conference Discussion
Stara: The animals were exposed, almost to the day, for 68 months. The other 2 months were
spent performing the pulmonary function and other tests, and moving the dogs to Davis at 70
months. During this time the beagles were in the chambers but not exposed to the auto
exhaust and to the other atmospheres.
Albert: Would you explain what the NOL + N02H and NOn + N02L are?
Stara: The concentrations are 0.2 of NO and 0.5 to 1 ppm of N02 in one instance, 1.5 to 2
ppm of NO and 0.2 ppm of NOa in the other instance.
Albert: These were based on the ratio of NO/N02 in raw and irradiated atmospheres, so this
was pulling out NOX as a pollutant and looking at it individually.
Stara: Exactly. These were the concentrations that appear in Table 3. The time line shows the
series of tests that were done in Cincinnati. They are indicated with an asterisk. The
remainder of the tests were done in California. About February 1969, we have separated the
dogs in chambers. There were other miscellaneous tests performed. For example, Dr. Lee has
done some sulfur determinations, and Dr. Johnson has studied the evoked potential (EEC)
response. There was also some clinical chemistry data available. Some immune response
testing was performed with rabies antigen at CDC.
Hueter: We weren't interested in the driving system. We were interested in the concentration
of pollutants in the atmosphere. We ran the engine any way we had to in order to get those
concentrations. The carbon monoxide level actually was the pollutant index controlling the
exposure levels.
Albert: How did you make the sulfuric acid?
Stara: The sulfuric acid was produced using fuming sulfuric acid, and then evaporating it into
the atmosphere of 55% humidity where it became reconstituted into sulfuric acid mist, with
an average particle size of 6 microns.
Question: At what relative humidity?
Stara: About 55%. We used 28 of the inhalation chambers. Ken Busch has selected at
random chambers for the various treatments according to Table 1.
Comment: How many chambers for each atmosphere?
Stara: Three chambers for each of the exposure atmospheres, five chambers for the control,
for a total of 26 chambers. Two of them were used for other purposes. Table 1 is a review
figure for the individual atmospheres and how many dogs were alive in which atmosphere
and which chamber. Figure 1 1 shows a diagram of one of the chambers. The exhaust and the
clean air supply was brought up from the top, and on the bottom the gaseous exhaust and the
liquid wastes were eliminated. This chamber is a useful chamber for inhalation studies, but it
would serve better for smaller animals like rodents, because they could be separated at three
levels. A 3 ft. x 3 ft. area is just too small for four beagles. A review of the maintained gaseous
concentrations is presented in Table 3.
38
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Busch: The NO is twice as high by itself as it was in the irradiated, but it's low. It was meant
to match, but it's a little bit high, 0.2 instead of 0.1.
Stara: Yes. The NO, N02, S02, ozone were supposed to be, as Dr. Hueter said, at
approximately the levels found in raw and irradiated auto exhaust, and that's why they were
selected.
Comment: They're nominal there. They're not actual concentrations.
Question: But it didn't work that way, did it? When you put that much S02 in with the
irradiated, you convert a lot of the sulfuric acid. We found out after a while that we had
something like 300 or 400 mg/m3. We were killing some guinea pigs and then we had to
reduce the S02 going in to get back to 100. Wasn't that true, Mr. Malanchuk?
Malanchuk: That's true. Again, as mentioned previously, the S02 is a nominal value that was
aimed at, and we adjusted the supplementary supply to try to maintain the exposure chamber
concentration at that level. And sometimes it would go up pretty high. That is, we wanted to
maintain the S02 overall concentration, with sulfuric acid really. Apparently S02 was getting
converted into sulfuric acid and we had to reduce the SO 3 flow from the supplementary
fuming sulfuric acid supply to try to maintain the concentration at the nominal level
Stara: As to the dogs, we had the four dogs at the low level of the chamber. Female beagles at
this age, 4 or 5 years old, in a small enclosure become rather irritable, perhaps also because
they were not mated. The animals were separated into pairs, after the leader problem was
solved. They were fed during the 8-hour rest period; they had water ad libitum. In early 1970,
we began to exercise the dogs during the 8-hour period ... and most of the dermatitis cases
and other cases were corrected. One of the points that I want to make in conclusion is that we
were fortunate that we kept the animals alive after the experiment, even though they were not
exposed in California, since we were able to use techniques for analysis of the tissues and
pulmonary function tests that were not available at the time when the exposures were
stopped. So I think it was quite fortunate that we had saved the animals and had them
transferred to California where many additional and new tests were performed. I just hope
that our decisions, which were of course uncertain at the time, were correct and that this
extensive study will result in useful data for EPA and the scientific community.
39
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2. EXPOSURE CHAMBER ATMOSPHERES.
SAMPLING AND ANALYSIS
M. Malanchuk
Chamber Atmospheres
The exposure study facilities are described in the preceding report. Details of the system for
delivering the exhaust emissions from the automobile engine to the animal exposure
chambers are given in that report, as well as the preparation for monitoring the atmospheric
components in the chambers.
In addition to the diluted exhaust gas, supplemental supplies of several individual gases and
of sulfuric acid aerosol were introduced to certain chambers. The gases — nitric oxide (NO),
nitrogen dioxide (N02) and sulfur dioxide (S02) — were metered from supply cylinders of
those gases diluted in air or nitrogen. The concentrations of these cylinder gases were
established at levels convenient for delivering amounts that would accurately attain the
desired levels in the exposure chambers. The flow-meter readings were checked continuously
and the flow rates adjusted infrequently to maintain the desired pollutant levels in the test
atmospheres.
Sulfuric acid aerosol was provided by the pressure of a stream of dry air flowing over the
surface of fuming sulfuric acid in a tightly enclosed Erlenmeyer flask. The sulfur trioxide
(SOs) vapor was transferred into the inlet duct at the top of the exposure chamber. The
reaction of the SOs with the water vapor of the duct air produced the sulfuric acid needed for
the chamber atmospheres. There was a total of nine chambers, each equipped with its own
acid generating system. The acid supply, approximately 30 to 40 ml in a 250 ml flask, was
renewed as needed to maintain a median value concentration of 100 jug H2S04/m3 in the
chamber.
Methods of Sampling and Analysis
Automatic continuous monitoring and manual sampling checks, both regular and occasional,
were carried out on the various exposure atmospheres (see Table 1). The automatic continu-
ous monitoring focused on the irradiated (I) chamber atmospheres for measurement of
carbon monoxide (CO), hydrocarbons (HC) and the nitrogen oxides (NO and N02) by
panel-mounted instruments (see Table 2). A combination of sampling tubing network mani-
folds and automatically controlled solenoid valves enabled the use of one analytical instru-
ment for each of these components in all the chambers. Ozone was continuously monitored in
the I-chambers by individual detection instruments mounted on the side of each chamber,
with automatic input of the electronic signal to the data acquisition system.
In addition, sulfur dioxide (SOg) was monitored continuously in the irradiated atmosphere
chambers that were supplemented with S02 (I + SO*) as well as in the S02-supplied clean air
(SO*) chambers and in the S02-supplied chambers containing the non-irradiated exhaust
emissions (R + SO*). All the automatically monitored concentration values were recorded
both on charts and on tape. The chart readings were used to maintain daily control over the
operations of pollutant gas supply to the chambers. The tape records were used for the
statistical evaluation of the data.
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Table 1. Major Atmosphere Components and Measurement Strategy
Treatment
Control (clean)
air (CA)
Non-irradiated
auto exhaust (R)
Irradiated
auto exhaust (I)
SO2 + H2SO4
(SOX)
Non-irradiated
auto exhaust + SC>2
+ H2SO4 (R + SOX)
Irradiated auto
exhaust +862
+ H2SO4 (I + SOX)
Nitrogen
oxides
(NOL + N02H)
Nitrogen
oxides
(NOH + N02L)
Chamber
2,9, 11,
21,25
5
12
23
14
16
22
4
10
20
6
18
24
7
15
17
8
19
26
3
13
27
CO
M
AM
A
A
A
AM
AM
AM
A
A
A
HC
M
AM
A
A
A
AM
AM
AM
A
A
A
NO
M
M
M
M
A
A
A
M
M
M
A
A
A
M
M
M
M
M
M
N02
M
M
M
M
A
A
A
M
M
M
A
A
A
M
M
M
M
M
M
03
A
A
A
A
A
A
S02
A
A
A
A
A
A
A
A
A
H2SO4
A
A
A
A
A
A
A
A
A
Key: A = Sampled and recorded automatically, continuously
AM = Sampled regularly and recorded manually
M = Sampled variably and recorded manually
Table 2. Pollutants Measured Automatically and Continuously
Component
Chamber type(s)
Principle of measurement8
Carbon monoxide (CO) I, I + SOX
Hydrocarbons (HC) I, I + SOX
Nitrogen oxides (NOX):
Nitric oxide (NO) I, I + SOX
Nitrogen dioxide (N02) I, I + SOX
Ozone (Os) I, I + SOX
Sulfur dioxide (SO2) I + SOX
Sulfuric acid (H2SO4) I + SOX
Non-dispersive infra-red, NDIR (U-108)
Flame ionization detection, FID (U-55)
Colorimetry (U-97)
Colorimetry (U-97)
Colorimetry (U-43)
Flame photometric detection, FPD
(U-133)
Ion Conductivity (U-20)
apage no. of instrument description in Air Sampling Instruments, 4th ed. (1972), published by
ACGIH, Cincinnati, Ohio, is provided in parentheses.
Analysis for peroxyacyl nitrates (PAN) in chamber atmospheres was fruitless. Measureable
concentrations of PAN were not detected in any of the exposure chambers, nor in several of
the irradiation chambers themselves.
Twice-a-day measurements were made on the non-irradiated atmospheres (one R chamber
and all three R + SOX chambers) for CO, HC, and NOX (NO and NOg). The manifold plus
42
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solenoid valve system was used to "cut in" the line samples to the same panel-mounted
instruments used for the I atmospheres. The readings were entered manually on a data sheet
for statistical processing. All the nitrogen oxide values were obtained by individually collected
bubbler samples treated for colorimetric measurement (1). Table 3 lists various components
and measurements that were handled on a non-regular basis during the study in order to get
a more comprehensive assessment of the exposure atmospheres. Procedural details are given
in the following discussion of the results of the measurements.
Analytical Results of the Study
Automatically Monitored Components
The mean concentration values for the major components during the 5 +-year period of the
study are listed in Table 4. A comparison of the data with the nominal concentration values
listed for the start of the study shows a satisfactory achievement of the intended levels. A
running account of the chamber atmosphere monitoring is shown in Figure 4 of Chapter 1. It
is evident that the concentrations were maintained reasonably constant throughout the study.
Specific Hydrocarbons
The gas chromatographic system to monitor specific organic components in the exposure
atmospheres was put into service 9 months after the start of the study and carried on to the
end. The system was used to measure various C2 to €5 hydrocarbons in 6 to 8 daily samples
collected from exhaust atmospheres after chamber concentrations reached equilibrium. Table
5 summarizes typical concentrations. Normal variations for these components are indicated in
parentheses.
Chamber samples were also analyzed for aromatic hydrocarbons. The two compounds
determined in this phase, and their typical concentrations in exhaust chambers, were benzene
Component
Hydrocarbons
Lead (Pb)
Particulate mass
Particulate size
Particulate anions
Sulfate
Nitrate
Halide
Acrolein
Formaldehyde
Table 3. Pollutants
Chamber type(s)
1, R, 1 + SOX,
R + SOX
1, R, 1 + SOX,
R + SOX
all
all
1, R, 1 + SOX>
R + SOx
1, 1 + SOX
1, 1 + SOX
Measured Periodically
Frequency
Daily, as of
May, 1966
Periodic
(weeks at a time)
Periodic
(weeks)
Periodic
(weeks)
One period of
several weeks
Periodic
(weeks)
6-mo. period
in 1966
Method of analysis
Gas chromatography
A.A. spectro-
photometry
Phototape sampler
Optical, light
scattering
Wagman, 1967 (2)
Saltzman, 1954 (1)
Bergmann, 1957(3)
Cohen, 1961 (4)
Sawicki, 1961 (5)
43
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Table 4. Pollutant Concentrations in the Exposure Chamber Atmospheres During the
Chronic Exposure Study (ppm [mg/m3], mean ± s.d.)
Group
1
2
3
4
5
6
7
g
Atmosphere
Control air (CA)
Non-irradiated
auto exhaust (R)
Irradiated
auto exhaust (I)
SO2 + H2SO4(SOX)
Non-irradiated A E
with
SO2+H2SO4(R + SOX)
Irradiated A.E.
with
SO2 + H2SO4(I + SOX)
Nitrogen oxides
(high NO2),
(NOL+N02H)
Nitrogen oxides
(high NO),
(NOH + NO2L)
Nominal
Carbon
monoxide
CO
49
97.5 + 100
(112.1 + 11.5)
945 + 196
(108.6 ±22.5)
98 4 + 1 3 8
(113.1 ±15.9)
94.8 ±19.8
(109.0 ±22.8)
100
Hydro-
carbons
(as CH4)
2.7
27.5 + 4.4
(18.0 ±2.9)
23 9 + 6 1
(15.6 ±4.0)
27 4 + 4 3
(17.9 ±2.8)
23.9 ± 6.0
(15.6 ±3.9)
24-30
Nitrogen
dioxide
N02
0.04
0.05 + 0.02
(0.09 + 0.04)
0 94 + 0 36
(1.77 ±0.68)
0 05 + 0 03
(0.09 ±0.06)
0.89 + 0.36
(1.68 ±0.68)
0.64 + 0 12
(1.21+0.22)
0.15 + 033
(0.27 ± 0.62)
0.5-1.0
Nitric
oxide
NO
0.04
1.45 + 0.42
(1.78 + 0.52)
0 19 + 029
(0.23 ±0.36)
1 51 + 0 44
(1.86 ±0.54)
0.19 + 0.29
(0.23 ±0.36)
0.25 + 0 06
(0.31 + 0.08)
1.67 + 021
(2.05 ± 0.26)
1.5-2.0
Oxidants
as
03
0.02
0.20 + 0 09
(0.39 ±0.18)
0.20 + 0.08
(0.39 ±0.16)
0.2-0.4
Sulfur
dioxide
S02
0.03
0.42 + 0.22
(1.10 + 0.57)
0 48 + 0 23
(1.27 ±0.61)
0.42 + 0.21
(1.10 ±0.56)
0.50
Sulfuric
acid
H2SO4
0.02 + 001
(0.09 + 0.04)
0 02 + 0 01
(0.09 + 0.04)
0.03 ± 0.01
(0.11 + 0.04)
(0.100)
-------
Table 5. Hydrocarbon Concentrations in the Exposure Atmospheres
at Equilibrium (ppm)
Treatment
Components
C2H4-C2He
C2Ha
C3H8
C3H6
i-C4Hio
n-C4H10
Butene-1
Isobutylene
Butadiene
i-C5H12
n-C5H12
R
1.04
0.36
0.02
0.29
0.05
0.21
0.02
0.02
0.02
0.27
0.15
(0.25)
(0.10)
(0.02)
(0.09)
(0.03)
(0.05)
(0.03)
(0.02)
(0.04)
(0.07)
(0.05)
I
0.86
0.34
0.02
0.15
0.04
0.20
trace
trace
trace
0.24
0.14
(0.19)
(0.07)
(0.02)
(0.06)
(0.02)
(0.05)
(0.01)
(0.01)
(0.01)
(0.10)
(0.06)
R +
1.CO
0.37
0.02
0.26
0.05
0.20
0.03
0.02
0.04
0.26
0.14
sox
(0.25)
(0.09)
(0.02)
(0.10)
(0.02)
(0.08)
(0.03)
(0.02)
(0.04)
(0-10)
(0.06)
I +
0.86
0.35
0.02
0.17
0.05
0.20
0.01
trace
trace
0.25
0.15
sox
(0.18)
(0.08)
(0.02)
(0.08)
(0.02)
(0.08)
(0.01)
(0.01)
(0.01)
(0.11)
(0.05)
(0.1 ppm) and toluene (0.2 ppm). Several other peaks were observed in the chromatogram, but
-generally at concentrations too low to be adequately quantified.
At various times analyses were made for other aliphatic hydrocarbons. Concentrations of
hexane, cyclohexane, heptane and octane were estimated at about 0.1, 0.1, 0.05, and 0.1 ppm,
respectively. Separations were poor among this group and the results should be considered
semi-quantitative only.
Atmospheric Paniculate Concentration and Size
Relative values of airborne paniculate concentrations and of particle size were obtained to
present a comparison among the different exposure atmosphere types. A phototape sampler
was used for indicating the mass concentration. It measured and recorded the light transmis-
sion through an area of filter paper (tape) on which a thin layer of particulate had been
collected. The decrease in transmission over the collection period represented the equivalent
of a concentration of airborne particulate. The phototape sampler was not calibrated to
indicate an absolute mass concentration but was used for a limited period for comparisons of
particulate loading among the chambers. The transmission values reported in Table 6 show
the final percent transmission after sampling 15 cubic feet over a 30-minute period onto a
14-mm-diameter spot, with the initial transmission adjusted to 100%. The data show that the
transmission of the I-chamber samples was the lowest (i.e., indicating the highest concentra-
tion of particulates), followed in order by R, SOX, NOX and clean air.
An optical particle sizer using the light scattering principle indicated that more than 90% of
the particles of any atmosphere type were smaller than 0.5 urn in diameter. The I atmo-
spheres bore 10 to 15 times as many particles as the R atmospheres. The CA chambers
contained only about one percent the number of particles found in the I-chamber atmo-
spheres.
45
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Table 6. Phototape Sampler Transmission Values
Atmosphere type Transmission (%)
Irradiated auto exhaust (I) 85.5
Irradiated A.E. + SO2 + H2SO4 (I + SOX) 88.7
Non-irradiated A.E. (R) 93.0
Non-irradiated A.E. + S02 + H2SO4 (R + SOX) 95.1
S02 + H2S04 (SOX) 97.8
NO + N02 (NOX) 99.4
Clean air (CA) 100.0
Analysis of the particulates collected by filters (6) gave the concentrations of anions shown in
Table 7.
Table 7. Anions Associated with Airborne Particulate (mg/m3)
Sulfate
Nitrate
Halide
R
0.37
3.3
20.2
I
5.9
581.
28.7
R + SOX
21.0
11.3
17.6
I + SOX
99.2
223.
32.7
Acrolein
Acrolein was determined by the method of Cohen and Altshuller (4), which utilizes
4-hexylresorcinol as the color-producing agent. The method measures concentrations as low
as 0.001 ppm. A total of 150 samples from I chambers 14, 16 and 22, and I + SOX chambers
7, 15 and 17 were analyzed in 1966 and 1967. The data are summarized in Table 8.
Table 8. Acrolein (ppm)
Treatment
1
1 + SOX
All samples
Min.
0.0090
0.0072
0.0072
Max.
0.0288
0.0306
0.0306
Mean
0.0198
0.0192
0.0195
Formaldehyde
Formaldehyde analyses were performed on 357 total samples from I chambers 14, 16 and 22
and I + SOX chambers 7, 15 and 17 in 1966, using the 2-hydrazinobenzothiazole method of
Sawicki et aL (5). Table 9 summarizes these data.
Table 9. Formaldehyde Concentrations (ppm)
Range
Treatment Min. Max. Mean
~j 028 UIO 0.57
I + SOX 0.26 0.88 0.49
All samples 0.26 1.00 0.53
46
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Atmospheric Lead Determinations
Atmospheres containing raw and/or irradiated auto exhaust mixed with SOX were analyzed to
determine chamber atmospheric lead concentrations during 1967, 1969 and 1970 (7). The
sampling plans for the three determinations were somewhat different from each other. In
1961 only total lead in four treatment atmospheres was measured by a direct method. In 1969
assays for both soluble and insoluble lead were performed and total lead concentrations were
calculated. In 1970, both calculated and direct assays were compared by means of paired
filters, but only two treatments were sampled.
Filter holders connected with calibrated critical pressure orifices were used to collect 16-hour
suspended-paniculate samples from the automobile exhaust chambers on two-inch GA-4 grid
Millipore filters having a pore size of 0.08 /j. Glass probes were introduced to the chambers to
sample at a point directly above the animals.
Both water and acid-lead extracts were analyzed by atomic absorption spectrophotometry.
Lead absorption was measured at a wave length of 283 p.
The 1967 data showed about 30% higher average calculated total lead concentrations in R +
SOX, compared to I + SO* A corresponding difference did not exist between R and I. All
four atmospheres exhibited large, positively correlated, concomitant day-to-day variations in
total lead concentrations.
The 1969 data showed about 50% higher concentrations of both soluble and insoluble lead in
the I + SOX and R + SOX treatments as compared to the respective concentrations in the I
and R treatments. Soluble lead was about 40% higher than insoluble lead in all four exhaust
treatments. A similar soluble.-insoluble ratio was observed for CA and SOX but was not
measured as precisely as for the other four treatments.
The 1970 data showed an almost equal division of total lead between soluble and insoluble
forms. No treatment differences existed for either of the lead forms, for their total, or for the
directly measured total lead concentrations. However, the directly measured total lead
concentrations were about one-third higher than the calculated total lead concentrations.
Averages of directly measured total lead concentrations were about the same in 1967 and
1970 for the I + SOx and R + SOX treatments. On the other hand, for the same two
treatments, the average calculated total lead concentration for 1969 was 37% higher than the
average calculated total lead for 1970. The difference appeared to be due entirely to the
soluble lead, which was 74% higher in 1969.
Figure 1 summarizes these conclusions. Averages are shown wherever components of the
average were statistically homogeneous.
47
-------
1967
Direct Total Lead
I + SOX
19.4 \— NS
+34% (")
1969
1970
R, I
R + SOX I + SOx
+54%
Soluble | 79
•l^MB
+39% +42%
Insoluble | 5.7 | 1~86
| +51%
Calculated Total I 13.6 I
+53% (")
R + SOX, I + SOX
+74%
Soluble
Insoluble
Calculated Total
Direct Total
(NS)
8.2
15~2~
Weighted
Average
+40% (")
+37% C)
+33% (")
(NS) = not statistically significant at P = 0.05
(*) = statistically significant at P = 0.05
(**) = statistically significant at P = 0.01
Figure 1
Pattern of differences in mean CAE study lead concentrations (ptglm3)
48
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References
1. Saltzman, B.E. 1954. Colorimetric microdetermination of nitrogen dioxide in the atmo-
sphere. AnaL Chem. 26: 1949-1955.
2. Wagman, J., R.E. Lee, and CJ. Axt 1967. Influence of some atmospheric variables on the
concentration and particle size distribution of sulfate in urban air. Atmos. Environ. 1:
479489.
3. Bergmann, J.G., and J. Sanik. 1957. Determination of trace amounts of chlorine in
naphtha. AnaL Chem. 29: 241-245.
4. Cohen, I.R., and A.P. Altshuller. 1961. A new spectrophotometric method for the deter-
mination of acrolein in combustion gases and the atmosphere. Anal. Chem. 33: 726.
5. Sawicki, E. 1961. The 3-methyl-2-benzothiazolone hydrazone test AnaL Chem. 33: 93.
6. Lee, R.E. 1971. Concentration and particle size distribution of particulate emissions in
automobile exhaust Atmos. Environ. 5: 225-237.
7. Barkley, N.P., 1972. The concentration of lead in automobile exhaust exposure chambers.
AIHA Journal 33: 67&683.
Asilomar Conference Discussion
Malanchuk: If you will refer to Table 6, the phototape sampler transmission values show a
progressive increase in the transmission value, which means that at the top of the list with the
lower transmission, that actually means a higher relative concentration of particulate mass.
You can see it stepwise there from 85.5 for the irradiated auto exhaust to 100.0 for clean air.
Tyler: The most particles are in the irradiated, and they literally grew there as you irradiated
it, pretty significantly over the nonirradiated.
Malanchuk: Right
Question: Is there a difference in the aging time in the different atmospheres? In other words,
is there more time for particulate growth in the irradiated?
Malanchuk: Yes. There was very little time for the raw exhaust particulate due to the direct
dilutant effect versus the irradiated particulate in the irradiation chambers.
Stara: We have realized this problem in our more recent studies, and we now have a mixing
chamber for the raw exhaust also, which gives us the same residence time for the various
chemical reactions in both raw and irradiated exhausts.
Lewis: Mr. Malanchuk, I have heard criticisms of the study, many of which were invalid and
some of which I consider valid. Can you make any kind of quantitative estimate as to how
much of the particulate was lost in our simulated system versus what one might encounter in
the atmosphere? Was it 10%, 50%, 80%, or what? Dr. Stara is nodding his head yes; maybe
he can answer that
49
-------
Stara: I don't know if Mr. Malanchuk momentarily recalls, but we have done a study of this
problem. The losses of paniculate from the mixing chamber or the irradiation chamber to the
animal chamber are between 10 and 20%.
Malanchuk: Right
Lewis: What you are trying to simulate is the urban environment where you have dilution as it
comes out of the tailpipe. Well, the criticism that I heard was that we didn't have enough
particulate, and intuitively I would think that we had more than would be found in the urban
environment
Question: How many changes per hour did you have in the chamber?
Malanchuk: Fifteen.
Comment: There's one point; maybe I missed it in your discussion of the exposure system, Dr.
Stara. It is my understanding that on an engine that is pulling weight, the exhaust would be
different than on an engine that is free-wheeling. I heard you mention something about the
simulated weight or wear. But was it actually pulling any weight?
Stara: Yes. The stationary dynamometer system that we employed attempts to simulate the
road conditions.
Hueter: Engine output, and that's why it was cycling, because you don't get much NOX in
certain modes of operation without cycling the engine.
Stara: For example, during steady speed this may be so. We had to use an approved engine
cycle which'decelerates and accelerates to produce auto exhaust which has the required
pollutant combination.
Hueter: Relative to lead content, I wondered, the concentrations were similar, but were the
particle sizes similar, and do you have some data on the ratio of organic/inorganic, which
were different?
Malanchuk: I don't know whether I have all the raw data here to readily answer your
questions. I think the report that we had written by Barkley et al. (7) shows a fairly detailed
discussion of this problem. I would refer you to that, to answer your question.
Lewis: I recall from the biological data that there was a difference in bone meal which we
attributed to particulate size, chemical composition, and solubility.
Malanchuk: As I remember it, there seemed to be a lower lead concentration in the SOX
chambers, wasn't there? Can you remember whether you saw that in the Barkley report? It's
as if the SO x had a chance to do a scavenging act, apparently, on the lead and bring it down.
Hueter: Although the animals in raw and irradiated exhaust received the same essential lead
exposure, measured as total lead, the animals receiving the lead in raw exhaust had higher
blood levels than those receiving irradiated exhaust. We didn't know if that was due to
particle size or due to chemical form.
50
-------
Stara: There are two or three future studies that have not been completed, even though for all
practical purposes I feel that the major study is completed. One of them is the bone lead
values at sacrifice. We still have the bone samples. As a matter of fact, Webster Jee from Salt
Lake City suggested a small amount of lead mapping in the bone to see the lead distribution
in the hydroxyapatite.
Albert: Could you explain what Figure 1 is about.
Malanchuk: These values and percentages are to show the difference between conditions that
were similar, and that the values are apparently significantly different, such as the 37%(*).
For instance, if you look at the 40%(**) on the extreme right-hand side, it says "statistically
significant at P = 0.01," meaning that between the soluble and insoluble forms of lead that
were analyzed in 1969, the 10.1 and 7.2 show that much difference, to that extent
Hueter: Right to the left of that you've got "R, I" and "R + SOX, I + SOX." So you say that
"R, I" means soluble/insoluble with 7.9/5.7. There wasn't any difference between R and I.
Malanchuk: The weighted average for the solubles (R and I plus the R + SOX and I +S0x) is
shown statistically to be significant at P = 0.1 (**) when compared to the composite value for
the insolubles, however. The R did not vary significantly from the I values in these particular
groupings of data, it would seem.
Hueter: We didn't have time to find out why. If lead comes out of the tailpipe, it's usually in a
form of chlorobromo-lead, either di-chloro, di-bromo, or chloro-bromo, something like that. I
don't know if the irradiation of that has an effect on changing the form before it gets to the
animal.
Bhatnagar: But you probably have more sulfate in the irradiated exhaust.
Albert: But was there more lead in the nonirradiated than in the irradiated atmospheres?
Hueter: Not significantly, according to this table here.
Albert: You can make that out?
Hueter: Well, it says R, I, 22.7, so apparently they were so close that they weren't significant.
Albert: What is the 22.7?
Hueter: f^g/m3,1 presume.
Malanchuk: Right.
Albert: Nonirradiated and irradiated. Is there a difference?
Malanchuk: No.
Hueter: Not in total lead.
51
-------
Albert: How can you tell? What about the holdup in the radiation chamber where you could
lose particles?
Hueter: The particle size at that point where it comes out of the tailpipe is pretty small.
Lewis: And it's diluted equivalent to the dilution to 100 ppm of CO.
Question: In the case of sulfate in the irradiated versus the nonirradiated atmospheres there
is a difference of 6 ^ig.
Lewis: That's what Mr. Malanchuk said. The sulfates seem to be scavenging lead.
Hueter: But yet if that's true, then more of it should be insoluble than soluble, and it's not.
Lewis: But we don't know what kinds of subsequent chemical reactions occurred within the
irradiation chamber.
Busch: It was significant that the value for R and I had enough measurement error to be
potentially equal to either the 19.4 or 25.9. There wasn't a statistical difference in either of
them. But the two extremes, the I + SOX and the R + SOX, were different.
Malanchuk: These samples were done in three different years — 1967,1969, and 1970 — and
we may have had differences in engine operations and fuel supply that may have affected the
lead and paniculate analytical scheme. So that, looking at the overall picture, I come up with
a couple of bare conclusions. One is that over that period of 1967-1970, and I am repeating
myself, the lead concentration apparently had no radical change in the gasoline supply during
that period of time. Take it for what it's worth. Apparently the SOX had a kind of scavenging
effect on bringing down the lead since there seemed to be a fairly good difference in the SOX
chambers as compared to those that did not have SOX supplement.
Lewis: Correct me if I'm wrong, but I think it's important that when you work for the
government, GSA wants you to buy on low bid, so we didn't always have the same supplier.
Isn't that right, Dr. Hueter?
Hueter: That's true. We didn't have storage facilities to store enough gasoline for 5 years.
Lewis: The blend changed through those years, as did other factors which affected the
composition of the gasoline used to generate the auto exhaust atmospheres.
Busch: The engine was overhauled and tuned up, rings and valves were replaced, the head
was taken off periodically and the valves ground — wasn't there a complete new engine
installed during the course of the study at one time? I can't recall.
Hueter: There were two engines. They served as alternates to one another. When one was
being overhauled, the other one was being run.
Busch: But we never purchased new engines, did we?
Malanchuk: No. Not for this study as far as I know.
52
-------
Lewis: Engine changes only occurred in the old initial study when things were changed
around for the new system, i.e., 1972.
Busch: What bothered me was that the NO seemed to be so stable around the target level
during the major part of the study and then dropped off some tenfold in the last couple of
years. I wondered if we could pinpoint why that happened. The N02 remained the same, but
the NO is lower. Could it have been an analytical error?
Malanchuk: Let me appeal to Dr. Stephens here, as an expert Aren't we put out yet to
explain the complex interactions among the N02, NO, oxidants and so forth in the
atmosphere? There is a very touchy condition where you have this equilibrium, N02, NO, and
ozone, and hydrocarbons involved. I think with a slight decrease in hydrocarbons or what
have you, you can have a pretty harsh change in the other components.
Busch: Are the 6-month averages plotted in the diagram (Figure 4 of Chapter 1)?
Malanchuk: I think they're more like about 41/2 or 5 months,
Busch: In the computerized data analysis, we were assessing the values each 6 months.
Malanchuk: This was the data that was handled especially by the group summarizing the
information for the final report.
Comment: Well, to establish that curve, I think that special statistical or data accumulation
studies were conducted.
Stephens: Well, I couldn't claim to be an expert as far as the dynamics between the gases are
concerned. We've had a great deal of experience with N02 compound exposures as well as
ozone. I do not claim any expertise in the dynamics between the two other atmospheres.
Lewis: I don't know what to attribute it to, but it's only in one of the eight treatments where
it's outstanding. That's the I+ SOX. In the other six treatments, it's not apparent. If you look
at Figure 4 of Chapter 1, which is the auto exhaust and auto exhaust + SO*, it's essentially
linear. If you look at the I, it's not nearly as abnormal, although the concentrations of oxides
of nitrogen are rising in the last phase of 1971. So whether it's an analytical mistake or
whether it's an engine or what, it's hard to say. But it would appear to be related to an
irradiation interaction phenomena.
Hueter: It couldn't be an engine because the other chambers also got the same exhaust from
the same engine. So it would have to be an analytical error or some other reaction occurring
in the system.
Lewis: But I think it's more of an artifact than a reality.
Hueter: You'll never explain it.
Bhatnagar: It's not an artifact if you start looking for all the chemical entities and free radical
species in the irradiated exhaust.
53
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Lewis: Yes, but to tie them into the NO levels is very difficult when you look at all other things
in common, the CO, the N02, etc.
Bhatnagar: Dr. Stara, I have a question for you. You said that in your later experiments, the
raw exhaust also passed through a chamber giving it the same time as irradiated exhaust. Is
there a difference in particulate concentration?
Stara; First of all, this phenomenon was not observed during this study. It was noted during
the studies of catalytic converter emissions. And in the latter instance, the particulate matter
analysis is completely different. The particles are inorganic sulfuric acid and sulfates rather
than the inorganic/organic particulate mix you find without catalytic converters. So I don't
think that the question is relevant to this study.
Hueter: Also, the irradiation there creates particulate matter. You always have more particles
in irradiated than in raw.
Stara: Yes.
Busch: If I could make one observation about the quality control on the atmospheric
measurements in the chambers; with respect to the CO concentrations, which I understand
were nonreactive, we used data during an 8-hour day, monitoring data. We had 5-minute or
10-minute readings, as I recall, automatically monitored, and they were plotted. A calculation
was made completely independent of the data. An independent mathematical model was
developed based on a differential equation. The differential equation showed that the rate of
increase in the CO was proportional to the rate of introduction of CO into the chamber. The
input concentration (it was monitored going into the radiation chamber), the flow rate, and the
volume of the chamber were known (it was 864 cubic feet, as I recall). Based on the
differential equation we obtained a mathematical model for the exponential increase in CO
concentration in the irradiation chamber. Assuming that there was instantaneous mixing and
that as the material enters it immediately mixes and is diluted with what was already in the
chamber, we could draw the mathematical curve which followed almost exactly through the
data. It was remarkable. It indicated to us that the engineering of the radiation chamber,
including the fans to mix the gases as they were introduced, was adequately done.
54
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3. CLINICAL SUMMARY OF BEAGLE STUDY.
PHYSICAL EXAMINATIONS, OCULAR EXAMINATIONS,
HEMATOLOGY EXAMINATION (POST-EXPOSURE)
AND BLOOD CHEMISTRIES
J.G. Orthoefer, J.E Stara, Y.Y. Yang and K.I. Campbell
Clinical examinations were periodically conducted by a veterinarian to assess the health
status of the animals. The results of these examinations were used to form a basis for therapy
and/or for inclusion or exclusion of functional data to be analyzed.
During the exposure period, the use of drugs was minimized, particularly drugs which might
change the animals' response to a pollutant, such as corticosteroids. Health disorders were
cared for immediately. The primary purpose of any treatment was to prevent clinical
disorders from compromising or complicating the toxicologic interpretations.
Body weights were taken on a monthly basis throughout the study. The body weights were
corrected on a body volume basis using girth size at the shoulders to determine an ideal
weight. No group differences in body weight were attributed to the treatment regimen.
However, for intervals of time throughout the study period certain animals had to be specially
fed in order to adjust the body weight to a more ideal weight. Cases of obesity and cachexia
were corrected primarily by these husbandry practices. This was accomplished by removing
the animals at night during the normal feeding time and allowing them to eat prescribed
amounts of food.
Dermatitis cases were seen throughout the exposure and post-exposure period. The cases
ranged from mild to severe (Figure 1). There were specific exposure trends in dermatitis with
a prevalence in the NOL + N02H group in 1969 and 1971. However, a relatively high
prevalence occurred in the control group. Therefore, it was difficult to attribute the dermatitis
to anything other than some unknown factor. No specific etiology was ever determined. The
dictates of the study demanded that four beagle dogs live in an area of 9 square feet. This
may have been more detrimental psychologically to the animals than exposure to pollutants.
By December of 1973, as shown in Figure 1, the dermatitis was of relatively minor
significance, indicating that removal from the chambers and placement in a less restricted
environment during non-exposure periods was beneficial. Since the animals were removed to
a different environment entirely (food, caging, disinfectants, etc.), the change in skin condi-
tion cannot be attributed solely to a lack of pollutant exposure. The cases of dermatitis were
vigorously treated after the cessation of the exposure, when time was available for therapeutic
baths, etc. Although higher incidences of dermatitis occurred in certain groups during the
exposure period, and these effects persisted in some groups to a minor degree even during
the recovery period (Groups 6 and 7, Figure 1), no attempt was made to attribute the
dermatologic differences directly to the pollutants.
Ocular examinations were performed during the routine physicals. Observations were made
of the external and internal structures of the eye in search of gross disorders. Two more
detailed examinations were conducted, one by Dr. Milton Wyman (Ohio State University)
near the termination of the exposure, and one by the University of California during the
post-exposure period just prior to euthanasia.
55
-------
Prevalence of Chronic Dermatitis (%)
^ K <•* *• 01
O O O o o
Prevalence of Chronic Dermatitis (%)
8 g § S
g
n
CD
o
CD
3
CO
_L
(?
o
CD
d
ST
co
N>
n
n
CO
CT
*
CO
s
_±J
IS)
co
* 1
01
O)
^i
oo J
1 _i
ts)
CO
Zl *
Ol
en
oo |
] -*
N)
w
^
Ol
o>
3 "^
oo
1
8
_i
1 &
^
c
Q^ "2 3
1 o o ~*^
1
1
1
-------
Epiphora (constant washing of the eye by tears) may have been related to the exposure
atmospheres. As shown in Figure 2, the dogs exposed to a few atmospheres (2, 3, 6) show a
prevalence of epiphora above that of the control group of dogs. This relationship did not
persist after the cessation of exposure.
The tabulated results of the detailed ocular examination prior to cessation of exposure are
shown in Table 1. Although some superficial effects due to close contact of the pollutants with
the eye were expected, no such effects were discerned. Permanent eye damage may have been
prevented by the constant washing of the eye associated with epiphora.
No association between exposure and oral disorders or otitis was observed. Dental calculi and
periodontal disorders were largely prevented by routine cleaning of the teeth by the
caretaking staff. Otitis externa was treated by the use of antibiotic ointments when necessary.
From the initiation of the experiment, hematological studies were conducted at approxi-
mately 6-month intervals. Measurements included red (RBC) and white (WBC) blood cell
601
50-
£. 40-
a
0)
o
c
30-
20-
10-
1. Control
2. Raw(R)
3. Irradiated (I)
4. SOX
5. R + SOX
6. I + SOX
7. NOL + NO2H
8. NOH + NO2L
November 1969
August 1970
June 1971
Figure 2
Prevalence of epiphora in experimental animals.
57
-------
Na
Table 1. Tabulation of Ocular Lesions, as Reported by Dr. Wyman
Exposure Lids
Con-
junct. Cornea Iritis
Lens Retina
Other
18
11
10
8
10
11
11
10
89
CA
R
I
SOx
R + SOx
l + SOx
NOL + NO2H
NC-H + NO2L
Total
3
2
1
2
4
4
2
2
3 3
2 2
1
2
3
1 2
1
3
1 2
2
2
4
6
4
1
Papill 1
1 Papill 1
Phthisis
bulb
1
1
Papill 1
Plus: a. Dental abscess-related inflammation — one dog (R + SOX)
b. Post-distemper-related retinal atrophy — one dog (NOn + NO2i_)
"Two dogs (one in SO* and one in R + SOX) were not accounted for in the report as having
been examined. They are assumed to have been in the "normal aging changes" category.
counts, hematocrit (Hct), hemoglobin (Hg), and the derived indices of mean corpuscular
hemoglobin concentration (MCHC), mean corpuscular volume (MCV^ and mean corpuscular
hemoglobin (MCH). The exposure hematology did not include differential cell counts.
Separate univariate analyses of variance of hematologic data were performed for each
measurement period Whenever significant treatment effects were detected, twelve defined
contrasts among the treatments were tested for nullity to reveal a possible cause of the effects.
During the exposure, groups exposed to auto exhaust (R, R + SOX, I, and I + SOX) had
higher mean Hct and Hg values than those exposed to no exhaust (CA, SOX, NOL + N02H,
NOn + N02l). The RBC of auto exhaust groups was higher throughout the study, even
though statistical significance was not obtained at every period. The comparison of Hct, Hg
and RBC under exhaust and no-exhaust conditions is shown in Figures 3 and 4. There was no
effect of auto exhaust on WBC, and no consistent effect on MCHC, MCV or MCH. The I and
I + SOX groups showed reduced RBC and Hg after exposure was terminated. No post-
exposure effect on any of the hematological parameters was detected in any of the other
treatment groups.
Table 2 presents a summation of the post-exposure hematology results. This work was done in
Dr. Jain's laboratory at the University of California, Davis. A more detailed post-exposure
hematology statistical work-up is presented in Appendix I of this chapter. The post-exposure
hematology included the differential cell count and differed in this respect from the exposure
hematology.
A set of 19 clinical chemistry parameters was measured on blood samples taken four times
during the study. These analyses were performed by Automated Analytical Laboratories, Inc.
(AAL) in Ventura, California. Samples were obtained in special tubes or bottles and shipped
air mail to AAL three times from Cincinnati, Ohio and once just prior to euthanasia from
Davis, California. Although there were some deviant individual values, these did not reflect
consistent or statistically significant group trends, and were not regarded as lexicologically
significant The group mean values for each of these tests are appended to this report (see
Appendix II).
58
-------
70
65
60
55
50
45
40
20
15
10
Red Blood Cells
(105 cells/mm3)
f(t) = 64.7 + 0.50t + 0.00019t3
Hemocrit
f(t) = 59.3 + 0.911 - 0.03312 + 0.00028t3
f(t) = 46.4 + 0.23t - 0.007112 + 0.00006t3
f(t) = 42.9 + 0.45t - 0.014t2 + 0.00012t2
Hemoglobin
(g/100 ml)
f(t) = 14.3 + 0.33t - 0.0097t2 + O.OOOOSt3
f(t) = 12.1 + 0.48t - 0.014t2 + 0.0001153
Exposure begins
10
20 30 40 50
Exposure Period (months)
60
70
Figure 3
The effect of exposure to auto exhaust on blood indices in the beagle dogs
pre-exposure and exposure. (Solid curves represent CO atmospheres, dashed are non-CO.)
^ = Non-exhaust
• = Exhaust
59
-------
75
70
65
50
45
20
17.5
15
Red Blood Cells
(105 cells/mm3)
Hematocrit
Hemoglobin
(g/100 ml)
80 90 100
Post-Exposure Period (months)
Figure 4
The effect of exposure to auto exhaust on blood indices in the beagle dogs post-exposure.
(Exposure ended at 68 months.)
^ = Non-exhaust
• = Exhaust
60
-------
Table 2. Certain Hematological Abnormalities in Beagles in
Relation to Different Treatments
Treatment
Period
and Nucleated
No. of dogs
Nov
1971
June
1972
Nov
1972
May
1973
Nov
1973
CA
I
l + SOx
R
R + SOx
NOX
sox
CA
I
l + SOx
R
R + SOx
NOX
sox
GA
I
l + SOx
R
R + SOx
NOX
so*
CA
I
l + SOx
R
R + SOx
NOX
sox
CA
I
l + SOx
R
R + SOx
NOx
sox
(18)
(10)
(11)
(11)
(10)
(20)
( 8)
(17)
(10)
(11)
(11)
(10)
(20)
( 8)
(17)
(10)
(11)
(10)
(10)
(20)
( 8)
(17)
(10)
(11)
(10)
(10)
(20)
( 8)
(17)
(10)
(11)
(10)
(10)
(20)
( 8)
RBC
38.9
40.0
18.2
45.5
60.0
75.0
25.0
17.6
33.3
27.3
27.3
10.3
35.0
0.0
23.5
30.0
27.3
30.0
30.0
35.0
0.0
35.3
20.0
18.2
30.0
20.0
25.0
0.0
23.5
40.0
18.2
20.0
20.0
20.0
25.0
Percentage of dogs with:
Plasma proteins
>6g%
5.5
0.0
27.3
27.3
10.0
5.0
12.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.9
0.0
9.1
20.0
0.0
5.0
0.0
<6 g%
0.0
0.0
9.1
0.0
0.0
0.0
0.0
5.9
10.0
9.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.0
18.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.0
0.0
Left shift
300 Bands
33.3
50.0
45.5
36.4
30.0
15.0
25.0
17.6
22.2
27.3
36.4
30.0
40.0
25.0
5.9
0.0
18.2
0.0
30.0
30.0
25.0
23.5
10.0
45.5
20.0
30.0
25.0
12.5
11.8
0.0
0.0
0.0
0.0
5.0
0.0
Basophils
<0.5%
66.6
50.0
45.5
54.5
70.0
60.0
25.0
55.0
44.0
45.5
72.7
90.0
65.0
50.0
76.4
80.0
45.5
60.0
90.0
60.0
75.0
76.5
60.0
63.6
70.0
80.0
65.0
87.5
67.7
50.0
36.4
50.0
50.0
50.0
37.5
Anaesthetic accidents and trauma resulting from fighting were the major causes of pre-
euthanasia deaths during the study. Tissue obtained from the dead animals was generally
inadequately preserved, thus preventing meaningful morphological examination. However, in
most cases the cause of death could be verified. Table 3 shows the number and causes of
pre-euthanasia deaths.
61
-------
Table 3. Date and Cause of Pre-Euthanasia Deaths
in the Chronic Auto Exhaust Study
Treatment
Control
Raw exhaust (R)
Irradiated exhaust (1)
sox
R + SOX
I + SOX
NOL+NO2H
NOH + NO2L
Month/year
of death
6/67
2/70
12/71
5/68
6/72
12/69
12/69
12/68
4/70
8/70
4/71
10/68
5/66
2/74
10/68
12/69
11/71
3/67
2/71
Cause of
death3
Ai
T
Ai
T
X
T
T
Ai
T
AT
A2
AI
X
X
Ai
T
T
Ai
Ai
&Ai = Anesthesia related to obtaining physiological data
A2 = Anesthesia related to surgical procedure for medical treatment
T = Trauma usually resulting from fights
X = Other
62
-------
Appendix I
Means by Treatment (Nov. 1971)
CA R I SOX R + SOx
TP
RBC
HG
PCV
MCV
MCHC
WBC
BAND
NEUT
LYM
MONO
EOS
Key:
TP
RBC =
HG
PCV =
MCV =
MCHC =
WBC =
BAND =
NEUT =
LYM
MONO =
EOS =
7.206 7.409 7.340 7.225
7.719 7.570 7.700 7.805
17.835 17.209 17.690 18.513
52.382 50.455 50.700 52.250
67.007 66.623 66.229 67.122
34.280 34.065 34.895 35.424
11594 11754 10350 10075
833 553 291 230
6595 6994 6051 5685
2743 2673 2504 2755
977 935 812 938
361 326 256 365
Total protein (g/100 cc)
Red blood cell count (x 106)
Hemoglobin (g/100 cc)
Packed cell volume (%)
Mean corpuscular volume
Mean corpuscular hemoglobin content
White blood cell count
7.450
7.641
17.880
51.300
67.137
34.879
12850
933
6644
2710
992
384
l + SOx
7.453
7.412
17.100
49.182
66.515
34.548
10772
267
6794
2379
1029
266
NOL +
N02H
7.100
7.324
17.270
49.900
68.203
34.557
12170
312
7111
2933
1033
672
NOH +
N02L
7.080
7.595
17.640
51.000
67.225
34.581
10940
172
5705
2943
808
387
Immature polymorphonuclear leukocytes
Mature polymorphonuclear leukocytes
Lymphocytes
Monocytes
Eosinophiles
Means by Treatment (June 1972)
CA R I SOX R + SOx
TP
RBC
HG
PCV
MCV
MCHC
WBC
BAND
NEUT
LYM
MONO
EOS
6.412 6.627 6.627 6.520
7.495 7.595 7.269 7.494
16.709 16.527 16.575 16.113
49.588 49.364 48.900 48.000
65.886 65.064 67.320 64.212
33.841 33.327 33.900 33.525
9458 10245 9670 8775
271 549 237 259
4907 5633 5888 4523
2691 2934 2293 2731
515 571 617 623
562 557 448 463
6.690
7.546
16.300
49.000
64.856
33.120
11190
309
6068
2608
651
588
l + SOx
6.673
7.220
15.523
46.591
64.609
33.391
10109
231
5774
2682
619
297
NOL +
N02H
6.730
7.095
16.175
47.500
67.030
33.940
12590
422
6966
2971
629
789
NOH +
N02L
6.560
7.143
15.645
47.100
65.580
32.940
11500
332
6602
3217
652
416
63
-------
Appendix I, cont.
Means by Treatment (Nov. 1972)
TP
RBC
HG
PCV
MCV
MCHC
WBC
BAND
NEUT
LYM
MONO
EOS
CA
6.976
7.771
17.544
51.588
66.482
33.971
10117
168
5177
2224
730
650
R
6.903
7.651
17.500
51.200
67.080
34.170
9070
186
5422
2305
581
476
I
6.980
7.219
16.675
49.200
68.170
33.830
8660
114
5115
1791
634
419
SOx
7.000
8.020
17.594
52.313
65.400
33.625
10137
210
5991
2740
674
403
R + SOx
6.980
7.248
16.525
48.100
66.400
34.280
11060
262
6672
2005
678
681
l + SOx
6.809
7.348
16.341
48.545
66.118
33.582
9081
207
5850
1829
680
451
NOL +
N02H
7.000
7.146
16.550
48.800
67.840
33.940
9640
266
5593
2505
600
587
NOH +
N02L
6.860
7.587
17.250
50.700
66.970
34.030
9600
214
5796
2467
684
385
Means by Treatment (May 1973)
RBC
HG
PCV
MCV
MCHC
WBC
BAND
NEUT
LYM
MONO
EOS
CA
7.543
17.044
50.206
66.659
33.924
9188
226
5357
2142
471
548
R
7.499
16.750
49.800
66.440
33.600
8720
195
5188
2228
364
575
1
7.442
17.389
50.889
68.389
34.133
8344
155
5447
1841
342
449
SOx
7.589
17.094
50.250
66.262
34.050
9900
384
4318
2309
436
620
R + SOx
7.347
16.800
50.100
68.230
33.500
10030
641
5377
1956
550
374
I + SOX
7.82
15.955
47.182
66.630
33.700
9581
341
5866
2779
523
393
NOL +
N02H
7.007
15.720
47.700
68.080
32.990
9920
346
5742
2521
561
549
NOH +
N02L
7.124
16.375
48.400
67.870
33.680
9100
221
5004
2910
505
364
Means by Treatment (Nov. 1973)
TP
RBC
HG
PCV
MCV
MCHC
WBC
BAND
NEUT
LYM
MONO
EOS
CA
7.071
7.352
16.353
48.353
65.776
33.782
10476
5935
6591
2423
522
693
R
7.290
7.462
16.450
48.500
65.100
33.900
9210
308
5549
2387
574
402
I
6.950
7.050
15.850
47.400
67.260
33.420
8460
206
5633
2459
491
334
SOx
6.975
7.545
16.531
48.750
64.712
33.875
10137
258
6078
2379
789
474
R + SOx
6.760
7.259
16.400
48.500
68.890
33.770
10450
504
7116
2107
697
416
l + SOx
6.955
7.145
15.636
46.727
65.500
33.418
9381
464
5773
2088
869
468
NOL +
N02H
7.050
6.787
16.325
46.100
68.040
33.240
9350
209
5471
2132
664
485
NOH +
N02L
6.950
7.174
16.225
47.600
66.260
34.080
11190
413
7175
2879
686
334
64
-------
Appendix II
Clinical Chemistry Summary (Jan. 1970)
Mean8
Calcium
Inorganic
phosphorus
Glucose
Urea nitrogen
Uric acid
Cholesterol
Total protein
Albumin
Globulin
A/G ratio
Total bilirubin
Alkaline
phosphatase
Lactic
dehydrogenase
S.G.O.
transaminase
Protein-bound
iodine
Ts uptake
Na
K
CO2
Cl
Triglycerides
CA
9.77
4.96
79.2
19.9
0.721
186
6.68
1.76
4.93
0.347
0.195
43.7
107
36.0
5.26
38.5
143
4.90
28.1
113
95
R
9.58
4.96
76.8
18.9
0.609
182
6.62
1.54
5.08
0.309
0.155
38.8
104
33.7
5.49
40.1
140
4.89
27.1
110
78
1
10.06
4.85
76.0
18.1
0.770
199
6.63
1.77
4.86
0.350
0.170
28.7
133
32.5
5.10
35.0
144
4.96
28.9
114
88
SOX R + SOx l + SOx
9.95
5.06
83.2
20.7
0.700
199
6.78
1.76
5.02
0.335
0.218
35.3
132
38.2
4.87
40.5
142
4.94
27.4
114
109
9.98
5.01
82.0
19.2
0.700
168
6.71
1.76
4.95
0.320
0.190
39.8
103
43.7
5.54
39.8
145
5.19
30.4
113
84
9.81
4.90
74.5
19.0
0.918
182
6.66
1.77
4.88
0.345
0.182
31.9
114
37.6
5.04
38.7
145
5.01
27.5
111
96
NC-L +
N02H
9.95
4.93
82.7
19.6
0.664
174
6.82
1.76
5.06
0.336
0.173
40.5
92
33.9
4.66
39.8
141
4.94
29.6
114
79
NC-H +
N02L
9.56
4.96
75.9
18.1
0.591
177
6.32
1.68
4.64
0.336
0.155
40.0
97
35.4
5.11
38.7
139
5.00
28.8
113
98
"Calcium — mg/100 ml
Inorganic phosphorus — mg/100 ml
Glucose — mg/100 ml
Urea nitrogen — mg/100 ml
Uric acid — mg/100 ml
Cholesterol — mg/100 ml
Total protein — g/100 ml
Albumin — g/100 ml
Globulin — g/100 ml
Total bilirubin — mg/100 ml
Alkaline phosphatase — milli-international units/ml
Lactic dehydrogenase — milli-international units/ml
S.G.O. transaminase — milli-international units/ml
Protein-bound iodine — f*g/100 ml
T3 — percent
Na — meq
K — meq
CO2 — meq
Cl — meq
Triglycerides — mg/100 ml
65
-------
Appendix II, cont.
Clinical Chemistry Summary (Sept. 1970)
Mean
Calcium
Inorganic
phosphorus
Glucose
Urea nitrogen
Uric acid
Cholesterol
Total protein
Albumin
Globulin
A/G ratio
Total bilirubin
Alkaline
phosphatase
Lactic
dehydrogenase
S.G.O.
transaminase
Protein-bound
iodine
TS uptake
Na
K
C02
Cl
Triglycerides
CA
10.23
4.79
103
16.78
0.556
73
6.83
1.46
5.37
0.278
0.306
50.6
83.1
37.5
6.18
41.1
146
4.88
37.2
114
50.9
R
10.23
4.90
95
17.91
0.518
95
6.74
1.49
5.25
0.278
0.225
50.0
68.0
33.5
5.88
41.5
146
5.04
36.0
117
68.8
I
10.56
5.26
103
15.20
0.49
76
6.65
1.49
5.16
0.291
0.210
42.0
75.0
33.5
6.42
43.5
145
5.20
38.0
118
76.1
NOL+ NOH +
SOx R + SOx I + SO, N02H NO2L
10.06
4.91
104
17.00
0.512
166
6.49
1.34
5.15
0.265
0.263
49.4
86.3
50.0
6.11
41.9
146
4.98
37.0
116
57.3
10.11
5.29
98
17.20
0.620
165
6.64
1.51'
5.23
0.292
0.280
46.5
55.5
32.0
6.22
42.0
146
4.92
37.2
116
79.2
10.39
4.73
100
14.18
0.455
180
6.39
1.40
4.99
0.285
0.277
41.4
45.0
31.8
5.09
42.0
146
4.86
35.9
116
58.5
10.44
4.77
103
14.36
0.518
146
6.97
1.34
5.64
0.248
0.255
46.8
55.5
34.5
5.16
42.1
145
4.68
36.5
117
52.7
10.15
4.99
115
21.0
0.600
178
7.00
1.56
5.45
0.292
0.255
45.5
73.2
50.9
5.86
41.5
145
5.03
38.4
117
96.9
66
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Appendix II, cont.
Clinical Chemistry Summary (April 1971)
Mean
Calcium
Inorganic
phosphorus
Glucose
Urea nitrogen
Uric acid
Cholesterol
Total protein
Albumin
Globulin
A/G ratio
Total bilirubin
Alkaline
phosphatase
Lactic
dehydrogenase
S.G.O.
transaminase
Protein-bound
iodine
TS uptake
Na
K
C02
Cl
Triglycerides
CA
10.33
3.57
78.3
10.61
0.867
179
6.72
1.544
5.17
0.267
0.272
50.6
113
37.2
5.41
41.4
145
4.81
37.9
104
74.9
R
10.49
3.74
76.4
9.09
0.573
212
6.76
1.518
5.24
0.264
0.282
50.9
120
34.7
5.21
41.5
145
5.00
38.6
103
86.1
1 SOX R + SO, l + SOx
10.35
3.81
78.5
9.80
0.680
165
6.36
1.480
4.88
0.250
0.260
43.2
111
35.4
4.79
41.9
146
4.89
38.8
106
63.0
10.13
3.74
81.9
10.25
0.775
185
6.59
1.438
5.15
0.250
0.214
44.4
108
34.0
6.25
41.4
146
4.78
38.9
105
75.5
10.49
3.61
76.0
10.20
0.880
175
6.86
1.530
5.33
0.250
0.280
52.7
128
40.0
5.52
41.7
146
4.84
38.0
105
81.2
10.53
3.94
76.8
9.64
0.995
178
6.59
1.527
5.06
0.264
0.318
40.9
123
36.1
5.01
40.4
147
4.83
38.5
106
69.3
NOL+ NOH +
NO2H NC-2L
10.25
3.74
75.9
10.82
0.782
156
6.79
1.500
5.29
0.264
0.373
56.8
149
39.5
5.09
41.5
146
5.05
38.4
105
70.3
10.50
3.81
74.5
11.10
0.860
160
6.89
1.550
5.34
0.250
0.280
50.8
126
40.8
5.48
41.7
145
4.78
37.8
104
65.0
67
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Appendix II, cont.
Clinical Chemistry Summary (Nov. 1973)
Mean
Calcium
Inorganic
phosphorus
Glucose
Urea nitrogen
Uric acid
Cholesterol
Total protein
Albumin
Globulin
A/G ratio
Total bilirubin
Alkaline
phosphatase
Lactic
dehydrogenase
S.G.O.
transaminase
T3 uptake
Na
K
Cl
CA
9.20
3.41
85
12.18
0.512
211
6.62
2.72
3.90
0.712
0.212
60.3
227
37.4
46.6
146
4.82
110
R
9.00
3.86
91
10.70
0.450
245
6.63
2.86
3.77
0.850
0.320
69.0
304
45.0
44.8
145
4.88
109
1
8.96
3.43
82
10.67
0.511
189
6.41
2.68
3.73
0.744
0.278
41.7
259
47.2
47.6
147
4.96
108
SOX R + SOx I
9.39
3.83
82
10.38
0.475
188
6.55
2.66
3.89
0.687
0.200
49.4
209
35.0
47.1
146
4.81
110
8.92
3.68
79
11.10
0.450
183
6.49
2.70
3.79
0.710
0.300
55.5
282
46.5
44.9
147
5.12
110
+ SO,
9.26
3.80
83
12.09
0.518
216
6.54
2.66
3.87
0.709
0.245
43.6
232
45.9
42.9
146
5.06
108
NC-L + NOH +
N02H N02L
9.040
3.30
86
11.10
0.440
189
6.60
2.59
4.01
0.650
0.270
47.5
190
44.5
45.8
145
4.85
111
9.010
3.72
81
12.00
0.460
198
6.79
2.71
4.08
0.700
0.430
84.5
243
46.5
42.2
147
4.93
112
Asilomar Conference Discussion
Hueter: This is like going back to a class reunion, being here with a lot of the people who are
here today, and I would like to make a few thank yous if that is appropriate, particularly to
two gentlemen who are no longer with us, Mr. Eugene Krakow and Charlie Punty, who were
heads of the laboratory at the time this study was started, and were instrumental in getting
support for this chronic study from our headquarters people. At that time, we were with the
U.S. Department of Health, Education, and Welfare. Many of you have been involved with
chronic studies in the past, and maybe some of you haven't, but I think we learned a lot in
conducting this. As Dr. Stara said earlier, we would do things a lot differently now if we did it
over again, but under the circumstances I think the study was done quite well. Mr. Hinners
and Joe Burkart, who were our engineers, put together an excellent facility that I don't think
exists anywhere else in the world. Mr. Krider and Malanchuk had the chore of doing these
routine monitorings of the atmospheres for many, many years. It's very difficult to find
people, scientists who would dedicate that much of their lives to this kind of a study. Most
people want a lot more visibility than one gets from this type of work. Of course, Ken Busch
pointed out the part that statistics played and we were fortunate in having him and his staff. I
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think the biologists involved should be complimented for being willing to cooperate the way
they did with the statisticians when the study was started, so at least we could get as much out
of it as possible under the circumstances. Drs. Lewis and Campbell and many other people
that were associated with the study devoted a lot of their time to something with an unknown
end product. I might point out, the question was raised before as to why the study was
stopped. The study cost us, I believe, about $250,000 a year to run. This included staff time
and supplies, that's to say nothing of what the facility cost to install, which I think was 11/2
million dollars. Our budget at that time was somewhere in the order of $900,000, so you can
see we were spending over 25% of our laboratory budget on this facility, and consequently we
were getting shot at every year when it was program planning time and budget time. What
have we found out, what do you see, have you seen anything, stop the study. So, it was a
constant battle from that point of view. The reason the study was stopped was in part that
kind of pressure and in part that at that particular time, the facility was being considered for
removal from Cincinnati and relocation in RTF in North Carolina. This meant a tremendous
disruption in the facility and the staff, so serious questions were then being raised as to
continuing the study. Compromises were then made, about where the animals would be
maintained. I'm not sure what my opinion was at that time, but I'm certainly glad that
whatever it was, it occurred the way it did. I might point out before this study was started
there were some very serious and detailed studies done with the chamber system. The dogs
were held in runs for a period of time to check them for their general health. They were then
put in the chambers for a period of almost 2 months to condition them to that type of living
facility before they were exposed to any atmospheres at all. The chambers themselves before
the dogs were even placed in them were characterized over a period of 6 weeks. We looked at
what concentration of pollutant was coming in at the top, what concentration was going out
the bottom. What were the chamber losses, in other words. We had different cage configura-
tions in the chambers, to look at that effect on the losses across the chamber of things like
ozone and NO* We then had different cage configurations in animal loadings, to look at the
effects of the animals on losses across the chamber. So that when we finally did start the
study, we could have a minimization at least of loss of what the animals on the bottom were
receiving relative to the animals on the top. So a lot of work went into this before the study
was even started. The reason these atmospheres were selected, of course, is pretty obvious.
We were interested at that time in auto exhaust, catalytic oxidation catalysts were not even
thought of at that time, and we were also interested in NOX as an individual pollutant because
it of course is a source of pollution from point sources as well as mobile sources. And with a
limited number of chambers, that was the reason we picked those eight atmospheres, which
include the clean air. We had done another study prior to this with auto exhaust using smaller
chambers and using all rodents, hamsters, rats and mice, in which we attempted on an
engineering basis to expose animals to four different levels of auto exhaust, irradiated and
nonirradiated, and cycling it diurnally over a period of 24 hours to simulate the type of thing
that happens, for example, in Los Angeles. This was extremely difficult to do from an
engineering point of view. There was a lot of overlap between the various levels that we
attempted to reproduce. That's why we decided on trying to maintain the exposure atmo-
spheres at a constant level over a period of 16 hours per day, 7 days a week. This is not
realistic from the point of view of city atmospheres, it was realistic from the point of view of
cutting down the variability of the study. We did, as Ken Busch pointed out, control about as
many variables as we possibly could. The windows in the room were blocked off so all animals
had artificial light. The visitors were not allowed to run in and out looking at the dogs, so we
controlled the amount of activity in the room. The chambers were operated under a half-inch
of negative pressure so that there was no leakage from the chamber into the room, and the
69
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room in which the chambers were located itself also had its own clean air supply, its own GBR
filters, its own air conditioning humidity capability, so when the doors were opened the
animals weren't exposed to a lot of pollutants which might have been coming in from the city
atmosphere itself. It was also necessary in order to maintain the temperature in chambers
since these were all stainless steel glass chambers. The dogs were, I believe, all within 2 weeks
of the same age when the study began.
Albert: Were these dogs abnormally healthy?
Hueter: No, I wouldn't say so.
Albert: You didn't lose a single one from disease over 9 years?
Hueter: They weren't exposed to very much where they should be getting disease.
Thurlbeck: Three died from disease.
Orthoefer: These were from inguinal hernias. These dogs had a very high incidence of
inguinal hernias; the hernia had strangulated, and the animal died.
MacEwen: Dogs on long-term experiments tend to get healthier with time because they are
not exposed to outside environmental influences and they don't get infections because they
are not exposed to other dogs. They see only the others in their chamber so they become
highly stabilized. In my experience, in our laboratory at Wright-Patterson, we get much
narrower ranges of normal values within the experimental groups maintained in chambers for
long periods of time where temperature, humidity and feeding times are relatively constant.
Stara: This is the end of this session. Thank you, gentlemen, for a fruitful discussion.
70
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4. COLLAGEN AND PROLYL HYDROXYLASE LEVELS IN
LUNGS IN DOGS EXPOSED TO AUTOMOBILE EXHAUST
AND OTHER NOXIOUS GAS MIXTURES
US. Bhatnagar
Summary
The effect of prolonged exposure to a variety of noxious environments on lung connective
tissues was examined in beagle dogs. The animals were exposed to raw and photochemically
reacted automobile exhausts, oxides of nitrogen and sulfur, alone and in combination with
exhausts for over 5 years, following which they were placed in ambient air for 2Vz to 3 years.
No significant differences were seen in the concentrations of collagen in exposed and
unexposed animals. Significant differences were seen, however, in prolyl hydroxylase levels
between control lungs and lungs exposed to irradiated exhausts (I), a high concentration of
nitric oxide plus a low concentration of nitrogen dioxide (NOn+NOa), and irradiated
exhaust plus sulfur oxides (I + SOX). Exposure to low nitric oxide plus high nitrogen dioxide
(NOL+N02H), to oxides of sulfur (SOX), to oxides of sulfur plus nonirradiated exhaust
(R + SOx) and to nonirradiated exhaust alone (R) produced smaller differences in prolyl
hydroxylase levels.
Introduction
Exposure of lungs to a variety of noxious gaseous and particulate chemical agents results in
tissue damage and impairment of pulmonary function. These changes in lungs are character-
ized by connective tissue growth and distortion of lung architecture (1,2). Although only
morphological evidence has been available, it has been assumed that these changes involve
increased deposition of collagen, the major protein component of connective tissues. Despite
mounting concern over respiratory disorders directly related to automobile exhaust emis-
sions, few systematic investigations have been made of the biochemical effects of the toxic
agents contained in internal combustion engine exhausts and resulting from the interaction
of these exhausts with the environment. The EPA-UC Davis study on the effect on pulmonary
function of nonirradiated and irradiated automobile exhausts and mixtures of oxides of sulfur
or nitrogen has been a major effort in elucidating this question. The major components of
this study were pulmonary function analyses and morphological studies. Analysis of data from
these investigations should yield invaluable information concerning the health effects of
automobile exhausts. The studies, however, originally did not include biochemical examina-
tion of pulmonary connective tissues. Biochemical studies were initiated during the sacrifice
phase of the EPA-UC Davis project.
The objective of this study was to analyze the collagen content and the ability to synthesize
collagen in the lungs of dogs exposed to a variety of noxious gaseous environments for over 5
years and subsequently maintained in ambient air for over 2 years.
Materials and Methods
Our studies were carried out using frozen samples of parenchymatous tissue provided by Dr.
J. Orthoefer. Because of difficulties involved in obtaining and transporting viable tissues
immediately after sacrifice, it was not possible to assay cellular protein and collagen synthesis.
71
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It was only possible to measure the colkgen content as a fraction of the total protein content
of the tissue. Changes in the collagen content of impaired lungs should be reflected in altered
ratios of collagen to total protein content
Since it was not possible to assay directly for collagen synthesis, we measured the levels of
prolyl hydroxylase, a key enzyme involved in the synthesis of collagen. Prolyl hydroxylase
levels generally reflect the potential of a tissue to synthesize collagen and are elevated under
conditions of enhanced collagen synthesis.
Details of the exposure of the beagle dogs to the various environments have been discussed in
Chapter 1. The tissues used in the present investigation were packed in dry ice and shipped
from the University of California at Davis to our laboratory at the University of California,
San Francisco. The tissues were allowed to thaw at cold room temperatures and were rinsed
in chilled distilled water to remove extraneous protein and other adhering materials. Portions
of the tissues were homogenized in 0.1 M TRIS HC1, 0.1 M KC1, at pH 7.4, and the
homogenates were centrifuged at 15,000 x G to obtain a supernatant which was used as the
crude prolyl hydroxylase preparation. Small portions of the tissues were hydrolyzed in 6 N
HC1 for the assay of protein and collagen content.
Prolyl hydroxylase activity was assayed using a substrate labeled with (3,4-3H2)-proline. The
substrate was prepared by incubating 10-day embryonic chick cartilage explants with the
labeled proline in the presence of 1.0 mM a,a' bipyridyl (3). Prolyl hydroxylase reaction was
carried out as described by Bhatnagar et aL (3) and Rapaka et aL (4). During the reaction,
tritium is released in stoichiometric proportions to proline hydroxylated. ^HHO is distilled
under vacuum, and radioactivity in the collected ^HHO is used as a measure of enzyme
activity. In these experiments the activity of prolyl hydroxylase is expressed as dpm ^HHO
released per mg of protein in the lung homogenate.
Protein and collagen contents were determined in hydrolyzates of the tissues. The tissues
were hydrolyzed in 6.0 N HC1, under N2, for 20 hours. The hydrolyzates were decolored with
activated charcoal and evaporated to dryness in a rotatory evaporator, under vacuum. The
residues were dissolved in 5 ml distilled water, and appropriate dilutions and aliquots were
used for the assays.
Protein content of the hydrolyzates was measured using the method of Rosen (5) by assaying
for the total ninhydrin reactive materials, using leucine standards. These data are expressed
in terms of leucine equivalents. Collagen was assayed on the basis of hydroxyproline content
using Woessner's method (6). Hydroxyproline is present only in collagen. Therefore a specific
measurement for hydroxyproline gives a measure of collagen content Since the objective in
these studies was to determine whether there was any change in the tissue content of collagen,
the ratio of collagen to total protein was considered. This is expressed in terms of the ratio of
hydroxyproline to total ninhydrin reactive material (in leucine equivalents).
Results
Tissue concentration of hydroxyproline was used as an index of collagen concentration, and
total ninhydrin reactive material was used as an index of total protein content Thus, the
experiment was designed to detect possible changes in collagen-proline ratios. No significant
72
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differences were found in the quotient hydroxyproline/total ninhydrin reactive material
(Tables 1 and 2); nor did the preliminary histologic and other morphological studies on these
tissues indicate major differences in the collagen content of exposed tissues.
As described, the relative levels of prolyl hydroxylase were determined in the tissues (Tables 3
and 4). Although no significant differences were observed in the collagen protein ratios
between tissues derived from dogs receiving different treatments, significant differences were
seen in the levels of prolyl hydroxylase. Lungs from animals exposed to irradiated exhaust
had the highest levels of prolyl hydroxylase. The nitrogen oxide environments and irradiated
exhaust in combination with sulfur dioxides also resulted in an increase in prolyl hydroxylase
activity. Sulfur oxides caused only minimal increases in the enzyme level and raw exhaust
alone and in combination with sulfur oxides did not significantly alter the enzyme levels.
Discussion
Our data indicate that there were no significant differences in the tissue concentrations of
collagen between the control and exposed animals. Crystal and colleagues (7,8) also failed to
detect significantly altered collagen concentrations in the lungs of patients with severe
symptoms of fibrosis. A possible explanation for this has been provided by recent studies on
the effect of short- and long-term exposure of rat lungs to ozone (9,10) and to nitrogen oxides
(11). In these studies, the rate of collagen synthesis in the lungs of animals exposed to the
toxic environment was significantly increased during the initial exposure. The initial spurt in
collagen synthesis was followed by increased synthesis of all proteins, until no difference could
be detected between the rates of collagen synthesis and of overall protein synthesis, indicating
that all tissue protein components were deposited at the same rates. After prolonged exposure
Table 1. Analysis of Variance for Hydroxyproline/Ninhydrin (X
Source of variation
Treatment
Error
Total
df
7
39
46
MS
3972
3151
F
1.261
P
0.295
"Hydroxyproline/Ninhydrin = collagen (specific activity)
"Orthoefer et al. (27)
Key: df = degrees of freedom
MS = mean square
F = "f" test
P = significance
Table 2. Mean Hydroxyproline/Ninhydrin (X 1Q-4) by Treatment
and the Results of Duncan's Multiple Range Test8
NOL +
N02H
NOH +
N02L
CA
R + SOx
l + SOx
SO,
425 388
381
370
363
344
344
341
aOrthoefer et al. (27)
73
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Table 3. Analysis of Variance for Prolyl Hydroxylase***
Source of variation
Treatment
Error
Total
df
7
39
46
MS
732441 1
543347
F
13.480
P
0.000
aOrthoefer et al. (27)
bSee Table 1 for key.
Table 4. Mean Prolyl Hydroxylase (dpm/mg) by Treatment and the
Result of Duncan's Multiple Range Test (a = 0.05)*
NOi +
I NO2L l + SOx NOzH SOx R "R + SOx CA
5351 3339 2970 2958 2030 1931 1680 1622
aOrthoefer et al. (27)
to ozone, there were no detectable differences in collagen concentrations between control and
exposed animals (10). These studies also revealed a marked capacity of the lung to repair itself
after the fibrogenic stimulus had been removed (9).
Although biochemical data are not available for the initial phases of the dog study, it is
possible that dog hung connective tissue underwent a course of biochemical changes similar to
those observed in the rat studies. Thus it is conceivable that during the initial exposure
period, the chronically exposed beagle kings underwent a concomitant increase in collagen
and non-collagenous proteins leading to increased tissue mass without chemically discernible
alterations in collagen concentrations. During the chronic exposure and subsequent mainte-
nance in "clean" air, any initial alterations in the connective tissue would have been
obliterated, as seen in the rat studies (9-11). This explanation does not preclude any
pathological alterations in pulmonary mechanics and other physiological parameters since
these properties are dependent on the macro- and micro-architecture of the alveolar wall,
which would undergo drastic changes as a result of both increased collagen and non-
collagenous protein deposition. Furthermore, any repair processes involving local deposition
of scar tissue and increased turnover of pre-existing connective tissue components would also
lead to altered mechanics without quantitative alterations in the tissue protein and collagen
contents.
The limited nature of these studies did not permit evaluation of qualitative differences in the
collagen contents of control and exposed animals. Collagen comprises a family of genetically
related proteins. Several different polymorphs of collagen have been described. Many of these
collagen species are tissue-specific. Type I collagen, the most abundant collagen species in the
body, is present in most tissues and organs, and is also a major component of pulmonary
connective tissue, being present in the large bronchii and blood vessels. It is synthesized by
the parenchyma (12,13). Lung mesenchymal cells in culture synthesize type I collagen (14X as
do parenchyma! cells of apparent epithelial origin (7, 15). Many cells of non-mesenchymal
origin also synthesize collagen (16,17), hence it may be presumed that some collagen may be
synthesized in the lungs by non-mesenchymal cells. Type II collagen, a component of
74
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cartilage, is present in trachea and bronchii as well as in other tissues (12). It may be
synthesized by chondrocytes in trachea! and bronchial cartilage. Type III collagen is present
in the lungs, but its structural location is not known. It is synthesized by hing mesenchymal
cells in culture (14). Type I and II collagens are also synthesized by lung epithelial cells in
culture (7,15). Type IV collagen, the principal collagen component of basement membrane, is
associated with alveolar septa. It may be synthesized by lung epithelial and endothelial cells.
Pulmonary parenchyma is a complex tissue with considerable variation from site to site.
Different parts of the parenchyma may contain different types as well as different amounts of
collagen. Variability may also be manifested in the collagen-associated structural proteins.
Measurements of collagen in the parenchyma tend to average out these variations, and thus
may mask experimentally induced effects. Changes in collagen in a small but crucially
important component, or destruction of collagen in one component with a commensurate
increase in another, may elude detection by chemical measurements, since overall collagen
content may be unaltered. Such changes may in some cases be identified by morphological
examinations. For example, the destruction of basement membrane collagen, which does not
have a clearly defined fibrous structure, and its subsequent replacement with scar tissue
collagen, which is organized as loose fibers, may give the appearance of increased collagen
deposition even though the net concentration of collagen may have remained unaltered.
Although it was not possible to measure directly the ability of lung explants to synthesize
collagen in vitro, it was possible to assay the tissues for their proryl hydroxylase content Proryl
hydroxylase is a critical enzyme in the pathway of collagen synthesis, hence its presence in a
tissue usually denotes a potential for collagen synthesis. In lungs (9, 10, 19) and other tissues
(20-22) engaged in enhanced collagen synthesis, the levels of prolyl hydroxylase are elevated.
Our study on prolyl hydroxylase levels revealed a marked difference between control tissues
and tissues that were exposed to irradiated exhausts (I), NOH + N02L, I + SOX, and
NOL+N02H- Exposure to SOX and to raw exhaust (R) also caused increased enzyme levels
although the difference was not as large. Surprisingly, exposure to R + SOX did not elicit any
change. In the absence of data on the rates of collagen and protein synthesis in the explants,
it is not possible to explain the significance of these findings. It may be speculated that the
tissues with the higher levels of prolyl hydroxylase were somehow more actively turning over
collagen and were in a dynamic state of repair and regeneration.
In order to obtain a clear understanding of the reasons for the differences in prolyl
hydroxylase content, it would be necessary to obtain correlations between the proh/l hydroxy-
lase levels and various mechanical and physiological parameters. Altered mechanical charac-
teristics accruing from the altered structure of the alveolar septa may result in a continuing
injury situation and a continual reparative response, leading to an elevated collagen turnover
rate. Higher collagen turnover rates may reflect a situation where both synthesis and
breakdown proceed at equal but elevated rates. This would explain increased prolyl hydroxy-
lase levels without any apparent changes in the net composition of the tissue.
An interesting aspect of these observations is that the highest proryl hydroxylase levels were
seen in tissues most likely to be exposed to ozone since both irradiated exhaust and oxides of
nitrogen can lead to the formation of ozone. Ozone and other oxidant gases generate
superoxide radicals in biological systems (23). Our studies have indicated that superoxide
radicals are an important reactive species in the prolyl hydroxylase reaction (24). Our recent
75
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studies suggest that the superoxide radicals induce collagen synthesis in a variety of cells
including fibroblasts of pulmonary origin in cultures (25, 26). These studies showed that
superoxide also induces prolyl hydroxylase in these cells. The implication of these observa-
tions is that the induction of pulmonary fibrogenesis may involve the generation of superox-
ide radicals. Generation of superoxide by oxidizing gases may thus be a part of their
fibrogenic effect on lungs.
Our studies raise several important questions:
1. What is the role of turnover rates of connective tissue macromolecules in pulmonary
fibrosis?
2. Are the differences in the mechanical and physiological characteristics of control and
exposed lungs attributable to morphological alterations involving all components rather
than connective tissue alone?
3. What type of biochemical changes occurred immediately after the initial exposure?
4. Since the experiment lasted several years, could the effects of aging be separated from the
pollution effects?
5. What kinds of changes occurred in the macromolecular content of the tissues? Alteration
in the types of collagen, elastin, etc., would lead to altered mechanical properties.
In concluding this report, it is suggested that serious consideration be given to a thorough
examination of connective tissue turnover and the chemical and physicochemical nature of its
components throughout the course of this type of a study. Since the mechanical and
physiological functions of the lungs are entirely dependent on the connective tissue compo-
nent of lungs, this point cannot be overemphasized.
References
1. Liebow, A.A. 1975. Definition and classification of interstitial pneumonias in human
pathology. Prog. Resp. Res. 8: 1.
2. Spencer, H. 1975. Pathogenesis of interstitial fibrosis of the lung. Prog. Resp. Res. 8: 33.
3. Bhatnagar, R.S., R.S. Rapaka, T.Z. Liu, and S.M. Wolfe. 1972. Hydralazine-induced
disturbances in collagen biosynthesis. Biochim. Biophys. Acta 271: 125.
4. Rapaka, R.S., K.R. Sorenson, S.D. Lee, and R.S. Bhatnagar. 1976. Inhibition of hydroxy-
proline synthesis by palladium ions. Biochim. Biophys. Acta 429: 63.
5. Rosen, H. 1957. A modified ninhydrin chlorometric analysis for amino acids. Arch.
Biochem. Biophys. 67: 10.
6. Woessner, J.E, Jr. 1961. The determination of hydroxyproline in tissue and protein
samples containing small proportions of this amino acid. Arch. Biochem. Biophys. 43:
440.
7. Hance, AJ., A.L. Horowitz, MJ. Cowan, NA. Elson, J.E Collins, R.S. Bienkowsi, K.H.
Bradley, S.D. McConnel-Breul, W.M. Wagner, and R.G. Crystal. 1976. Biochemical
approaches to the investigation of fibrotic lung disease. Chest SuppL 69: 257.
8. Fulmer, J.D., and R.G. Crystal. 1976. E 419 in R.G. Crystal, ed. The biochemical basis of
pulmonary function. Marcel-Dekker, New York.
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9. Hussain, M.Z., C.E. Cross, M.G. Mustafa, and R.S. Bhatnagar. 1976. Ozone-induced
pulmonary fibrosis: synthesis of collagen in lungs of rats exposed to ozone. Life Sciences
18: 897.
10. Cross, C.E., K. Reddy, M.Z. Hussain, L.W. Schwartz, M.G. Mustafa, and D.L. Dungworth.
1972. Lung adaptation to low levels of ozone on long-term exposure. Am. Rev. Resp. Dis.
113: 102.
11. Hacker, A. 1975. Effects of nitrogen dioxide on collagen metabolism. Ph.D. thesis,
U.C.LA
12. Bradley, K.H., S.D. McConnell-Breul, and R.G. Crystal 1974. Lung collagen composition
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Asilomar Conference Discussion
Bhatnagar: The data that we have provided were based on all the tissues we received and I
had no idea which animals were which. Mr. Yang did the statistical analysis.
Orthoefen I think it has to do with the duplicate samples.
Bhatnagar: That's right.
Albert: Could you give us a rough idea of how many animals there are, for instance, in the
irradiated group?
Bhatnagar: I'm very sorry, I don't have those figures. I asked for summarized data, and that's
what I have. For the total collagen content as well as the prolyl hydroxylase, our analysis,
based on simple statistics, matched the analysis widi a limited number of samples. We did
analyses of all the samples we had, and eliminated some of the samples because they were too
far out of the range.
Kleinerman: Did you know the identity of each sample you analyzed?
Bhatnagar: No, we had no idea. They were done totally blind. In fact, for the longest time I
had no idea of what these results meant although we had all the data. It took an awful lot of
time to generate data on 86 animals and doing each one in triplicate or quadriplicate and so
forth. We did these analyses on two different occasions. The result of all that was that, even
after doing all this work, we had no idea what we had done, because we did not have the code.
I was unable to get the code.
Stara: The blind on analytical work was done on purpose after discussion to see if there would
be some valuable data coining. As far as the 47 dogs, I will send the information within weeks
to each of you.
Lewis: Dr. Hueter and I, and others in the laboratory, were interested in this type of
biochemical reaction 10 years ago or more, and at the time we analyzed tissues from animals
in the first auto exhaust studies and got the same kind of results you did and so did many
other colleagues in the field. That's 10 years ago. The hypothesis was at that time that it's not
an increase in the content of collagen or elastin, but it's a physical/chemical rearrangement or
cross-Unking to hydrogen bonding, amino end groupings of h/sine, other amino acids, etc. I'm
quite surprised that you looked at it in this fashion rather than from a physical/chemical
fashion.
Bhatnagar. It would be very nice to look at it that way, but from the amount of tissue that we
got, there wasn't very much we could do.
McLees: I don't know how you look at it in a physical/chemical fashion. It's very tough. Most
of mat work has been done, say with something like tendon, of that nature, and that's a very
indirect kind of thing. It is possible to look at cross-links reasonably directly with this tissue
and it could be done probably now with the tissue using chlorohydride reduction, that is,
introducing tritiated tritium into the reducible cross-links and subjecting that to alkaline
78
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hydrolysis and passing it over a specific ion exchange cross-link analyzer. The trouble with
that is that these animals, I take it, are fairly old, as far as animals go, and most of the
reducible cross-links are physiologically reduced as part of aging, so I'm not sure what you
would get from this even with that, but that's do-able and you could at least probably tell if
the cross-links are more and more abnormal.
Lewis: Is the hypothesis of increased cross-linking with aging of collagen and elastin the
current one accepted, or are there other more tenable hypotheses in acceptance today?
McLees: I don't know how to answer that; it depends on who's making the hypothesis. I would
say, I guess my thought is that there are not enough data at the molecular level to allow you
to choose between one hypothesis and another.
Bhatnagar: One of the problems with lung collagen is that it is comparatively unstable. It does
turn over quite rapidly, much more rapidly than some other collagens in the body. So over
this period of time, we had some new collagens synthesized. If there were any difference in
cross-Unking and so forth, introduced during the exposure part of the study, you probably
would not expect to see them, at the end, anyway.
Lewis: We had an extremely difficult time demonstrating increased collagen deposition in
animals exposed to material like coal dust, much less trying to demonstrate it with this type of
exposure. I'm talking about 10 milligrams per cubic meter loads, like that Dr. Lee, have you
any experience with collagen analysis in your work?
Dr. Lee: No.
Question: Are there any more questions?
Tyler I would like to ask one, please. Ninhydrin reaction. It's been a long time since I thought
about it, but plasma proteins react also, don't they? So that if you corrected these essentially
for the hematocrit for the amount of blood left in the tissues, which would relate to the
protein left in the tissues in the bloodstream, perhaps then your collagen index now is
structural collagen against total protein, and the total protein is going to vary with how much
blood was left in the lung at the time the animal was killed. So if you correct for the amount of
blood left in the lung at that time it was killed by hemoglobin concentrations, much the way
Staub does, then you would perhaps make more sense out of the collagen index because it
would be a true index instead of an index which is influenced by the amount of blood present
Bhatnagar: We'll talk about that, but I would think it is corroborated by the two or three
other studies where they did not find any changes in the collagen concentrations, as in studies
from Dr. Crystal's lab at the Heart and Lung Institute and from the studies that Dr. Hussain
did, just looking at the rates of collagen synthesis. They also seem to suggest that there are
possibly mechanisms which will bring the concentrations of collagens down to normal I'm
talking about the concentrations now. There may be qualitative changes in collagens which
could not be seen in other studies.
Albert: Do you know any conditions of the lung where collagen turnover has been
accelerated?
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Bhatnagar: Perhaps Dr. McLees can answer that.
McLees: Not that I know of, but after you remove the lung, it seems to lay down more
collagen at a very rapid rate.
McLees: Well, that's true, but everything else is turned on with that. The whole lung grows
after you do an embinectomy and so it's kind of everything going, but even so, the turnover,
when we look at, say, people with idiopathic fibrotic lung disease, using labeled collagen and
lung exponents, their rate of synthesis of collagen is normal compared to controls in biopsy
specimens. These are people with a classic physiologic picture of lung fibrosis, classic
pathologic picture, and with biochemical data to go along with that. We don't find that it's
increased. I raise this question because the fraction of collagen that would turn over rapidly
would be the soluble fraction probably, and our experience has been that the stuff is so
insoluble in the adult lung that we get, you know, 1 % or half a percent that is extractable with
salt and acetic acid under those conditions. I wonder about that I dare say I'm uncertain....
Except in these situations where everything goes up, collagen synthesis will go up along with
total protein synthesis, but that's a little bit different situation.
Albert: That's not turnover, is it? That's just an increased deposition.
McLees: Right, but in the synthesis, I'm not aware of any circumstance where the turnover
number you'd look at that we've done would show that. I wanted to come to this point
because I think one of the important things in this study is where those biopsies of the lungs
came from.
Orthoefer: They all are left diaphragmatic lobes.
McLees: One of the problems is that ratio of milligrams of collagen per milligram of dry
weight will depend on how much airway tissue there is in the biopsy specimen. If you take
that from the periphery of the lung, you are going to get a different ratio than if that biopsy
comes up closer in to the large airways.
Orthoefer: I basically prepared all these and I clipped the major airways out, and on some of
them, Dr. Bhatnagar was interested in seeing what amount of collagen was being clipped out
with these, so I packaged them up separately and mailed them down to him. There should
probably be some data on this. Actually, I did them all as close to the same way as possiBle. It
is possible that a small airway was left in one and not in another, but I tried to get the major
airways out of the lung.
McLees: But in answer to your question, I think the problem is tied up in this. It's a
denominator problem. If you take a situation much more impressive than this in terms of
pathology, where our pathologists will tell us that the lung is loaded with fibrosis, the
biochemical analysis on that done in many different fashions always shows that the collagen
content is normal biochemically. I think that is a problem of biochemical analysis and not
knowing what to choose as a denominator. The trouble is collagen makes up so much of the
lung in itself that it could be very, very difficult to pick up a change in collagen content when
you're selecting a denominator such as per milligram dry weight or ninhydrin or what have
you.
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Orthoefer: Apparently all proteins are turned on — the total ninhydrin increases as does the
collagen.
McLees: Yes, but the same thing is true of dry weight or whatever you choose. That's the
problem. I guess the other question I have refers to hydroxyproline, though. That is maybe
another question because in many illnesses the serum level of that protein is increased
substantially. For example, in hepatitis, probably you have all-time record highs of the prolyl
hydroxylase in the serum and it's been analyzed in a series of 80 different diseases and it's a
very nonspecific protein elevated everywhere. That could be a problem here, if you couldn't
get rid of the serum from the lung tissue. Were the lungs perfused when they were taken out?
Orthoefer: No.
Bhatnagar: The only processing of the lungs that was done in terms of cleaning them was that
in my lab they were cleaned as thoroughly as they could be cleaned up.
Kleinerman: What does that mean?
Bhatnagar: What that means is that they were washed in many, many changes of buffer and
homogenized in buffer for the purpose of doing the hydroxylase. Of course, we had to take as
much of the tissue as possible, for the purpose of determining the hydroxyproline. What we
tried to do was to take most of the insoluble material, that was not soluble in buffers that
would not solubilize collagen.
Question: But you had to pull hydroxylase.
Bhatnagar: We used that wash, too, because there was obvious debris attached to these tissues
and they were not clean. We tried to wash out as much of the material as possible. I don't
know if when one washes these things by rinsing them thoroughly, one gets into the alveoli
and gets rid of everything out of them.
Orthoefer: What do you mean by debris?
Bhatnagar: When you cut these lungs up, there was a lot of stuff, just minute chunks of tissue
that were adhering to the pieces of lung; also, there were precipitated proteins, blood, and
stuff that came off very easily when we washed these tissues. We tried to get rid of as much of
the adhering material as we could.
McLees: I would like to take the Chairman's prerogative of asking two more questions, if I
may. One of them is that I am concerned a bit about the equation of increased prolyl
hydroxylase activity with increased collagen turnover, you've shown an increase in enzyme
activity.
Bhatnagar: That was not an assumption, that was a speculation. I tried to explain why one
might see more of it.
McLees: But it could be a denominator problem there also. Or it could be a specific activation
of the enzyme that would have nothing to do with its catalysis.
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Bhatnagar: Why would that occur in particular groups?
McLees: I have no idea.
Bhatnagar: I think these differences were real.
McLees: I am not arguing that the differences were real. I'm arguing about the interpretation
of the difference. In other words, the fact that the enzyme activity is increased, to me means
just that. I guess what I have more trouble with is the extrapolation from that fact to the fact
that the turnover or the synthesis of the collagen is increased.
Bhatnagar: But any alternative speculations on that are quite welcome.
McLees: I offer two others. One is that your expression per unit something, ppm per
milligram or something, that per is changing. The other is that one could just as easily
speculate that this is an enzyme that has many cofactors, and in fact it has a co-substrate, that
alterations in one of those factors ...
Bhatnagar: Yes, the way this work was done, the only thing we took was a dialyzed extract;
you don't really have cofactor problems here.
McLees: Well, that you know about.
Bhatnagar: Any cofactors that could be associated with the enzyme that we don't know
about?
McLees: Because you're using very impure preps.
Bhatnagar: We are using a very impure prep, but the cofactors of the enzyme are small
molecular weight compounds and they are dialyzed out.
McLees: I understand that; what I'm really getting at is that what you add back, you're
assuming that you dialyze them away from the enzyme. What I'm just really saying is a more
general fact: that we know of situations where enzymes are activated. It could well be that this
enzyme is activated by one of these agents and you're just seeing an increased activation.
Bhatnagar: Well, that agent would have to come along with the enzyme from the lungs. If it is
there, then it is still causing an activation of the enzyme and increased activity.
McLees: The reason I would like to make this point is that there are cases in lung associated
with increased prolyl hydroxylase activity that as far as we can tell are not associated with
increased turnover of collagens, and that's true in some pathologic states.
Bhatnagar: It would have to be a nondialyzable macromolecular factor that travels through all
the steps.
McLees: I have no idea of mechanism, I just know that there are investigators who have
reported -increased prolyl hydroxylase activity in pathologic pulmonary states without
increased turnover of collagens, or increased amounts of collagens.
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Albert: What kind of state?
McLees: Some of the fibrotic lung disorders, and I'm sorry, I can't give you off the top of my
head the specific pathologic state, but in lung fibrosis, my guess is that it's one of the
idiopathic pulmonary fibroses.
Orthoefer: Aren't you talking about the same thing?
McLees: No, I don't think so. That's the other point here. We sort of assumed here that we
are talking about pulmonary fibrosis as the pathology in this disorder. Is that true or is it not?
If this is emphysema, then that is probably not a fair comparison at all.
Orthoefer: We're speculating on remodeling.
Comment: What is the pathology, do you see pulmonary fibrosis pathologically?
Orthoefer: No, it's not a major thing.
Dungworth: It's not a major feature of the lesion, no. It's an amount of increase in fibrous
tissue around small airways that we haven't really got a good clue on yet because we haven't
got any quantitative data. It's not the major feature of what we see.
McLees: Is the physiology compatible with fibrosis?
Comment: No.
McLees: Then I have to say I'm not sure that you are dealing with a fibrotic lung process.
Bhatnagar: I guess that's why there is no difference in collagen.
McLees: Well, in the human state, we are dealing with it and we still don't see that, but we
have to take something as a base for lung fibrosis and I think it is important to establish. The
base we use now, at least in humans, is physiology and pathology, and biochemically, as I say,
we're not able to ...
Orthoefer: I don't think we can here either.
Bhatnagar: May I ask a very naive question? This is something that's completely foreign to
me. I don't really understand lung structure and morphology. What exactly does constitute
fibrosis? What does one see in a lung when one says it's a fibrotic lung? I see that term in a
lot of places and I've tried to understand the pictures that come with that, but I don't
understand the pictures.
McLees: I'm sure there are people like Dr. Kleinerman who could answer this better than I,
but...
Kleinerman: Maybe we ought to leave this for the time when we are discussing pathology, but
I think, in general, one would see fibroblasts and the deposition of collagen in a number of
83
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areas. The deposition of collagen as defined morphologically by stains and morphologic
characteristics.
Bhatnagar: Using morphologic techniques one does not really see basement collagen in the
same way as one sees fibrous collagen. Early events in lung injury remove the basement
membrane. What I would be able to measure biochemically as collagen, you will not be able
to see. If this were replaced with a few fibers of type I collagen, for instance, you would see
more collagen morphologically, even though there may be less collagen or no difference in
the collagen content, as we saw here.
Kleinerman: We would be able to tell because basement membrane looks different than other
types of collagen. It is possible that there could be a subtle change from one type to the other
that wouldn't be distinguishable morphologically, but really, it's quite striking, the difference
in basement membrane structure from the usual collagea
Bhatnagan This is what I wanted to know. When we see fibres is we don't necessarily relate
that to increased thickness of the basement membrane. Instead one sees the periodically
banded collagen fibers.
Kleinerman: You can see it in that area and in other areas as well.
Bhatnagar One other possibility that I did not mention here is that perhaps it is possible that
in lesions, if one would call these things lesions, a part of the alveolar wall disappears, for
instance. When we have increased collagen in one area and a total loss in another, then a
technique like mine, where I can just take a fragment and hydrotyze it, may not be able to
detect quantitative and qualitative differences.
Comment That's right
Bhatnagan So there can be a number of different reasons why we didn't see these differences.
Orthoefen Isn't it also true that basement membrane collagen is more compact, but you just
looked at total collagen? Therefore, if you take a basement membrane which is more
compact, a smaller unit of this would probably equal a large unit of collagen.
Bhatnagar. It certainly doesn't show up like nice fibrous collagen and other types which may
form loose aggregates of fibers.
McLees: The problem here is that basement membrane collagen has never been biochemi-
cally found in lung. No one has isolated the basement membrane collagen from lung to my
knowledge.
Bhatnagan It seems to be there though.
McLees: We can't find it, and I mean we have looked.
Bhatnagar: What criteria have you used?
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McLees: We've used specific isolation techniques for type IV collagen; we've used
3-hydroxyproline analysis and in the scheme that we've used, we've isolated types I, II, and
III collagen from lung in an inclusive way where we keep material balance, and we're not able
to distinguish it
Bhatnagar: We didn't see any 3-hydroxyproline either and I wondered if the basement
membrane collagen of the lungs is somewhat different.
McLees: Well, that may be, but if it is different it certainly does not meet the criteria where we
normally classify it as a type IV collagen.
Bhatnagar: So little is known about what goes on in the lung anyway.
McLees: Well, that's true, but the point I wish to make is that in terms of basement
membrane collagen as normally defined, (a highly glycosalated 3-hydroxyproline-containing
moleculeX there's no evidence supporting its presence in lung. Now, there may be a different
type of basement membrane collagen that's not been isolated.
Kleinerman: Even when you look in the large airways, is that true?
McLees: We have looked everywhere. We have looked everywhere for that molecule and we
believe there is a basement membrane collagen, we think that we may have it, I don't know.
Lee: This might muddy the water a little more, but Dr. Bhatnagar mentioned about the work
of Hussain et aLl on the altered collagen metabolism following chronic exposure of rats to
ozone, and recently Tierney's group down at UCLA ran similar experiments and obtained
very similar data. Furthermore, these workers supplemented the experimental animals with
vitamin E at 500, 50 and 10 ppm, and observed protective effects.
Bhatnagar: Were they looking at ozone or nitrogen oxide?
Lee: Ozone at 0.8 and 0.5 ppm for 4 days.
^Hussain, M.Z. et al. 1976. Ozone-induced pulmonary fibrosis: Synthesis of collagen in lungs
in rats exposed to ozone. Life Sciences.
85
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5. THE EFFECTS OF AIR POLLUTANTS ON MEMBRANE LIPIDS
G. Rouser and R. Aloia
Abstract
The effects on membrane lipids of long-term exposure (68 months) of beagle dogs to raw and
irradiated auto exhaust and various mixtures of oxides of nitrogen and sulfur followed by
exposure to clean air (2l/2 to 3 years) were studied. No changes were found in brain or heart;
an indication of small changes in liver phospholipids was apparent, and clear-cut changes
were observed in the lung. Phosphatidyl ethanolamine from lung was reduced from 19.0% of
the total phospholipid to 17.2% and some of the minor acidic phospholipids were elevated.
No change was found in the levels of phosphatidyl choline or the other major phospholipid
classes. Since the range of phosphatidyl ethanolamine in 15 lung samples was 17.7 to 20.3%,
it is clear that the mean value for the experimental groups is outside of the normal range of
variation. A mechanism for production of apparently permanent changes other than at the
genetic level is not apparent.
Introduction
The effects of pollutants on membrane lipids that are essential for membrane function are
important to define since membranes control the passage of substances into and out of cells,
and function in impulse transmission. In principle, effects can be evaluated by analysis of
whole organs or isolated membranes and organelles. In practice, analysis of whole organs is
the only way that a large series of animals can be examined with reasonable effort, and it has
the advantage that membrane lipids are not altered by isolation procedures. In this report,
the results of a study of the effects of pollutants on the phospholipids of brain, heart, liver and
lung are presented.
Materials and Methods
Beagle dogs were maintained and exposed to polluted air as described elsewhere (1). Organs
from the animals were removed immediately after sacrifice and stored at -20 °C. Lipids were
extracted with chloroform/methanol, and nonlipid contaminants were removed by Sephadex
column chromatography. Phospholipids were separated by two-dimensional thin-layer chro-
matography, and their molar amounts determined by phosphorous analysis as previously
described (2,3,4).
Results and Discussion
The data for heart lipids (Table 1) show that the controls and the experimental groups are
closely similar. The small range of variation of the values shows the high degree of
reproducibility of the analytical methods that is also shown by the standard deviations (in the
1 to 2% range for major components). Phospholipids of brain (data not shown) do not show
changes in the exposed animals and the range of variability is slightly less than that for heart
lipids.
87
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Table 1. Heart Llpids(Mean Values)*
Experimental groups
Lipidb
TL
TPL
PC
LPC
PE
LPE
SPH
PS
PI
DPG
LDPG
PA
Misc
Control
range
5.12-10.49
1.78- 4.44
38.9 -42.7
1.0 - 1.8
28.2 -29.7
0.8 - 2.0
3.7 - 5.2
2.3 - 3.6
3.6 - 4.3
7.6 -10.1
1.5 - 2.4
0.3 - 0.8
3.7 - 6.2
R
6.79
2.03
42.1
1.6
28.6
2.5
4.6
2.7
4.0
8.2
2.1
0.4
3.3
I
6.29
2.12
40.1
2.1
29.3
1.5
4.1
2.4
4.2
6.8
2.3
0.3
6.9
SOx
5.93
2.06
41.6
1.6
29.0
1.4
4.1
3.6
3.8
10.7
1.7
0.4
3.1
R + SOx
6.35
2.14
41.5
1.4
28.8
1.6
3.9
2.7
3.8
9.7
2.1
0.3
4.1
l + SOx I
6.91
1.66
40.1
1.7
28.9
1.4
4.4
2.5
4.3
9.5
1.8
0.9
4.6
NOL + N02H
4.86
2.19
40.3
1.8
29.6
1.3
4.6
2.3
3.9
10.4
1.8
0.4
3.5
NOH + NOa.
4.76
2.12
40.7
1.7
29.0
1.2
4.3
2.5
4.2
10.3
1.9
0.3
3.9
All
experimental0
5.98 ±0.86
2.04 ±0.1 8
41.0 ±0.8
1.7 ±0.2
29.0 ±0.3
1.6 ±0.4
4.3 ±0.3
2.7 ±0.4
4.0 ±0.2
9.4 ±1.4
2.0 ±0.2
0.4 ±0.2
4.2 ±1.3
All
control*1
7.52 ±1. 73s
2.25 ±0.78
41.1 ±1.4
1.4 ±0.3
29.1 ±0.6
1.2 ±0.3
4.3 ±0.5
2.8 ±0.3
4.0 ±0.2
8.9 ±0.8
1.8 ±0.3
0.6 ±0.2
5.0 ±0.8
Quantities expressed in all tables:
TL = total lipid = % fresh weight
TPL = total polar lipid = m moles/100 g fresh weight
Individual lipid classes = mole % values based on recoveries between 97 and 101%
"Abbreviations used:
TL = total lipid; TPL = total polar lipid; PC = phosphatidyl choline; LPC = lysophosphatidyl choline; PE = phosphatidyl ethanolamine;
LPE = lysophosphatidyl ethanolamine; SPH - sphingomyelin; PS = phosphatidyl serine; PI = phosphatidyl inositol;
DPG = diphosphatidyl glycerol; LDPG = lysodiphosphatidyl glycerol; PA = phosphatidic acid; LBPA = lysobisphosphatidic acid;
Misc = miscellaneous
cMean and standard deviation of all experimental groups.
dMean and standard deviation of all control groups (n = 10).
eSignif leant difference at 2.5% level between mean values of control and experimental groups (TL; Student's t-test).
-------
Liver lipids (Table 2) present a less clear-cut situation. The mean values for experimental
groups 1 and 2 (R and I) in particular suggest a small effect on phosphatidyl ethanolamine.
Each sample in these groups was analyzed separately. Although the results do not show any
obvious differences, some of the phosphatidyl ethanolamine values are below the lowest value
found for controls. Also, a number of minor components could be seen more prominently on
thin-layer chromatograms of the experimental groups. Additionally, the total lipid fraction
(phospholipids and neutrals) was increased significantly in the experimental groups (Table 2\
In contrast to the other organs, the phospholipids of lung showed definite differences between
controls and all experimental groups. The percentage of phosphatidyl ethanolamine is 90%
of the control mean value. The values for minor acidic phospholipids in general, and in
particular the levels of lysobisphosphatidic acid and phosphatidyl glycerol (significant at the
1 % level; Table 3), are increased. The changes are relatively specific since values for the other
major lipid classes were not changed. It should be noted that the mean value for phosphatidyl
ethanolamine of the experimental groups (17.2%) is outside the normal range of variation.
A mechanism for the production of permanent changes other than alteration at the genetic
level is not apparent It seems probable that the production of some particular molecular
species of phosphatidyl ethanolamine in lung is impaired. In this regard it is interesting to
note that exposure to ozone was found to decrease linoleic acid and increase arachidonic acid
in lung lipids (5). The changes are similar to those produced by vitamin E deficiency (6,7). A
logical next step is the determination of the fatty acid composition of each of the phospholip-
ids of lung. It is to be noted that analysis of pure membrane preparations and organelles
might disclose changes that are not apparent from whole organ data.
References
1. Stara, J., K. Busch, R.H. Hinners, and J.K. BurkarL 1979. Study overview, rationale,
experimental design, experimental facilities, and exposure atmospheres. Chapter 1 of
this book.
2. Rouser, G., G. Kritchevsky, and A. Yamamoto. 1967. Golumn chromatographic and
associated procedures for separation and determination of phosphorized and glycolipids.
Pages 99-162 in G.V. Marinetti and Marcel Dekker, eds., Lipid chromatographic
analysis, voL 1.
3. Rouser, G., S. Fleischer, and A. Yamamoto. 1970. Two-dimensional thin-layer chromato-
graph separation of polar lipids and determination of phospholipids by phosphorous
analysis of spots. Lipids 5: 442-444.
4. Rouser, G. 1973. Quantitative liquid column and thin-layer chromatography of lipids and
other water-soluble substances, elution selectivity principles, and a graphic method for
pattern analysis of chromatography data. J. Chromatographic Sci. 2: 60-76.
5. Menzel, D.B., J.N. Roehm, and S.D. Lee. 1972. Vitamin E: The biological and environ-
mental antioxidant J. Agr. Food Chem. 20(3): 481-486.
6. Witting, L. 1967. Effects of antioxidants deficiency on tissue lipid composition in rats.
FV. Peroxidation and interconversion of polyunsaturated acids in muscle phospholipids.
Lipids 2: 109-113.
7. Bernhard, K., S. Leisinger, and W. Pedersen. 1963. Vitamin E und Arachidonsaure-
Bildung in Der Leber. Helv. Chim. Acta 46: 1767-1772.
89
-------
Table 2. Liver Lipids (Mean Values)"
Experimental groups
Ljpid"
TL
TPL
PC
LPC
PE
LPE
SPH
PS
PI
DPG
LDPG
PA
Misc
Control
range
4.04- 7.22
2.74- 4.04
45.9 -50.4
1.5 - 2.5
24.9 -27.4
0.6 - 1.0
4.1 - 5.7
2.3 - 4.1
6.2 - 8.3
3.4 - 4.6
0.1 - 0.6
0.6 - 1.1
1.9 - 3.0
R
6.02
3.56
50.2
1.9
24.7
0.5
4.6
3.0
7.7
3.9
0.5
0.7
2.2
,
5.80
3.86
49.3
1.7
27.2
0.2
4.6
2.5
7.6
4.2
0.1
0.4
2.2
sox
5.54
3.59
49.5
1.8
25.6
0.4
4.7
3.8
7.1
3.9
0.6
0.7
2.0
R + SOx
6.86
4.02
48.2
1.8
25.2
0.8
4.8
3.8
7.8
4.0
0.7
0.9
2.0
l + SOx
6.70
3.68
50.9
1.6
24.4
0.4
4.5
3.7
7.8
3.2
0.4
0.7
2.4
NOL+NO2H
6.94
3.56
49.3
1.8
25.3
0.0
5.1
3.7
7.8
3.8
0.6
0.7
2.0
NOH + N02L
6.34
3.82
48.6
2.0
25.5
0.6
4.4
3.8
7.4
3.6
0.7
0.8
2.0
All
experimental
6.31 ± 0.55
3.73 ±0.18
49.4 ±0.9
1.8 ±0.1
25.4 ±0.9
0.5 ±0.2
4.7 ±0.2
3.5 ±0.5
7.6 ±0.3
3.8 ±0.3
0.5 ±0.2
0.7 ±0.2
2.1 ±0.2
All
controld
5.63 ±0.79"
3.56 ±0.41
48.4 ±1.5
2.0 ±0.3
26.0 ±1.0
0.7 ±0.1
4.6 ±0.4
3.6 ±0.5
7.4 ±0.5
3.8 ±0.4
0.4 ±0.1
0.8 ±0.2
2.3 ±0.4
aQuantities as for Table 1.
bAbbreviations as for Table 1.
cMean and standard deviation of experimental groups.
dMean and standard deviation of control groups.
eMean values of control and experimental groups are significantly different at the 5% level (TL; Student's t-test).
-------
Table 3. Lung Lipids (Mean Values)3
Experimental groups
Lipid»
TL
TPL
PC
LPC
PE
SPH
PS
PI
DPG
PA
LBPA
PG
MiSC
Control
range
1.57- 4.01
1.12- 2.60
33.6 -47.1
1.0 - 2.1
17.7 -20.3
13.9 -18.3
8.5 -10.6
1.8 - 3.3
0.8 - 1.3
0.7 - 2.3
1.4 - 5.2
2.1 - 3.9
1.4 - 3.9
R
2.74
2.08
44.5
1.2
16.9
14.8
8.2
3.3
0.9
0.7
3.8
3.1
2.6
I
2.78
2.10
41.4
1.4
17.5
16.1
9.1
2.7
1.0
1.5
3.7
3.7
2.1
SOX R + SOX
2.63
2.07
37.9
1.3
18.0
16.9
9.7
2.6
1.1
1.2
3.6
4.7
3.1
2.73
2.11
44.1
1.4
17.1
15.2
8.1
3.4
1.1
0.7
3.4
3.5
2.1
l + SOx NOL+NO2H
2.84
2.17
39.9
1.3
17.8
17.1
9.3
2.7
1.0
1.8
3.3
4.0
1.9
2.99
2.29
43.0
1.4
16.6
15.7
8.8
3.1
1.1
0.9
3.5
3.8
2.2
NOH + NO2L
2.97
2.29
43.0
1.3
16.7
15.7
8.7
3.3
1.0
1.2
3.6
3.5
2.1
All
experimental0
2.81
2.15
42.3
1.3
17.2
15.8
8.8
3.1
1.0
1.1
3.6
3.8
2.3
±0.13
±0.11
±2.3
±0.1
±0.5
±0.8
±0.6
±0.3
±0.1
±0.4
±0.2
±0.5
±0.4
All
controld
2. 78 ±0.50
2.05 ±0.41
42.0 ±3.1
1.3 ±0.2
19.0 ±0.8e
15.9 ±1.3
9.1 ±0.5
2.8 ±0.4
1.0 ±0.1
1.4 ±0.5
2.9 ±1.2
3.0 ±0.5'
2.0 ±0.6
aQuantities as for Table 1.
bAbbreviations as for Table 1.
°Mean and standard deviation of experimental groups.
dMean and standard deviation of control groups.
eSignificant difference at the 0.1 % level between mean values of control and experimental groups (PE; Student's t-test).
'Significant difference at the 1 % level between mean values of control and experimental groups (PG; Student's t-test).
-------
Asilomar Conference Discussion
Lee: As you noted already, in the initial phase of this study, not much biochemical work was
included, for two good reasons. First of all, even though our facilities might be the best in the
world, they still are limited, and we could not include sufficient numbers of animals to allow
serial sacrifice to do biochemical work. Secondly, perhaps more importantly, a lot of
information that's available now, at low level of exposure to various pollutants, mainly ozone
and NOs, down to 0.1 ppm levels, was not available back in 1964 and 1965. I think, even
though some of the techniques might have been available, there was not enough communica-
tion between basic researchers and applied researchers and the techniques were simply not
being used. It would have been ideal if the funds and facilities were available to accommodate
serial sacrifices to examine various changes throughout the duration of exposure. In the
recent days the biochemists are talking to morphologists, and morphologists are talking to
biochemists. I literally mean that, and a lot of the credit goes to the Davis School, SRI, and
UCLA groups. They found biochemical changes at very low concentrations, mainly I guess
the SRI group saw morphological changes at very low concentrations, they now can relate the
two different changes. In the past, when the biochemists gave you some kind of dose-
response, people in Washington, or even in Cincinnati, came back and said, "What does that
mean?". Still we were told to do the dose-response studies. I think this kind of difficulty is
gradually disappearing. At least, I hope so. Getting back to Dr. Bhatnagar's findings in terms
of collagen synthesis and Mustafa's finding and Hussain's work with ozone exposure, there is
initial depression. I'm talking about some of the subcellular marker enzymes that we
investigated. They showed initial depression and then increased coincidental with cellular
proliferation at about day 3 or 4. I think it's very important to include this kind of parameter
for any kind of future studies.
Since the time is getting quite late, I'm not going to go into any kind of specific recommenda-
tion. Hopefully, I will have a chance to insert my recommendation during the last day's
session, but I hope that all the presentations during the next two days will point to additional
studies of low-level, long-term effects. I wish that a group of experts like the present company
would plan that study. Hopefully, we could get stable funding for the entire period, so that the
researchers wouldn't have to worry about funding every year, thus enabling them to
concentrate on high quality research. I think I'll stop right there.
Stara: I wonder if you would mention the small experiments you performed at the beginning
of the study; I mean the sulfur content of the blood and the vitamin C.
Lee: During the early part of exposure of the dogs, namely, three sampling periods,
December 1965 and April 1966 and September 1967, we looked at total sulfur content of the
blood and vitamin C, in the hope that we could use them as indices of change in sulfur
metabolism or oxidative metabolism, but we did not see any difference among the groups and
we discontinued the effort One interesting aspect, a very small sidelight, was that we shipped
six blood samples from these dogs, three controls and three N02-exposed blood samples, to
John Rowlands at Southwest Research Institute. We sent the samples completely blind and he
segregated them into two different groups based on his EPR analyses. The control group did
not give any kind of triplet signal and the NC"2-exposed groups gave clear triplet signals. With
only that much information we did not get funding to continue that type of work and had to
stop.
92
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Kleinerman: Would you explain the significance of these preliminary findings? What do you
think they mean?
Lee: You mean this EPR signal? I think it means that N02 exposure does cause the free
radical formation and EPR techniques were able to pick it up.
Rouser: Dr. Lee showed me some of the lipid data that he selected. What was that on, rats?
Lee: Yes, the kind of PE change Dr. Rouser mentioned earlier in conjunction with that
particular experiment. Actually, about the same time we exposed some rats to N02 at 1 ppm
for 2 weeks and then we washed the lungs out and looked at the lipid profile of the
endotracheal lavage, as a quick means of looking at the change in surfactant There was a
definite drop in PE and increase in phosphotidyl choline. The latter change was not seen by
Dr. Rouser. The marked drop in PE in both experiments agreed.
Rouser: Yours was actually larger by quite a bit than we
Lee: Yes.
McLees: Another interesting ramification of that is that the spin label which was used is the
nitroxide radical which is NO. It may be that the EPR signal is coming from degradation of
the N02 that's attached to the lipid. Maybe it is generating an in vivo spin label with that.
Orthoefer: Would you expect a membrane lipid change in anything other than the lung in
normal animals that do not have some overt disease?
Rouser: No, as a matter of fact, Dr. Lee had to spend, as I recall, roughly 3 years talking me
into doing the study at all because I felt that there was only one way that we could see a
permanent change and that would be to affect the genetic apparatus. I felt that this didn't
seem too likely in organs generally, and there was a slight possibility that the low levels used
over long periods might do the job because of the overall importance even of a negative
result, and particularly since Dr. Lee was so persistent, I finally agreed to do the research.
Furthermore, I was so certain there would be no effect that I didn't bother even to look at the
data sheets to examine them for possible significance except to make sure that my technicians
had done all their jobs correctly. I was quite surprised that there was anything. I didn't
anticipate finding anything at all. I had no basis, no reason to think so, and I'm still a bit
surprised. My big question at this point, though, is how long really does it take to make such a
change? I suspect that in the growing animal, and this is why I would emphasize the
developmental aspect, these changes would occur over a shorter time period. I neglected to
mention a very important point — that these are time-coordinated. It appears from studies of
severe protein deficiency that in brain you can induce a deficiency and then can put an
animal back on a sufficient diet and it seems to regain everything. However, it does not regain
the morphologic and perhaps not the proper functional features, so that developmental
changes may be significantly affected. I think that's one of the reasons I'd like to see them
studied. Now I'm curious, how long does it take to produce a change that's going to be
permanent? In other words, expose the animals for short periods of time and then let them go
for a while and see what happens. I really think now that perhaps it doesn't take that long.
You have to realize, too, that all of the organs undergo development changes. Not just in
93
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terms of weight increases but the composition of all the major components is grossly different.
DNA and RNA drop down to about 20% of the starting value in the fetal tissue, the protein
changes, the lipid goes up always in each organ — the total amount of polar lipid — and
these seldom level off absolutely flat. In the human brain, this thing just continues at a
progressively decreasing rate throughout life, you see, and so there are really very large
changes. Our problem has been how to analyze the data. When you plot them incorrectly,
there is a huge data spread, which you don't see when you use the correct function. I think we
can analyze the data much better now.
Lee: Another thing that we should have looked at was the age-related fluorescent pigments on
which Dr. Tappel's group at the University of California at Davis had been working. Perhaps
this was due to Tappel's preference to grants over contracts, but the fluorescent pigments, at
least, in three different organs (heart, brain and testes), should have been investigated.
Stara: I just wonder if I could ask the three of you, Dr. Orthoefer, Dr. Lee, and Dr. Bhatnagar,
what tissues do we have left? We have still some of the tissues, right? You have it frozen, or
what is the situation with the tissues that were submitted for biochemical examination to your
laboratory?
Bhatnagar: We have a very small amount of tissue, if any at all, and not from all the animals.
Rouser: We actually have a goodly supply of tissues. We took organs which had not been
analyzed. I had made this mistake before of not taking everything. We have aorta and so on,
and we have quantities of everything that we had run so that there is anything that's
preserved. We had to put these in a -20 °C box. Now that means that many enzymes are not
going to be present, unfortunately.
Stephens: The changes that you observed in the lipids in the lungs were in the phospholipid
fraction, were they not? The lamellar bodies in the type 2 cells have, I believe a considerable
amount of phospholipids in them. Does someone who has worked with the lamellar bodies
have specifics?
Rouser: I know what's been reported about this lipid composition.
Stephens: They do have phospholipid components.
Rouser. Oh, yes. Well, there are two reports; one of them is correct, I think It says that there
is a lipid controlling ratio 4:1 similar to that in mice. The other one says 12:1 but I'm quite
certain that there's just no way that that could be correct. I think that 4:1 though is quite
probably correct. Of course, the lipid class composition has not to my knowledge been
reported.
Stephens: I just wondered if you would speculate on what, or if you have speculated on what
the factor is that causes the change in the lung. Could it be a change in the synthesis of the
phospholipid component of the lamellar body?
Rouser: Yes, it could be. The only mechanism I can visualize ... I've racked my brain, I've
talked to every geneticist, read every metabolic regulation guide around our place, and the
94
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only mechanism I can visualize is one affecting the genetic apparatus; that is, to make a
permanent change.
Albert: Couldn't you just get a difference in the balance of the cell types?
Rouser: Briefly, in quick passing, I realize I referred to that, and said that I feel that this is
most likely excluded by the types of numbers we got because in every case of that type that I
know of, there is change in all of the phospholipid classes. In other words, when certain
membranes do in fact have more sphingomyelin and some less phosphotidyl choline (PCX the
fact that the PC and sphingomyelin values in those animals were all exactly the same rules
out, for practical purposes, a variation in the relative proportion of membranes of a single
cell. You have, however, hit upon one of the very obvious possibilities as one of the things that
can happen to you in whole organ analysis. I might add there is one more thing where there
could be changes that are significant. For one particular membrane that we would miss ... it
may very well be the case, for example in lipids, that maybe what we're seeing in liver is a
change that would be prominent in say, endoplasmic reticulum or in the mitochondria, and
can't prove to be significant in whole organ analysis.
Stephens: The changes you saw were equal in all experimental animals?
Rouser: In the lung they were essentially the same, yes.
Stephens: It didn't matter what the exposure was, the change was essentially the same.
Rouser: They may have differed slightly. But by my estimate, I don't think it would be worth
the time and effort to analyze the individual samples to show the exact nature of that range.
Stephens: Many of us have speculated, of course, about the nitrous acid formed from N02 in
exposed animals and its potential to cause genetic change, but if all of the differences in the
experimental exposures produce essentially the same kind of change in the lipid component
of the membranes, then the change must be relatively nonspecific.
Rouser: Yes, from the standpoint of the cause, but from the standpoint of effect, I would
suspect that if a particular set of fatty acid combinations that for some reason simply are not
made in the proper manner (and of course this is something we could show, by determining
the fatty acid composition and the molecular species distribution of these lipids) — it's almost
certain that it would have to be a single type of membrane lipid. The levels are just so, cut
down to the same amount and I suspect that it's a particular type of phospholipid that
somehow gets wiped out However, is it significant physiologically? The only kind of answer
we could give to that, of course, is that we don't know. But we would expect on the basis of
what we know about the specificity of lipid protein-interaction that it very likely could be
significant to cause definite changes in structure/function relationships.
McLees: Our time is long past up. Thank you very much.
95
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-------
6. PULMONARY AND CARDIOVASCULAR PHYSIOLOGY
STUDIES DURING EXPOSURE
T.R. Lewis and WJ. Moorman
A limited amount of data exists for cardiopulmonary responses to well-defined exposure to
automobile exhaust or simple mixtures of other predominant air pollutants. Furthermore,
most investigations into the effects of air pollution have emphasized major anatomic,
subsystem failure, as denoted primarily by pathological lesions, instead of altered ranges of
performance. The studies which have dealt with urban air pollution in Los Angeles or
extreme air pollution episodes are of limited value in accurately assessing responses because
of the absence of essential analytical data regarding exposure. These exposures are also
difficult to interpret as a result of the inability to deal with the human factors of age, sex,
smoking, occupational exposures, genetics, nutrition, etc. Animal experimentation is a
necessary part of health effects data because of the controlled, analytical approach.
Swann and Balchum (1) reported significant increases in total expiratory flow resistances
when guinea pigs were exposed to oxidant levels of either 0.59 mg/m3 (approximately 0.3
ppm) or when approximate atmospheric concentrations of 45.8 mg/m3 (40 ppm) carbon
monoxide, 2.25 mg/m3 (1.2 ppm) of oxides of nitrogen and 10.6 mg/m3 (16 ppm) of
hydrocarbons were present. Severe air pollution episodes were reported by Wayne and
Chambers (2) to produce transient increases in pulmonary resistance in aged guinea pigs, but
no demonstrable chronic or cumulative effects. These responses were most evident on two
successive days when the oxidant level exceeded 0.98 mg/m3 (0.5 ppm). Emik and co-workers
(3) found that guinea pigs reared in filtered air were significantly more responsive to a
challenge dose of 0.98 mg/m3 ozone than their experimental counterparts reared in ambient
air.
Hueter et aL (4) measured total airway resistance in guinea pigs every 16 weeks for 20 months.
These guinea pigs were under a 7-day-per-week, 16-hour-per-day exposure regimen to either
diluted natural or simulated photochemically reacted auto exhaust; however, the total airway
resistance measurements were performed while the animals were breathing room air. No
alterations in airway resistance were demonstrable. Murphy (5), however, reported elevations
in airway resistance while guinea pigs were breathing low concentrations of auto exhaust. The
elevated airway resistance disappeared when the animals were breathing room air. Murphy (5)
also reported elevated carboxyhemoglobin values in animals exposed to irradiated auto
exhaust versus auto exhaust and also in rats and mice breathing a mixture of carbon
monoxide and ozone versus carbon monoxide alone. This observation of an apparent increase
in carboxyhemoglobin when oxidants are present warrants consideration.
Although heart disease is the leading cause of mortality in the United States (6) and air
pollutants are at historically high levels (7), no well-controlled experimental studies have been
performed to assess the chronic cardiovascular effects of long-term exposure to ambient levels
of air pollutants. Individual components of ambient air pollution have been demonstrated to
have cardiovascular effects: carbon monoxide inhalation has been associated with myocardial
ischemia and infarction (8, 9); ozone inhalation with discrete myocardial nuclear lesions in
mice (10); and sulfur oxides with human myocardial infarction (11). These physiological
investigations were initiated due to lack of objective studies to assess cardiopulmonary
performance in animals subjected to long-term, low-level exposure to simple and complex
mixtures of air pollutants.
97
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Methods
General experimental design features such as chamber design and function, number of
experimental subjects, test atmospheres, etc., have been described in previous papers. The
atmospheres are presented in Table 4 of Chapter 2.
Pulmonary function testing was performed prior to initiation of the exposure regimen and at
18, 36 and 61 months of exposure. Cardiovascular status studies were conducted following 48
and 54 months of exposure to the test atmospheres. Pre-exposure pulmonary function values
were determined on anaesthetized dogs utilizing a capacitance respirometer. Variables
measured were respiration rate, tidal volume and inspiratory and expiratory flow rates.
Dogs were tested at 18 months of exposure for single-breath diffusion capacity, pulmonary
compliance and total expiratory resistance (DL, CL, and RL) employing the methodologies
and conditions reported by Vaughn et aL (12). These same tests were performed with the
addition of single-breath nitrogen washout and all lung volumes at both 36 and 61 months of
exposure. At 61 months tests for maximum breathing capacity (MBC), peak expiratory flow
rate (PEF), pulmonary membrane diffusion (DM), and pulmonary capillary blood volume (Qc)
were added. The 18- and 36-month studies were performed on anaesthetized dogs using
positive pressure introduction of test gases via a syringe. At 61 months all directly performed
volumes and capacities as well as MBC and PEF were obtained by use of a plethysmograph-
respirator. In this system the introduction of test gases and inspiratory and expiratory
maneuvers are done passively, i.e., air enters and leaves the lungs by mechanical action on the
chest and not on the airway. Details of the experimental methodologies and conditions for
pulmonary function testing at 36 and 61 months can be found in the article by Lewis et aL
(13).
In order to assess the cardiovascular status of each beagle dog, three electrocardiograms were
performed on different days within a 3-week period. Because of the variability of ECG's on
any one beagle, resting EGG changes were not considered significant unless they occurred
repeatedly in all three ECG's in a given sampling period. Furthermore, only those EGG
abnormalities which persisted from the time of appearance were considered to be significant
An attempt was made to obtain an objective interpretation of the EGG data. A randomly
selected group of all resting ECG's from 31 dogs was interpreted independently by a canine
electrocardiographic consultant, D.K. Detweiler, University of Pennsylvania.
Other tests to determine cardiovascular status including vectocardiograms, post-exercise
(swimming) ECG's, selected blood pressures and phonocardiograms were also performed.
Details of experimental methodologies and conditions can be found in the publication by
Bloch et aL (14). Two other tests on cardiovascular status of beagles were conducted. Details
of the methodologies for determining the relative incidence of widened QRS complexes and
blood rheology can be found in other papers prepared by Bloch et aL (15, 16).
Results
At 18 and 36 months of exposure, no statistically significant treatment effects were detectable
by the analysis of variance for any pulmonary function variable. The pulmonary function
means by treatment and variable for 18 and 36 months are given in Tables 1 and 2. At 61
98
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Table 1. Means and Standard Deviations of Body Weight and Pulmonary Function Tests — 18 months
Atmosphere
CA
R
I
SOx
R + SOx
l + SOx
NOL+NO2H
NOH + N02L
No. animals
19
12
12
12
11
12
12
12
Weight (kg)
9.6
±2.0
11.5
±2.7
10.3
±3.2
10.7
±2.9
10.0
±2.9
11.5
±2.3
10.2
±1.8
11.0
±2.4
DL (ml/mm Hg/min)
6.95
±1.36
7.19
±2.09
6.58
±1.48
7.33
±1.74
8.49
±2.30
6.89
±1.20
6.71
±0.85
7.53
±1.01
CL (ml/cm H2O)
37.3
±11.6
37.0
±12.8
33.5
±10.0
35.7
±11.9
41.7
±13.4
36.9
±11.5
37.3
±7.6
34.6
±5.3
RL (cm H2O/l/sec)
2.15
±0.95
2.32
±1.56
3.06
±1.99
2.73
±1.54
1.99
±1.42
2.74
±1.30
2.49
±1.27
2.71
±1.20
Key: DL = Diffusing capacity for lung
CL = Compliance of lung
RL = Resistance of lung
-------
Table 2. Pulmonary Function Means by Treatment and by Variable at 36 Months of Exposure
CA R 1
CL dyn (ml/cm H2O) 46.6 32.1 41.9
CL st (ml/cm H2O) 46.0 44.2 48.9
CL /ml/cm H2O\
FRC\ ml / 0.15 0.13 0.14
RUoo/cm H2O/liter\
\ sec / 1.28 1.67 1.20
DLco/ml CO/min\
\ mmHg / 7.28 7.44 7.28
DLco /ml CO/min/mm Hg\
TLC \ ml / 0.008 0.007 0.008
RV/TLC(%) 20.8 19.3 19.6
FRC(ml) 314 332 305
ERV(ml) 132 137 124
RV(ml) 190 195 181
VC(ml) 770 837 755
TLC (ml) 952 1029 939
1C (ml) 636 697 634
N 19 11 11
aStandard deviation for replicate animals (experimental error).
bThe value was too small to be presented.
Key: CL dyn = Dynamic compliance of lung
CL st = Static compliance of lung
CL Specific compliance divided by
FRC ~ functional residual capacity
RL2oo = Resistance of lung measured at 200 ml/sec flow
DLco = Diffusing capacity of lung for carbon monoxide
TLC
so.
43.3
46.9
0.12
1.15
7.59
0.007
20.5
344
143
209
781
1035
691
11
R + SOx
39.6
45.9
0.12
1.45
7.47
0.008
20.2
319
129
197
805
992
663
10
RV/TLC =
FRC
ERV
RV
VC
TLC
1C
N
NOL + NOH +
1 + SOX N02H N02L
40.6 49.6 41.6
43.9 52.9 45.9
0.13 0.14 0.13
1.33 1.75 1.32
7.38 7.06 7.39
0.008 0.007 0.007
20.1 19.5 19.4
302 358 310
119 156 118
186 202 192
802 864 811
982 1067 1003
679 708 693
12 12 11
SD(E)*
12.2
11.4
0.03
0.09
1.27
b
3.5
61
47
33
114
154
117
Residual volume divided by total lung capacity
(hyperinflation index) expressed as percent
Functional residual capacity
Expiratory reserve volume
Residual volume
Vital capacity
Total lung capacity
Inspiratory capacity
Number of animals per treatment
-------
months of exposure the ANOVA did reveal significant differences in functional status among
treatments. The pulmonary function means by treatment and variable are given in Table 3.
Those dogs receiving auto exhaust (R and I) had higher diffusing capacities than those
receiving mixtures of oxides of nitrogen (9.91 vs. 8.13 ml CO/min/mm Hg/ml). A significant
increase in the pulmonary capillary blood volume was demonstrated in dogs receiving auto
exhaust when compared to those not receiving auto exhaust (19.8 vs. 17.2 ml, respectively).
The difference here is the result of the higher concentration of CO in the auto exhaust
treatments. Dogs receiving auto exhaust, auto exhaust plus oxides of sulfur, and NOn +
N02L demonstrated moderately elevated residual volumes (267, 280, 258 ml vs. 232 for CA).
The irradiated auto exhaust treatment produced less uniform distribution of inspired air,
whereas the oxides of sulfur appeared to improve distribution (1.8% N2 vs. 1.4% Na, and
1.1% N2 vs. 1.4% N2, respectively).
A comparison of the incidence of individual dogs functioning at levels at the lower spectrum
of normality based on clinical criteria revealed that dogs receiving NOL + N02H had lower
diffusing capacity values than the controls after 36 months of exposure. The incidence of
lower diffusing capacities for the exposed NOL + N02H versus control dogs was 5 of 12
versus 0 of 18, respectively. This was the first treatment-related effect to be statistically
identified in this study.
The following results of 61 months of exposure were found to be statistically significant when
the clinical entries and subsequent chi-square analyses were employed. Dogs that received
irradiated auto exhaust (I and I + SOX) had higher total expiratory resistances than their
comparable controls (CA and SOX), 9 of 21 vs. 0 of 28, respectively. These data were
statistically significant at the 0.0001 probability level. A decrease in peak expiratory flow rate
was noted in dogs receiving the NOL + N02H treatment when compared to CA subjects (5 of
11 vs. 1 of 18, respectively), P = 0.018. Analysis of the DLCO values did not detect treatment
differences and was again probably due to the failure to compensate for the biological
variation inherent in the experimental population. The DLCO was adjusted to a percent of the
predicted value which was markedly different from dogs receiving NOL + N02H- The
respective frequencies were 0 to 18 for CA dogs and 5 of 11 for NOL + N02H dogs, P =
0.0004. The prediction formula employed for DLCO was the one proposed by Giammona and
Daly (13) in which DL<,O = 0.0058 TLC + 1.7. Such an adjustment is similar to DLc0/TLC
ratio and the results are essentially identical. Dogs receiving R + SO* had a higher mean
RV/TLC ratio at 61 months of exposure than dogs in CA (6 of 10 vs. 1 of 18, respectively),
P = 0.003. This result is smaller than the mean in the three treatments R + SOX, R, and NOH
+ N02L obtained by analysis of variance.
The incidence of abnormal ECG's, static and post-exercise, are reported in Table 4. Consis-
tently positive static EGG diagnoses were made of one dog exposed to clean air and irradiated
auto exhaust during both sampling periods and in one dog exposed to oxides of sulfur alone
and in combination with auto exhaust in the second sampling period. Since the left axis
deviation could be explained on the basis of mitral insufficiency, the abnormality of the EGG
of the dog exposed to clean air was discounted from consideration. Hence, there was an
incidence of 4.1 % (3 of 73) of consistent EGG abnormalities in dogs exposed to air pollutants
versus 0 of 18 in controls. One dog exposed to irradiated auto exhaust and auto exhaust had
bradycardia, a diagnosis with nonspecific implications. The findings of LAD and elevated
101
-------
Table 3. Pulmonary Function Means by Treatment and by Variable at 61 Months of Exposure
CA R I
CL dyn (ml/cmJH2O) 35.7 36.3 35.8
CL st (ml/cm H2O) 38.8 43.9 45.0
CL /ml/cm H2O\
FRC \ ml / 0.10 0.09 0.10
RL200 (cm HgO/literX
\ sec / 2.94 2.87 3.14
DLco /ml CO/min\
\ mmHg/ 8.63 10.40 9.36
DLco /ml CO/min/mm Hg\
TLC \ ml / 0.008 0.009 0.009
Dm / ml \
^min/mmHg^ 25.3 16.6 20.8
Qc(ml) 16.6 19.8 18.6
RV/TLC(%) 21.0 22.3 20.3
"Standard deviation for replicate animals (experimental error).
Key: CL dyn = Dynamic compliance of lung
CL st = Static compliance of lung
CL
RL2oo = Resistance of lung measured at 200 ml/sec flow
DLco = Diffusing capacity for carbon monoxide
— ~- = Diffusing capacity divided by total lung capacity
TLC
Dm = Diffusing capacity of pulmonary membrane
Qc = Pulmonary capillary blood volume
SOx R + SOX I + SO,
34.3 40.2 32.9
41.5 48.9 47.7
0.10 0.10 0.09
2.85 2.48 2.53
9.31 9.71 8.74
0.008 0.009 0.007
23.1 24.4 24.1
18.0 20.2 20.5
18.0 24.7 21.8
capacity
NOL +
N02H
41.0
48.3
0.10
2.72
7.67
0.007
22.1
18.5
20.0
NOH +
N02L
38.6
41.9
0.10
2.73
8.89
0.008
20.4
16.5
22.0
SD(Ep
12.0
15.9
0.03
0.85
2.63
0.002
7.1
5.5
4.0
RV/TLC = Residual volume divided by total lung capacity (hyperinflation index)
-------
Table 3. Pulmonary Function Means by Treatment and by Variable at 61 Months of Exposure (cont.)
MBC (liter/sec)
PF (liter/sec)
N2 washout %
FRC (ml)
ERV (ml)
RV (ml)
VC (ml)
TLC (ml)
1C (ml)
N
Standard deviation
Key: MBC
PF
N2 wash- =
out %
FRC
ERV
RV
VC
TLC
1C
N
CA R 1
34.8 38.5 35.3
110 125 111
1.44 1.46 1.77
347 414 369
142 152 149
232 267 220
922 1,000 926
1,102 1,188 1,087
727 775 719
18 11 10
for replicate animals (experimental error).
Maximum breathing capacity
Peak flow
Percent rise in nitrogen between 5 and 7 times
SOX R + SOX
35.3
108
1.14
370
159
212
966
1,144
773
9
the dead space
37.3
115
1.40
418
138
280
943
1,139
734
10
l + SOx
36.7
114
1.43
376
137
239
999
1,166
790
11
NOL +
N02H
32.3
100
1.34
402
167
235
969
1,183
782
11
NOH +
N02L
34.5
112
1.57
408
149
258
962
1,175
767
11
SD(E)a
5.0
17
0.49
79
44
56
158
182
132
(nitrogen washout - index of uniformity of distribution)
Functional residual capacity
Expiratory reserve volume
Residual volume
Vital capacity
Total lung capacity
Inspiratory capacity
Number of animals per treatment
-------
midprecordial P-waves of a dog exposed to oxides of sulfur were not collaborated by similar
QRS-and-P-loop changes in the corresponding VCG, although LAD had been present on the
previous VCG-.
These P-wave and QRS-axis changes suggested right atrial and left ventricular abnormalities
in depolarization, the causes of which were not apparent. Positive findings on post-exercise
ECG's were distributed between two animals, one exposed to R + SOX and one to NOL +
N02H- All of the abnormalities noted were mild and subtle. Three animals, the exception seen
in R + SOx, demonstrated ventricular premature contractions. Although this type of ar-
rhythmia after exercise may imply ischemia, the meaning of VPC's in dogs has not been well
defined.
To summarize, the incidence of both static and exercise EGG abnormalities is 8.2% (6 of 73)
vs. 0 in controls. This suggests air pollutants can be an etiologic agent inducing cardiac
electrophysiologic damage.
There was also an association between air pollutant exposure and positive VCG abnormality
in a dog exposed to clean air, that of LAD attributable to an acquired mitral stenosis. This
could be discounted as was its EGG. The number of other dogs per treatment that developed,
or consistently had, diagnoses of VCG abnormality were as follows: clean air, none; R, none; I,
three; SOX, two; R + SOX, none; I+SOX, one; NOL + NC-2H, three; NOn + N02L, three
(Table 5). This incidence of 16.4% abnormalities in air pollutant-exposed dogs versus 0% in
control dogs is striking. The VCG diagnoses of abnormality were not identical for dogs within
Table 4. Summary of the Electrocardiograph^ Data
Incidence
Treatment Pre-Exercise Post-Exercise Period Finding
CA
1
sox
R + SOX
NOL+NO2H
1/18
1/10
1/19
1/10
0/11
1/18
0/10
2/9
1/10
1/11
1,2
1,2
2
2
LAD
bradycardia
LAD, precordia
P-waves
bradycardia
VPC
3 of 73 = 4.1% resting
6 of 73 = 8.2% post-exercise
Table 5. Summary of Vectorcardiographic Data
Treatment
CA
NOh + NO2L
NOL+N02H
R
I
l + SOx
R + SOx
sox
Incidence
0/18
3/11
3/11
0/11
3/10
1/11
0/10
2/9
Finding
mitral stenosis
RVH
12 of 73 = 16.4%
104
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an air pollutant treatment group, except for irradiated auto exhaust where right ventricular
hypertrophy, ranging from questionably present to mild, was consistently present
Another study was designed to assess in each of the eight treatment groups the relative
incidence of cardiac arrhythmias, which could represent air pollution-related cardiac
pathophysiology. Electrocardiographic telemetry was performed for a 13V6- to 15-hour
diurnally identical period on each living dog (93) exposed for 4 years. An arrhythmia-
monitoring system identified widened QRS complexes (W-QRS-C) and recorded them on a
bar graph. Planimetry area of the bar graphs divided by time, in hours, resulted in a
Widened-QRS-Complex Index (Table 6). The mean W-QRS-C indices were similar (deviation
less than 8.6%) for dogs in clean air, auto exhaust, oxides of sulfur, high nitrogen dioxide and
auto exhaust plus oxides of sulfur.
The NOn + NOaL treatment mean was markedly but not statistically lower than the above
treatments by approximately 72%. Dogs exposed to irradiated auto exhaust alone and with
oxides of sulfur had mean W-QRS-C indices greater than controls by 31 and 42% respec-
tively. Analysis of variance on pooled individual mean W-QRS-C indices of dogs exposed to
irradiated auto exhaust and on their corresponding controls resulted in a significant probabil-
ity of 0.057. The latter strongly suggests that exposure to irradiated auto exhaust may cause
an elevated Widened-QRS-Complex Index.
A third study was an assessment of the effects of 4 years of exposure to air pollutants on blood
viscosity changes resulting from alterations in blood cellular or extracellular composition.
Measurements employing the Wells-Brookfield cone-plate microviscometer revealed no signif-
icant differences in blood viscosity among any of the eight treatment groups, despite
significant elevations of carboxyhemoglobin in the four auto exhaust exposures fTables 7 and
8). Exposed treatment mean methemoglobin ranged from 0.36 to 1.29%; control values were
0.33%. There were no statistical differences detected among the eight treatment means for
methemoglobin concentration (Table 8).
Comment
Due to the ubiquitous nature of nitrogen and sulfur oxides and auto exhaust (per se and
photochemically reacted) in the ambient air of urban communities, the chronic car-
Table 6. Mean Widened-QRS-Complex (W-QRS-C) Indices for
Air Pollutant-Exposed Groups of Dogs
Treatment W-QRS-C Index
CA
R
|a
SOX
R + SOx
1 + SOxa
NOL+NO2H
NOH + NO2L
x ± S.E.
3.4 ± 0.6
3.5 ± 0.7
4.5 ± 0.6
3.1 ± 0.9
3.2 ± 0.7
4.9 ± 0.5
3.4 ± 0.8
2.4 ± 0.8
and I + SOx, P = 0.057
105
-------
Table 7. Mean Canine Blood Viscosity (Centipoise)
Treatment
CA
R
1
SOX
R + SOx
I + SOX
NOL+NO2H
NOH + NO2L
Hct%
x±S.D.
45±5
48±4
46±5
43±5
48±3
46±4
43±4
42±5
23.0
(6 rpm)
JT±S.D.
8.5+1.9
8.3 ±2.0
7.9 ±1.0
7.6 ±1.9
8.2 ± 2.2
8.7 ±1.9
7.3±1.5
7.2 ±1.6
230.0
(60 rpm)
x ±S.D.
4.6 ±0.8
5.0 ±0.6
4.7 ±0.5
4.7 ±0.5
4.9 ±0.6
5.1 ±0.8
4.6 ±0.5
4.4 ±0.5
Table 8. Average Carboxyhemogtobin and Methemoglobin Concentrations
In Canine Blood (%)
Treatment
CA
R
I
SOX
R + SOx
l + SOx
NOL+NO2H
NOH + NO2L
Carboxyhemoglobin
1. 95 ± 0.45«
10.56 ±1.37
9.30 ±1.41
1.86 ±1.04
10.50±1.10
9. 74 ±1.26
1.82 ±0.99
2.18±0.61
Methemoglobin
0.33±0.29a
0.36 ±0.42
0.89 ±0.74
1.29 ±0.41
0.69 ±0.87
0.71 ± 0.85
0.66 ±0.86
0.98 ± 0.45
Standard deviation
diopulmonaiy changes reported in this study as resulting from long-term, low-level exposure
to such pollutants denote serious potential health hazards to the populace of certain
communities. Findings provide new research avenues for epidemiologists, clinicians and
lexicologists to pursue as they assess the health hazards of air pollution.
On the other hand, the experimental atmospheres did not produce readily detectable,
dramatic toxic responses following an exposure duration of 5 years. A key question is, are
5-year inhalation exposures to ambient or near-ambient levels of air pollution of sufficient
duration to demonstrate or predict suspect, insidious human diseases? Five-year lexicological
investigations are rare due to their expense and the impatience of investigators, their
administrative supervisors and funding entities. However, if one is to qualitatively and
quantitatively generate animal model systems to duplicate or simulate human responses
demonstrated or suggested in epidemiological studies, such long-term studies will be
required. The more dramatic, non-allergenic responses to air pollution in humans occur after
40 to 60 years of exposure.
The results presented in this paper stem from studies performed 6 to 9 years ago. Since these
studies were conducted, two new concepts of lung function have become very relevant in
assessing pulmonary impairment resulting from exposure to airborne contaminants (17). The
first rektes to the importance of small airway function, both from the standpoint of site of
action and as the earliest indicator of response. The second is concerned with the defense
106
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mechanism of the lung to protect the lung parenchyma against proteolytic enzymes released
by cells and macrophages within the lung. Air pollutants, both paniculate and gaseous, lead
to an increase of these cells and macrophages in the lung in response to the deposition of
particulates and irritation of the respiratory mucosa.
Chronic obstructive lung disease (COLD) has been increasing at an alarming rate as is
evidenced by the number of deaths attributed to it (18). A 25% increase was noted from 1965
to 1966 alone (19). An important point with respect to COLD is that the site of the obstruction
is in the small airways (20). The best test for evaluating small airway obstruction is the analysis
of maximum expiratory flow-volume at small lung volumes of helium isoflow. None of the
procedures of this analysis were employed on the dogs during the exposure phase of the
study. Many of these procedures were making their debut at the termination of our role in
this study.
Pulmonary function tests used to evaluate airway response would include: total expiratory
resistance, peak expiratory flow, maximum breathing capacity and nitrogen washout. Those
treatments associated with airway response are auto exhaust, auto exhaust plus SOX,
irradiated auto exhaust, NOL + N02H, and SOX. Those tests applicable to evaluating
alveolar, parenchymal response would include: diffusing capacity, compliance and the lung
volumes.
Treatments associated with alveolar, parenchymal response include auto exhaust, auto
exhaust plus SOX, NOL + N02H and NOn + N02L- One can only conclude that the
pollutant atmospheres were not selective in affecting either airways or parenchyma but, in
essence, affected both. Tests to evaluate small airway disease, however, were not available.
This study did demonstrate that exposure to air pollutants will produce the following aspects
of lung disease: pulmonary hyperinflation, non-uniform distribution of inspired air, reduced
diffusing capacity and elevated respiratory resistance. The cardiovascular responses indica-
tive of disease include: cardiac arrhythmias, ventricular axis deviation, bradycardia and
premature ventricular contractions.
Two important features of response to air pollutants were demonstrated in the study. First,
exposure to the air pollutants resulted in a gradation of individual responses even within the
same treatment. The incidence of individuals responding was the most sensitive means of
detecting treatment effect. Secondly, the exposed individuals, at the pollutant concentrations
investigated, demonstrated a decreased ability to perform vital functions and not an absolute
failure of a subsystem component resulting in death or marked morbidity.
References
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of effects of Los Angeles atmosphere on laboratory rodents. Arch. Environ. Health 16:
871-885.
3. Emik, L.O., et al. 1971, Biological effects of urban air pollution: Riverside summary.
Arch. Environ. Health 23: 335-342.
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4. Hueter, EG., et d. 1966. Biological effects of atmospheres contaminated by auto exhaust.
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Asilomar Conference Discussion
Lewis:... And there's no reason to believe that you can't have an impaired dog even though
she is in clean air. She was a small dog, as I remember, and could have been impaired
somewhat genetically, or during the testing procedure there were possibilities of aspiration,
pneumonias, and things of this nature, since aspiration can occur while recovering from the
anesthesia, etc. So you can induce artefacts or pulmonary disease just in your methodology.
Thurlbeck: Why are you interested in lower compliance rather than higher compliance?
Lewis: You mean in terms of specific compliance? We were looking for fibrosis and things of
that nature.
108
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Thurlbeck: Not for hypercompliance?
Lewis: We didn't have any. Hypercompliance usually precedes chronic obstructive lung
disease, and in our studies, when we perform pulmonary function tests sequentially and get
hypercompliance, it's indicative that we have to continue the exposure; that the ultimate
result is going to be chronic obstructive lung disease, small airway involvement, etc. But we
do look at it. However, the clinical significance of hypercompliance is a little difficult to
interpret. It may relate to emphysema.
Albert: You didn't use each animal as a control to look at sequential changes, did you?
Lewis: I thought I brought this out but I'll restate it. We didn't have baseline information to
do that.
Albert: Well, you did it at 18 and 36 months.
Lewis: Well yes, sequentially in that sense, however, I don't know what that would have told
you.
Albert: I'd just look at the data.
Lewis: Well, I'll tell you, other factors are involved in measurement of pulmonary function;
the same values in the same dogs aren't repeated from day to day. In itself, the same test can
be quantitatively different from one testing period to another.
Nettesheim: You said before that the best control is the animal itself.
Lewis: That's right That's the best control we have, but we don't have absolute control.
Busch: The pre-exposure data would have to have been taken when the animals were
puppies?
Lewis: Four to six months of age.
Busch: So there probably wouldn't have been good correlations between the later values and
the very young.
Lewis: I think that from our experience in this and other studies, the regression lines differ.
Compliance would go up; resistance would go down. That type of phenomenon is associated
with aging, particularly from puppyhood into maturity.
Gillespie: The peak flows from the NOL+N02H group were greater than the controls. Is that
right?
Lewis: No, they were lower. What we're talking about is peak flows, above 80 liters per
minute. The absolute criterion is what we used.
Gillespie: That was then measured during expiration?
109
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Lewis: Maximum expiration, yes. So, the most dramatic effects obviously are the N02-high in
terms of pulmonary impairment There are other effects which appear to be physiologically
significant and interpretable in different ways.
MacEwen: If you use the SOX groups as part of your control for comparison in the first set up
at the top, why don't you do it in the bottom when you're comparing the R + SOX? For your
RV/TLC.
Lewis: The comparison is the summation of two groups up at the top, and down at the bottom
one group is merely compared against another group. Such contrasts reflect differing
components of factorial design. This result was the only one found in terms of one treatment
versus another treatment
Hueten The question is, is it the R or the SOX that is causing the effect? You've got the
R + SOx together.
Lewis: I don't know, Dr. Hueter, they are working together and producing an additive effect
This was 6 years ago. This was the most dramatic comparison observed and it appeared to be
an additive effect I think we have some material which supports this in another fashion.
RV/TLC ... it's residual volume data where three treatments were significantly different from
the irradiated, and they were R and R + SOX, and N02-low treatments. Let me look back
here, if I can pick it up very rapidly. "Dogs receiving auto exhaust plus oxides of sulfur and
those receiving N02-low demonstrated elevated residual volumes: 280, 266, and 258 ml
versus 232." These results were from the analysis of variance. So there appears to be an
association ... the one that's common to all three treatments — the auto exhaust, the auto
exhaust + SOX, and N02 -low — is the concentration of nitrous oxide.
Busch: In the RV/TLC, how many in the raw were outside the range?
Lewis: I don't have those data. That was the smallest sample size group of the eight
treatments. We only had eight at that point in time, and that made it difficult in an individual
comparison because we were losing degrees of freedom, but the incidence data were helpful.
Busch: In the 9 of 21 in the first line, could the figures for I and I+SOX be pooled? Were
they comparable?
Lewis: They were comparable.
Thurlbeck: Is the same animal the exception; for example in R + SOX ?
Lewis: Not necessarily, and unfortunately I don't have the raw data to differentiate between
them anymore.
Albert: What magnitude of bradycardia are you talking about?
Lewis: I'll have to look that up for you later to try to follow in the time frame. Vectorcardio-
grams (Table 5).... These are incidence data once again. Dr. Bloch seemed to feel that the
one control cardiac incidence in the 1/18 was due to the diagnostic congenital mitral stenosis
and could be scientifically excluded. Thus, the respective incidence in each of the treatments
110
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was: 3/11 in the N02-low, 3/11 in the NOz-high, 0 in the raw, 3/10 in the irradiated, 1/11 in
the I+SOx, 0 in the R + SOX, 2/9 in the SOX treatment, for a total of 12/73 in the seven
exposure groups versus 0/18 or an incidence of 16%.
MacEwen: What happened to the rest of the dogs? Why 73? Weren't there 90-some at this
point? What happened to the other 20 dogs? Were the data not used?
Lewis: Add the controls into this and you come up with 91 at this point in time.
MacEwen: Well, the controk were listed above.
Lewis: Right, but there's 0/18 versus 12/73. This was the contrast that was made. We were not
able to pick out, say, any one of the seven exposure treatments that induced more cardiovas-
cular abnormalities than any other, but air pollutants per se had this incidence versus the
clean air dogs.
Albert: The RVH is right ventricular hypertrophy?
Lewis: Yes, and the reason I put that on the slide was that the only treatment that appeared to
have a similar abnormality within it was the irradiated treatment, and all three of these dogs
had evidence of right ventricular hypertrophy.
Albert: Was that confirmed at autopsy?
Lewis: By measurement and weight of the total heart right and left ventricles. The autopsies
were done several years later by the Davis group.
Thurlbeck: What about mitral stenosis?
Lewis: It was confirmed by other types of tests, but not by autopsy tests.
Orthoefen The right ventricle and left ventricle plus septum were weighed separately. If the
three of ten animals listed were outstanding, they would have shown up. I can't say without
knowing what animals, but according to a series of tests that were run, there was no statistical
significance as far as treatment is concerned in the organ weights.
Lewis: Your answer doesn't exactly answer his question. Dr. Albert wants to know whether
these particular dogs had right ventricular hypertrophy.
McLees: Did they have wedge pressure or anything like that done?
Lewis: Yes.
McLees: Were they abnormal?
Lewis: There was one abnormal wedge pressure; the other two dogs did not have abnormal
wedge pressures.
Ill
-------
McLees: The problem that is giving me and other people trouble is that the electrocardio-
gram is probably the least specific way of measuring RVH. If you have data to corroborate it,
that's one thing, increased pulmonary vascular resistance or pulmonary artery pressure,
something like that, or autopsy data What criteria were used?
Lewis: EGG, VCG, and pulmonary cardiograms. The problem that I have is that I left EPA,
and then Dr. Bloch who was a 2-year PHS Commission Corps Officer left, and he took the
data with him to write the paper. As a result, I think we've lost the identity of individual
values. We have only the summary statistics. I don't have the data, and I don't know whether
they could be retrieved. I've lost track of Dr. Bloch, and do not have his present address.
These studies were conducted 6 or 7 years ago.
Orthoefer: Wouldn't right ventricular hypertrophy show up as an increased heart weight as
compared to the total body weight?
Dungworth: I think the point they're making, though, is when you had it analyzed, the data
were lumped to see if there were differences between any treatment groups and controls. The
Question is whether for those three specific dogs there would be a significantly increased
weight of the right ventricle or whole heart.
Orthoefer: If those three dogs could be singled out, then you could have it done.
Dungworth: Then you have the raw data.
Albert: Why can't they be singled out?
Lewis: We have to know the individual identity of each of the three dogs. All I have is the
summary data, and Dr. Bloch had the raw data, when you're a physician moving from place
to place, I would say this might be the first thing you threw out.
Orthoefer: By inspection, we could look at three dogs in that particular group and say, these
three dogs look like they had high right ventricular hypertrophy.
Busch: I don't think you associate these effects with any particular component anyway. It
doesn't seem to be associated with NO2 because it doesn't appear in the raw exhaust, which
has N02- It isn't associated with auto exhaust per se.
Lewis: We didn't make any association. We just said that if you were exposed to air pollutants
you had this incidence compared to our filtered clean air animals.
Albert: Yes, but it would be nice to be able to say that the animal that had the severe
morphologic damage of the lung showed the severest abnormalities in pulmonary function
and they were the ones that had the right ventricular hypertrophy, etc.
Lewis: I couldn't concur with you more. This is one of the problems of running a chronic
study and moving facilities and people.
... I want to point out the word average. When Dr. Bloch did this, we had a 16-hour exposure
to the treatment atmospheres. He took the carboxyhemoglobin values every 6 hours, 40
112
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determinations and averaged these. So these are not peak values. This is how the values were
derived, and these values were what one would expect. Animals exposed to auto exhaust had
higher carboxyhemoglobin. That's the only significant figure here. We also did a methe-
moglobin. This was an average of the methemoglobin prior to the initiation of the exposure
and the methemoglobin at midnight, which was 16 hours later. These are the values here,
once again slightly elevated in any group other than controls but not statistically significant.
Hueter: Why was it that way?
Lewis: I don't know, maybe it's an artefact. I don't have any reason. It could be the way the
numbers fell out or it could be a sulfhydryl-type reaction. I have no physiological explanation
for it.
Busch: Trent seems to have made a point about the incidence approach being more sensitive
than the analysis of variance. We never recommend taking means of animals that have been
affected along with animals that have not. The only time it is appropriate to compare means
is when the variance remains the same, the standard deviation remains the same for the
treated as it does for the control group, so you can see that the animals respond similarly but
retained their similarity among themselves. In the case where there are some animals that
respond at a given point in time and others that do not, then what you've done is what should
be done. As a matter of fact, I suggested that yesterday for the biochemical data; to take an
approach like that instead of comparing the means.
Lewis: You have to have a handle on each dog as an individual. You can do it by covariance,
which is a convenient means, or you can do it by incidence data, but to use a variable like
body weight or just to compare treatments where you've got a big dog who has a high TLC
and another smaller dog with a low TLC, you can have the same variability within treatments,
but you increase the variability in your error terms because one dog has 700 and another has
1000.
Busch: Yes, but you need a high correlation between the pre- and post-values, and I don't
believe there would have been a good correlation here because of the long time lapse and the
change in the aging effect.
113
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-------
7. REVIEW OF THE CARDIOVASCULAR AND PULMONARY
FUNCTION STUDIES ON BEAGLES EXPOSED FOR
68 MONTHS TO AUTO EXHAUST AND OTHER AIR POLLUTANTS
/ R. Gillespie
Summary
This review includes the results of physiology studies on the cardiovascular and pulmonary
systems of groups of beagles exposed for 68 months to low levels of auto exhaust, SOX, NO,
N02 and combinations of auto exhaust and SOX. Cardiovascular and pulmonary function
measures were made on each beagle at intervals during and following the exposure period.
There were a few individuals in each group (including one in the control group) with some
evidence of cardiovascular dysfunctioa There is no substantial evidence that the cardiovascu-
lar dysfunction recorded was caused by air pollution exposure. Some of the abnormalities
were most likely congenital.
Periodically during the exposure period and 2 years after the termination of exposures (2YR),
investigators in Cincinnati or at UC-Davis measured the beagles' arterial blood gases, lung
volumes, chest wall and lung compliance (Ci, Ccw), dead space (Vp), frequency dependent
compliance (Cdyn/f), carbon-monoxide diffusing capacity (DLc0), and respiratory system, chest
wall, and pulmonary resistance (Rrs, Rcw, Rpul)- We compared the 2YR values made at
UC-Davis with those from studies during and immediately after (TE) the 68-month exposure
made by another research group in Cincinnati The 2YR values of the control group were
similar to their TE values and to those of other healthy beagles studied in the UC-Davis
laboratory. All exposure groups had pulmonary function and structural differences from the
control group, and these groups, but not the controls, had more functional abnormalities at
2YR than at TE.
In general, auto-exhaust (R and I) exposure appeared to injure airways and parenchyma,
whereas SOX, NO, and N02 caused injury to parenchyma. Combinations of auto exhaust and
SOX did not appear to augment specific functional losses caused by each single species of
pollutant The functional abnormalities correlate well in most instances with structural
changes in the exposure groups. Our studies support the hypotheses that: 1) exposure to low
levels of specific air pollutants produces pulmonary injury and loss of pulmonary function,
and 2) the functional loss continues following termination of the exposure.
Introduction
A multidisciplinary study of the effects of exposure to auto exhaust and other pollutant
mixtures upon the cardiovascular and pulmonary systems of beagles was undertaken begin-
ning September 1965. It was designed to enable study of the effects upon the pulmonary
system of long-term (68 months), low-level exposure to air pollutants. The exposures were in
the chambers at the Environmental Research Center, Cincinnati (1, 2).
Cardiovascular and pulmonary function studies were done periodically on the beagles by the
investigators in Cincinnati during exposure and immediately after termination of exposure.
115
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About 6 months after the termination of exposure, 86 of the original 104 beagles were
transported to Davis, where we studied the pulmonary function and cardiovascular function
of 71 of them about 2 years and 3 years after the termination of exposure. Those not studied
had pulmonary lobectomies. After these studies were complete, the animals were killed and
the morphology of their lungs was studied. Our aim was to evaluate the cardiovascular and
pulmonary function of these beagles for evidence of residual effects of the air pollutants 2
years after exposure. This paper presents our findings and correlates them with earlier
measurements upon these beagles during the exposure period.
Materials and Methods
Animal exposure: The air-pollutant exposure protocol and technique was described in detail
by Hinners and co-workers (1) and Hueter and co-workers (3). The exposure facility was
located at the National Environmental Research Center, Cincinnati. In brief, 104 female
beagle dogs were assigned to stainless steel and glass exposure chambers which contained
four dogs each. Beginning at 6 months of age a control group (group 1) of 19 dogs was
exposed to filtered, temperature- and humidity-conditioned clean air, and the remainder of
the dogs breathed pollutants for part of each day. Thos§ in the exposure chambers were
divided into seven treatment groups and exposed 16 hours each day to one of the following
pollutants or mixtures for 68 months: group 2, raw auto exhaust (R); 3, irradiated auto
exhaust (I); 4, SO*; 5, R + SOX; 6,1 + SOX; 7, N02 (high cone.) + NO flow conc.>, 8, NO
(high cone.) + N02 G°w cone.) (see Table 4 in Chapter 2).
Cardiovascular Junction, Cincinnati: Bloch and co-workers (4) described the methods they
employed to evaluate the cardiovascular function of the beagles at age 4V£ and 5 years during
the period of exposure. In brief, they recorded electrocardiograms (EGG) at rest and after
swimming exercise and devised a grading system to evaluate the graphs. They recorded
vectorcardiograms at rest on all dogs and phonocardiograms on those with some evidence of
cardiac dysfunction. They measured venous, right ventricular, pulmonary and systemic
arterial pressures on all the beagles under pentobarbital anaesthesia.
Cardiovascular function, UC-Davis: Seventy-one beagles ranging in age from 8*/4 to 8V2 years
were studied after being fasted for 24 hours. They weighed between 7.25 kg and 12.47 kg.
They had previously been divided into eight treatment groups as shown in Table 4 of Chapter
2. We studied them 36 months after the termination of exposure.
They were anaesthetized intravenously with sodium pentobarbital (50 mg/ml) and intubated
with a 9-mm endotracheal tube. A 5-mm Swan-Ganz flow-directed right heart catheter was
introduced into the femoral vein and passed into the right ventricle, pulmonary artery, and
pulmonary wedge pressures. A 7-F Goodall-Lubin standard wall catheter was placed in the
femoral artery and advanced approximately 15 cm into the aorta. Systolic, diastolic and mean
arterial pressures were recorded. The recording system was calibrated daily against a mercury
manometer, and the transducers were leveled with the base of the heart.
Cardiac outputs were measured using the indicator-dilution method in which we used
indocyanine green dye. We injected 1.25 mg (approximately 0.13 mg/kg) dye into the
pulmonary artery followed immediately by a 5-ml flush of heparinized saline. Arterial blood
was pulled through a cardiac output computer cuvette by means of a constant flow syringe
116
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puller. Cardiac output was recorded digitally on the cardiac output computer and the dye
dilution curve was simultaneously recorded. The cardiac output computer was calibrated
electrically before each measurement and this electrical signal was checked against standard
dilutions of indocyanine green in whole blood.
From the blood pressures and cardiac outputs, the following information was obtained: heart
rate (beats/mm), stroke volume (ml), cardiac index (L/min/kg3'4), total peripheral resistance
(dynes/sec/cm"5), total pulmonary resistance (dynes/sec/cm"5) and left ventricular work (L/mm
Kg/min-1):
Pulmonary function, Cincinnati: Vaughn and co-workers (5) and Lewis and co-workers (6)
.described in detail their methods to study pulmonary function after 18, 36 and 61 months of
exposure. In brief they measured lung compliance, subdivisions of long volume, pulmonary
resistance, single-breath, carbon-monoxide diffusing capacity, maximum breathing capacity
and peak expiratory flow. From these measurements they calculated ratios for compliance/
lung volume and diffusing capacity/lung volume.
Pulmonary function, UC-Davis: The pulmonary function techniques we employed in this
study have been reported by us in detail (7, 8, 9), and only a brief account will be given here
for each.
The beagles were delivered to the laboratory, weighed, and given a physical examinatioa The
pulse and respiratory rate were recorded, as were any comments of special interest resulting
from the physical examination. Chest measurements (width, depth, and length) were made
and a venous sample was taken for packed cell volume (PCV) and hemoglobin (Hb) analysis.
The beagles were anaesthetized intravenously with thiamylol sodium1, 8 mg/kg, and intuba-
ted with the krgest possible diameter endbtracheal tube (8-9 mm I.D.). An esophageal
balloon2 10 cm long (containing 0.5 ml air) on PE 200 tubing was placed in the esophagus
and attached to a pressure transducer3 for measuring pleural pressure. The dogs were
ventilated during most of the study with a constant volume ventilator.4 The ventilatory
volume and rate were adjusted to give end-tidal C02 concentrations of approximately 5%,
measured by an infrared CC<2 analyzer.5 Deep breaths at 5-minute intervals to a trans-
pulmonary pressure (Pip) of 30 cm H20 prevented the fall in lung compliance reported in
anaesthetized dogs (10,11).
We used a volume displacement plethysmograph6 specifically designed for dogs connected to
a wedge spirometer^ to measure slow changes in lung volume. The plethysmograph was
converted to a pressure box to record fast lung volume changes using a highly sensitive
'Surital, Parke-Davis
2Anode Rubber Plating Co., no. UC-516
SStatham, PM131-TC
4Harvard, model 614
5Beckman, model LB1
6K-plastix, San Francisco
7Med-Science Electronics, model 270
117
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pressure transducer8. We have previously described the performance characteristics of our
measuring apparatus (8). All data were recorded by an 8-channel oscilloscopic recording
system^.
Soon after giving anaesthesia and prior to mechanically ventilating the beagles, we collected
data to calculate physiological dead space volume (¥D) and dead space/tidal volume (VD/Vr)
ratios. We collected expiratory gas in a bag in a rigid box. Spontaneous tidal volume and
breathing frequency were recorded by a wedge spirometer attached to the rigid box. At the
midpoint of the 3-minute expired gas collection, we anaerobically collected an arterial blood
sample from the femoral artery. We measured10 arterial blood Po2, PcCfe, and pH immedi-
ately after collection, and corrected our values to the dog's deep rectal temperature. The
mixed expired sample was analyzed for 02 and C02 concentration with a Scholander gas
analyzer11. We used the conventional formula to calculate VD/VT (12).
We measured resistances of the respiratory system using the method of Mead and Witten-
berger (13). Sinusoidal flow oscillations were generated by a speaker system attached to the
endotracheal tube. Following a deep breath, the dog's lungs were allowed to return to resting,
end-expired volume (FRC), and we oscillated the airway at 3 to 5 Hz. We recorded data for
total pulmonary resistance (Rpu|), total respiratory resistance (Rrs), and chest wall resistance
(Rcw) by recording transpulmonary, airway, and pleural pressure, respectively, versus flow on
an x-y oscilloscope. Flow rates were measured with a pneumotachograph12 attached to a
pressure transducer13. Endotracheal tube resistances were subtracted to determine Rpu), Rrs.
and RCW-
We measured pulmonary diffusing capacity with the single-breath carbon-monoxide test
(Pico) (?. 9, I4)- We rapidly injected a volume of test gas (0.3% CO, 0.5% Ne, balance air)
equivalent to the dogs inspiratory capacity (1C) into the dog's lungs, allowed a 10-second
breath-hold, then collected an alveolar sample for analysis14. We calculated DLCO using the
formula of Ogilvie et aL (15). We repeated these measurements after each beagle had
breathed oxygen-rich gas for three breaths and 10 to 15 minutes. From these measurements
we calculated pulmonary capillary blood volume by the technique of Roughton and Forster
(16).
We recorded quasistatic lung and chest wall pressure-volume curves using techniques
reported earlier by this laboratory (7,8). We inflated the dog's lungs until FTP = 30 cm IfcO,
and allowed them to relax to FRC. Then we recorded volume on the y-axis and Ppr on the
x-axis as we slowly inflated the lungs to FTP = 30 cm H20, slowly deflated them to Ppr =15
cm FfeO, and allowed passive reinflation to FRC. The pressure-volume trace was nearly
horizontal from FTP = 10 to -15, and this volume was taken to be residual volume (RV).
SStatham, PM5
^Electronics for Medicine, model DR8
10Blood Gas Tension Analyzer, model 113, Instrumentation Laboratories
^Scientific Instruments
12Sims Instruments, Fleish model 1 (60 1/min)
13Statham, PM5
14Micrc-Tek, Tracer, Respiratory Gas Analyzer, model 150
118
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Chest wall volume-pressure curves were recorded using the same maneuvers with volume
plotted against pressure across the chest wall (Pew)-
We measured end-expiratory lung volume (FRC) following an inflation-relaxation maneuver
with two techniques: 1) the nitrogen equilibration technique, FRCN2 (7,8), and 2) the Boyle's
law technique, FRCRL (7, 8). In the first, following a lung-inflation-passive-deflation maneu-
ver, we ventilated the dog manually 15 times with a volume of Oa equivalent to its 1C. This
was done by using a syringe to move the oxygen in and out of the dog's lungs. The fraction of
N2 in the syringe at the end of the test was analyzed15, and the FRCN2 calculated (7).
In the second technique, done immediately prior to the first, we measured the plethysmo-
graph volume and airway pressure changes while the dog, beginning at FRC, breathed
spontaneously against an obstructed airway. We calculated FRCuL from slope of the
volume-pressure record (8).
We measured dynamic compliance (Cdyn) at 5, 10, 20, 30, 40, and 50 breaths/minute. We
compared the slopes of the plot of the Cdyn against the frequency for each group. The greater
the slope, the greater frequency dependency of lung compliance.
We compared the pulmonary function values of these beagles to those of a group of healthy
beagles studied in our laboratory using the same techniques (7, 8, 9) and, where techniques
permitted, to their own values measured 2 years earlier in Cincinnati (6).
Pulmonary function values for the 8 groups were evaluated statistically with Duncan's
multiple range test. Changes in individual's values within each group between termination of
exposure (TE) and 2 years after termination of exposure (2YR) were evaluated with the paired
Student-t test. We accepted the 0.05 probability level as being significant when comparing
values from the different groups.
Results
Cardiovascular function, Cincinnati: The cardiovascular studies done during the course of
exposure were described by Bloch and co-workers in three papers (4, 17, 18). The results of
their studies are summarized in Tables 1 through 8.
No specific abnormal function can be attributed to air pollution exposure from their studies.
There were no trends within groups or within individuals to suggest progressive cardiovascu-
lar dysfunction in the exposed beagles. There were individual dogs with cardiac dysfunction
in various groups (including an individual in the control group). Table 8 shows the hematocrit,
carboxyhemoglobin and methemoglobin values recorded by Bloch and co-workers (4) during
the period of exposure.
Cardiovascular function, UC-Davis: There was no difference in the body weight (range of
means 9.2 to 10.2 kg), PCV (range of means 37.3 to 40.9%) or Hb (range of means 14.2 to 15.8
gm%) of the eight groups of beagles. The results from the cardiovascular measurements at
UC-Davis 3 years after termination of exposure are in Tables 9 through 11. There was no
^Nitrogen Analyzer, Med-Science, model 105
119
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Table 1. Grading System for Electrocardiograms3
ECG grade
0
I-A
I-B
II
Meaningb
Within normal limits
Abnormal and nonspecific (even for heart disease),
but questionable abnormality
Abnormal and nonspecific (even for heart disease),
not questionable abnormality
Abnormal and probable heart disease of nonspecific
kind
Gradation
III Abnormal and probable heart disease
IV-1
IV-2
IV-3
IV-4
Abnormal and possibly compatible with
heart disease
Abnormal and definitely compatible with
heart disease
Abnormal and suggestive of
heart disease (more than compatible, though not
yet conclusive)
Abnormal and diagnostic of
heart disease (conclusive)
1 +
4 +
2 +
4 +
3 +
4 +
4 +
4 +
aBloch et a/. (4)
bThe type or types of heart disease is inserted in blank spaces in sentences IV-1 through IV-4.
Table 2. Some ECG Interpretation Grading Criteria3
ECG grade
assigned Associated abnormalities'3
0 (a) P-wave notched in V1
(b) ST-segment elevated or depressed less than 0.1 mv
(c) QRS/T-angle greater than 90" in one plane only
I-A (a) Bradycardia or tachycardia
(b) ST-segment elevated 0.1 to 0.15 mv
(c) QRS/T-angle greater than 90° in frontal and horizontal planes
(d) Increased right or left precordial R-waves
(e) Increased right or left precordial S-waves
(f) Combinations of (b) and (c)
I-B (a) P-wave 0.3 to 0.35 mv
(b) T-wave inverted or peaked in V1-6
(c) ST-segment elevated 0.15 mv or more
(d) ST-segment depressed 0.1 mv or more
II (a) P-wave 0.35 mv or more
(b) P-wave greater than 0.04 sec
(c) Mild arrhythmia (other than sinus arrhythmia)
(d) Sinus pauses of 1.8 sec or more which are at least double the
average R-R interval
(e) Right- or left-axis deviation
III (a) Severe arrhythmia0
(b) Combinations of I-A, I-B, II
aBloch et a/. (4)
bThis list includes only most commonly encountered abnormalities which are not unequivo-
cally associated with definite cardiac pathology.
This could also be designated as grade IV in some cases.
120
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Table 3a. ECG, VCG, and Other Diagnoses, by Sampling Period*
Static ECGs
Treatment
Clean air
R
1
SOX
R + SOx
1 + SOX
NOi. + NC-2H
NOh + NO2L
1a
II
I-A
I-A
0
II
I-A
0
I-B
I-B
0
0
0
0
0
0
0
I-A
II
III
0
0
I-A
0
0
1b
II
0
0
I-A
0
I-A
0
0
I-A
0
I-A
0
0
0
0
0
0
I-A
0
0
0
0
0
0
1C
II
I-A
I-B
0
0
I-A
0
I-B
0
II
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2a
II
I-A
0
0
0
I-A
I-A
X
I-A
0
II
0
I-A
0
I-A
I-A
I-A
0
I-A
.0
0
I-B
0
I-A
I-A
2b
II
I-B
0
0
0
I-A
0
X
0
0
II
0
0
I-B
0
I-A
I-A
0
I-B
0
0
0
0
0
0
2c
II
I-A
I-A
0
0
I-A
0
X
I-A
0
111
0
I-B
I-A
I-A
I-A
I-B
I-B
0
0
1
0
0
0
aBloch et al. (4)
121
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Table 3b. ECG, VCG, and Other Diagnoses, by Sampling Period3
Vectorcardiogramsb
Treatment
Clean air
R
1
sox
R + SOX
1 + SOX
NOL + N02H
NOH + NO2i
ECG
rating0
S
D
S
S
D
S
S
D
S
S
D
D
1
LAD
WNL
r^WNL
WNL
~WNL
M/VNL
WNL
WNL
?Mild RVH
M/VNL
LAD
Possible mild RVH
?Mild RVH
LVH vs VCD
WNL
WNL
WNL
WNL
~LVH, mild
^variant WNL
WNL
WNL vs mild RVH
~WNL
WNL vs mfarct
2
LAD
WNL
WNL
Mild RVH
? Mild RVH
WNL
"> Mild RVH
X
Possible Mild RVH
WNL
WNL
WNL
? Mild RVH
Mild to moderate LVH
WNL
? Postinfarct
WNL vs IVCD
Mild LVH
Moderate LVH
RVH (? mild)
WNL
WNL
Probable old posterior
infarct
~mild RVH
vs fibrosis
WNL
LAD
aBlochefa/. (4)
"Within normal limits, WNL; right ventricular hypertrophy, RVH; mean systemic arterial hyper-
tension, SAH; diastolic systemic arterial hypertension, DSAH; ventricular conduction defect,
VCD; left ventricular hypertrophy, LVH; intraventricular conduction defect, IVCD; left axis
deviation, LAD; animal dead, X.
cSame ECG abnormality throughout each period, S; differing ECG abnormality within same
period, D.
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Table 3c. ECG, VCG, and Other Diagnoses, by Sampling Period"-»
Treatment
Clean air
Postexercise
ECGs,
sampling Catheterization Phonocardiographic
period 2 diagnoses diagnoses
Mitral insufficiency
R
1
SAH
SOx
II
II
R + SOx
I-A( + )
Mitral stenosis
1 + SOx
NOi + NO2H
Pulmonic stenosis
II
SAH & DSAH
NOH + NC-2L
aBloch et al. (4)
bMean systemic arterial hypertension, SAH; diastolic systemic arterial hypertension, DSAH.
123
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Table 4. Selected Blood Pressures (mm Hg, ± 1 SO)8
Treatment
Clean air
R
1
SOx
R + SOx
1 + SOx
NOi + NO2H
NOH + NO2L
Pulmonary
wedge
2.43
±2.56
2.68
±2.57
2.23
±1.30
2.33
±2.71
3.67
±4.17
1.75
±2.74
1.05
±1.64
1.22
±1.92
Individual Mean
pulmonary pulmonary
wedge artery
8.50= 17.84
± 5.14
19.91
± 4.54
17.65
± 3.72
16.56
± 2.75
13.25 19.82
± 3.75
-4.75d 17.50
± 5.79
18.28°
± 3.36
17.07
± 3.56
Systolic
pulmonary
artery
26.72
± 6.56
28.91
± 5.34
27.15
± 6.07
25.78
± 3.87
29.05
± 4.39
27.91
± 10.53
26.70°
± 3.44
27.05
± 6.06
Individual
systolic Mean
pulmonary blood
artery1' pressure
40.5 122.4
± 15.2
130.9
± 10.6
120.9
± 16.9
122.3
± 20.2
124.0
± 13.3
50.0 123.8
± 14.1
124.8
± 19.2
42.0 121.8
± 12.8
Individual
blood
pressure
157.50
86.25
163.25
aBlochefa/. (4)
"Significant (P<0.05).
°Data from ten animals.
dData from different animals in same treatment.
-------
Table 5. Phonocardiographic and Physical Abnormalities8
Treatment Findings on individual dogs Diagnosis
Clean air Mitral area: early systolic Mitral insufficiency
decrescendo murmur
SOx LJL. ...
R + SOx Mitral area: mild to late diastolic Mitral stenosis
murmur with presystolic
accentuation
I + SOx ^ ...
NOu + NO2H Unable to pass catheter through Pulmonary stenosis
pulmonary valve
NOH + NO2L ._._. ^l^__
aBloch et al. (4)
125
-------
Table 6. Dog Illnesses During Observation Period3
Diagnosis Sampling period Treatment Findings
Probable myocardial 2 R + SOX Dog weak, eyes shut; cardiovascular, 81,82 within
infarction normal limits without murmur; ECG, ST: t1.5 mm in
Vi, 1 mm in V2, T-waves inverted and peaked, Vi.e
QRS/T-angle > 90° in horizontal plane only; these
changes were not present on static ECGs from pre-
vious sampling period; ECG 5 days later, changes per-
sisted with ST: t 2 mm in V1t 1.5 mm in V2; QRS
abnormally slurred, Vs-e; T-waves notched, 2,3, aVL,
aVF; animal gradually recovered
Myocardial infarction After 2 I + SOX Dog weak, moves little; faint Si, S2; ECG, pathologi-
cal Q-waves, 1, aVL, Vs, Ve, V-IQ; T-waves inverted and
slightly coved, 1, aVL; VCG decreased anterior forces,
terminal QRS-delay, QRS-loop open; enzymes, LDH,
and SGOT statistically significantly elevated (P<.05);
ECGs 3 and 7 days later, pathological Q-waves de-
creased in size; T-waves" reverted to upright; animal
gradually recovered
aBloch et al. (4)
-------
Table 7. Dog Deaths During Observation Period8' b
Sampling
period Treatment
1 NOL + NO2H
1 I
1 I
1 Clean air
2 SOX
2 SOX
After 2 NOH + NO2L
After 2 SOX
ECG premortem
6 days prior, compatible
with schemia
Compatible with myocardial
infarction
WNL (6 leads)
8 days prior, WNL
Pathological
heart findings
Gross, not
remarkable
Gross, not
remarkable
Gross, not
remarkable
Gross, not
remarkable
Histological, not
remarkable
Gross, heart muscles
replaced by probable
connective tissue
Gross, WNL
Gross, WNL
Likely cause of death
Trauma
Trauma
Trauma
Trauma
Trauma and probable
myocardial infarction
Failure to recover from
anesthesia, chronic
neurologic disease
suspected
Aspiration pneumonia,
secondary to diagnostic
procedure
Renal shutdown with
septicemia
aBloch et al. (4)
bWithin normal limits, WNL.
-------
Table 8. Average Hematocrit, Carboxyhemoglobin, and Methemoglobin Levels3
Treatment
Clean air
R
1
sox
R + SOX
1 + SOX
NOL + NO2H
NOH + N02L
Hematocrit (%)
45.2
48.1
45.9
42.8
47.7
46.3
42.6
41.9
SD Carboxyhemoglobin (%) ' SO
4.6
3.8
4.7
5.5
3.3
4.5
3.8
4.8
1.95
10.56
9.30
1.86
10.50
9.74
1.82
2.18
0.45
1.37
1.41
1.04
1.10
1.26
0.99
0.61
Methemoglobin (%) SD
0.33
0.36
0.89
1.29
0.69
0.71
0.66
0.98
0.29
0.42
0.74
0.41
0.87
0.85
0.86
0.45
aBloch et al. (4)
-------
Table 9. Cardiovascular Function of Each Group 3 Years after Termination of Exposure
Groups
Heart rate
Stroke volume
Cardiac output
Cardiac index'
1
Control
166.7
±5.8 (12)
9.9
± 0.7 (12)
1.6
± 0.06 (12)
0.31
±0.02 (12)
2
R
174.0
±11.1 (10)
9.6
± 0.5 (10)
1.6
± 0.06 (10)
0.29
± 0.02 (10)
3
145.0
± 9.3 (4)
13.0
±2.5 (4)
1.8
±0.18 (4)
0.33
±0.03 (4)
4
sox
170.6
± 8.0 (8)
10.1
± 0.6 (8)
1.7
±0.09 (8)
0.33
±0.03 (8)
5
R + SOx
159.4
± 9.4 (9)
10.8
± 0.9 (9)
1.6
±0.06 (9)
0.28
±0.01 (9)
6
l + SOx
140.0
±11.0 (11)
10.7
± 0.8 (11)
1.5
± 0.07 (11)
0.27
± 0.02 (11)
7
NOL + NO2H
161.7
±14.7 (6)
8.3
± 0.9 (6)
1.3
± 0.06 (6)
0.25
± 0.02 (6)
8
NOH + NO2L
152.5
±6.0 (10)
9.3
±0.6 (10)
1.4
±0.07 (10)
0.26
±0.01 (10)
-------
Table 10. Mean Intravascular Pressures of Each Group 3 Years after Termination of Exposure (mm Hg ± 1 SD;
number of dogs in group is in parentheses)
Groups
Site3
Pao-s
Pao-d
Pa5
Ppa-s
Ppa-d
PpS
Pwedge
Prv-s
Prv-d
Prv
1
Control
161.4
±5.9
108.5
±4.6
127.2
±4.3
27.9
±2.7
12.8
±2.0
18.9
±2.3
6.1
±2.0
29.6
±2.9
-1.22
±0.53
10.5
±1.3
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
2
R
175.4
±6.1
118.5
±3.7
139.8
±4.3
31.2
±3.3
14.9
±2.0
20.2
±2.6
2.8
±1.0
31.6
±2.7
0.04
±0.43
10.5
±1.3
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
3
I
148.2
±7.8
101.1
±3.7
119.5
±5.0
24.5
±9.5
10.2
±5.2
15.6
±7.3
-0.1
±1.5
25.9
±9.6
-2.93
±0.54
7.7
±4.4
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
(4)
4
SOx
165.8
±5.7
113.2
±4.1
136.0
±4.7
26.4
±2.4
13.2
±2.2
18.6
±2.1
2.0
±1.3
28.0
±2.2
-2.77
±0.36
9.2
±1.2
(8)
(8)
(8)
(8)
(8)
(8)
(8)
(8)
(8)
(8)
5
R + SOx
162.2
±6.9
103.8
±4.6
124.4
±5.5
27.8
±1.6
11.1
±1.2
18.4
±1.3
2.1
±0.9
31.0
±1.8
-0.35
±0.49
10.8
±1.2
(9)
(9)
(9)
(9)
(9)
(9)
(9)
(9)
(9)
(9)
6
l + SOx
162.7
±5.6
107.1
±3.4
127.6
±4.7
22.8
±3.4
8.7
±2.5
14.7
±2.8
2.0
±1.0
24.5
±2.6
-1.53
±0.52
1.5
±1.4
7 8
NOL+NO2H NOH + NO2L
(11)
(11)
(11)
(11)
(11)
(11)
(11)
(11)
(11)
(11)
157.8
±5.3
103.2
±4.8
123.8
±4.9
28.7
±4.2
12.2
±3.4
19.5
±3.8
5.5
±2.5
34.8
±4.1
-2.28
±0.69
11.4
±2.4
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
156.6
±6.1 (10)
104. 3
±4.3 (10)
123.6
±5.1 (10)
26.6
± 3.1 (9)
12.1
± 2.3 (9)
18.3
± 2.5 (9)
3.5
±1.7 (9)
29.0
±3.3 (10)
-0.48
±0.95 (10)
9.2
±1.7 (10)
aSite of pressure measurement: aortic systolic (Pao-s), aortic diastolic (Pao-d), aortic mean (Pao), pulmonary artery systolic (Ppa-s),
pulmonary artery diastolic (Ppa-d), pulmonary artery mean (Ppa), pulmonary artery wedge (Pwedge), right
ventricular systolic (Prv-s), right ventricular diastolic (Prv-d), right ventricular mean (Pfv).
-------
Table 11. Mean Values and Standard Errors of Peripheral Vascular Resistance (Rper), Pulmonary Vascular Resistance (Rpv) and Left Ventricular
Work (WLv) for Each Group 3 Years after Termination of Exposure (resistance in dynes-sec/cm-5 and work in 1 mm Hg/min; number of dogs in
group is in parentheses)
Groups
Rper
(x10-1)
RPV
WLV
1
Control
650.7
±44.7 (12)
641.3
± 52.6 (12)
206.1
±13.1 (12)
2
R
693.3
±25.4 (10)
856.6
±90.4 (10)
230.4
±16.3 (10)
3
1
571.8
± 75.9 (4)
749.5
±45.8 (4)
219.8
±33.3 (4)
4
S0x
658.4
± 62.3 (8)
782.9
± 122.9 (8)
233.2
± 16.7 (8)
5
R + SOx
605.2
±45.2 (9)
787.8
±71.6 (9)
209.5
±15.5 (9)
6
l + SOx
734.8
± 60.0 (11)
695.3
±124.3 (11)
185.2
± 12.4 (11)
7
NOL + N02H
787.2
± 76.2 (6)
944.8
±225.8 (6)
159.0
± 8.4 (6)
8
NOH + NOzi
724.1
±53.2 (10)
801.4
±62.9 (9)
177.5
±17.5 (10)
-------
difference in any of the groups' values except for left ventricular work. Beagles exposed to
nitrogen oxides had lower cardiac work values than did those exposed to SOX and raw auto
exhaust
Pulmonary function, Cincinnati: Investigators in Cincinnati had previously measured the
pulmonary function of these beagles after 18, 36 and 61 months of a 16-hour per day, 7-day
per week exposure regimen. After 18 months, Vaughn and co-workers (5) found no differences
in DLCO , CL^,, , or total pulmonary resistance in these beagles. After 36 months, Lewis and
co-workers (6) measured a lower DLc0/TLC (total lung capacity) ratio in the groups that
breathed NOL + N02H than in control beagles, and after 61 months (near the end of
exposure), they reported the following:
1. Dogs exposed to auto exhaust without addition of SOX had higher mean DLc0/TLC and
DLCO values than those receiving mixtures of NO and N02 (0.009 versus 0.008 ml/min/mm
Hg and 9.91 versus 8.13 ml/min/mm Hg respectively), and the NOa-high had lower
CO /TLC ratio than control dogs.
2. Dogs that received auto exhaust had greater pulmonary capillary blood volumes than
controls (19.8 and 172 ml, respectively).
3. Dogs receiving R + SOX, R, and NOn + N02L had larger mean RV values (280, 267 and
258 ml, respectively) than those receiving I + SOX, NOL + N02H, control I, and SOX(239,
235, 232, 220 and 212 ml, respectively).
4. Dogs that breathed I had a larger mean nitrogen washout than control dogs (1.77% and
1.44% respectively), and the SOX values were lower than the control group (1.14% and
1.44%, respectively):
5. Dogs that breathed I or I + SOX had a greater Rpui (expiratory) than control and SOX
groups.
6. A low peak flow rate was reported for the N02-high group when compared to controls.
7. These authors adusted the DLCO value to a percent of predicted, and found that none of
the controls (0 to 18) were below 80% of their predicted value and that 5 of the 11
N02-high dogs were. Their prediction formula (19) was DLCO — 0.0058 TLC + 1.7.
8. Six dogs that breathed R + SOX had a larger mean RWTLC ratio than predicted, while
only one control dog had a larger ratio.
Pulmonary function, UC-Davis: There was no difference in the body weight (range of means
9.2 to 10.2 kg) (Table 12), PCV (range of means 37.3 to 40.9%), or Hb (range of means 14.2 to
15.8 g %) of the eight groups of beagles. Table 13 shows the blood gas and pulmonary
function values related to ventilation during a period of spontaneous breathing soon after
induction of anaesthesia. The PacOa of groups R, R + SOX, I + SOX, and N02-low was
significantly greater than that of the control group. The same groups and SOX had greater VD
(Table 13) and VD than the controls. SOX had higher respiratory frequency, and R and
N02-low had significant acidosis.
132
-------
Table 12. Body Weight (WT), Blood Gases (pHa), Breathing Frequency (f), Tidal Volume (VT), Minute Volume (Vmin), Percent Dead Space (%VD),
Dead Space (Vo), and Dead Space Ventilation (Vo) of Beagles (mean value ± 1 SE; number of dogs in group is in parentheses)
Groups
1
Control
WT(kg)
Pao2 (mm Hg)
Paco2 (mm Hg)
pHa
f(breaths/min)
VT (ml)
Vmm (ml/min)
%VD
VD (ml)
VD (ml/min)
9.2
± 0.4
84.7
± 5.5
37.9
± 2.0
7.371
± 0.018
21.9
± 3.9
78.5
± 5.8
1662
± 300
58.4
± 2.6
45.1
± 3.1
954
± 168
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
2
R
9.9
± 0.4
77.1
± 4.9
46.8
± 1.4
7.322
± 0.009
16.6
± 1.6
108.3
± 9.5
1708
± 127
67.0
± 1.6
72.0
± 6.4
1146
± 105
0)
(9)
(9)
(9)
(9)
(9)
(9)
(9)
(9)
(9)
3
I
10.0
± 0.6
99.4
± 9.8
40.7
± 2.6
7.364
± 0.016
20.2
± 2.8
101.1
±7 .4
1970
± 146
57.3
± 4.4
57.5
± 5.2
1135
± 143
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
4
sox
10.0
± 0.3
85.0
± 5.6
41.8
± 2.1
7.303
± 0.010
23.7
± 3.5
111.1
± 8.0
1039
± 393
63.0
± 2.5
69.6
± 5.0
1588
± 257
5
R + SOx
(8)
(9)
(8)
(8)
(7)
(8)
(7)
(8)
(8)
(7)
9.4
± 0.5
74.3
± 4.5
47.0
± 2.0
7.333
± 0.013
19.6
± 2.5
98.2
± 8.9
1768
± 158
68.4
± 1.6
66.6
± 5.4
1221
± 129
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
6
l + SOx
10.2
± 0.4
83.8
± 4.5
45.3
± 1.8
7.364
± 0.017
17.2
± 1.2
102.2
± 5.8
1749
± 152
63.7
± 2.6
62.4
± 3.3
1104
± 95
7
NOi + N02H
(11)
(11)
(11)
(11)
(11)
(11)
(11)
(11)
(11)
(11)
9.5
± 0.4
96.0
±11.1
41.2
± 2.5
7.348
± 0.023
27.0
± 5.0
88.6
± 6.8
2244
± 339
64.8
± 3.3
56.8
± 3.5
1319
± 264
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
8
NOH + N02L
9.7
± 0.3
73.2
± 5.8
44.8
± 1.4
7.325
± 0.010
18.5
± 2.9
93.9
± 4.4
1787
± 250
66.2
± 2.3
65.3
± 3.1
1198
± 187
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
-------
Table 13. Total Dead Space of Control Group Compared with
Exposed Groups 2 Years after Termination of Exposure
Total dead space
(ml)
Control 45.1
R 72.0
SOX 69.6
R + SOx 66.6
NOH + NO2L 65.3
I + SOx 62.4
Figure 1 shows Rrs, Rpu], and RCW of the beagles. The R + SOX group had higher Rre, Rpu],
and RCW values than any of the other groups whose values were similar.
The DLCQ °f all the groups was similar, but when normalized with TLC, the ratio DLc0/TLC
was smaller in all groups exposed to air pollutants than that of the control group (Table 14).
The mean pulmonary capillary blood volume (Vc ) was 15.6 ± 2.0 ml in the control group
and 17.1 ± 4.3 ml in the exposed groups. There was no difference between any of the groups'
Vc values.
Table 15 shows the mean values of inspiratory and expiratory quasistatic lung and chest wall
compliances (Cij , CLE , andCcwE )• The CCWE was similar in the control and R + SOX groups,
but in all other exposed groups, CCWE was IGSS than that of the control group.
Figure 2 shows the mean values for total lung capacity (TLC) and the lung volume
subdivisions (VC, FRC, and RV) for each group. The mean TLC was similar in all the exposed
groups, but the SOX, R and N02 -high groups had significantly larger TLC than that of the
control group. There was no difference in the FRC measured by the N2 -equilibration or the
Boyle's law technique, and there was no difference between the FRC of the various groups.
Inspiratory capacity (1C) was greater than that of the controls in the SOX and R groups. The
RV was similar in all exposed groups, and was greater than that of the controls except I +
SOX.
Figure 3 shows the relationship between dynamic compliance (Cdyn) and frequency (f) for
each group. There was no difference in the slope Cdyn/f for the control, I + SOX, R + SOX, I
and R groups, but the SOX, NOL + N02H and N02L groups had greater changes in Cdyn with
increasing f than did the controls. The differences appear to be due to the high Cdyn at low
frequencies in the SOX, NOL + N02H and N02L •
Tables 16, 17 and 18 show the pulmonary function values for the control beagles in this study
compared to those from a group of healthy beagles previously studied in this laboratory. Most
values are very similar.
Table 19 shows the mean pulmonary function values for each group done immediately after
termination of exposure (TE) (6) and our values (2YR) done 2 years after termination of
exposure. We have included all comparable values for each group for the two test periods,
although techniques were not always identical Values for a group that appeared to have
changed between TE and 2YR are underlined in Table 19. We compared each dog's TE lung
134
-------
1
CA
110-
100-
90-
80-
70-
Resistance
(xlOOO) 60 •
(cm H20/l/sec)
50-
40-
30'
20-
10-
\
i
I
2
R
|
I
i
3
J
I
i
4
SOX
6
£
5 6
R + SOx l + SOx
7
NOL +
N02H
I
f
f
i
Jl
i
i
x
i
8
NOH +
N02L
f
I
RS P CW RS P CW RS P CW RS P CW RS P CW RS P CW RS P CW RS P CVi
Figure 1
Mean values with standard errors for respiratory (rs), pulmonary (p), and chest wall (cw)
resistances for each group of beagles.
135
-------
Table 14. Pulmonary Diffusion Capacity (DLco) and DLCO to Total Lung Capacity (TLC) Ratio (DLCo/TLC)
for the Beagles (mean value ± 1 SE; number of dogs In group Is In parentheses)
Groups
1
Control
DLCO 12.3
(ml/mm Hg/min) ±0.6 (12)
DLCO/TLC
(X1000)
Table 15.
11.3
±0.7 (12)
Inspiratory and Expiratory
2
R
12.4
±0.7
9.1
±0.6
Lung and
3
I
10.5
(8) ± 0.7 (5)
8.7
(8) ±0.9(5)
Chest Wall Quaslstatic
In group is
4 5
sox R + sox
13.3
±1.0 (8)
9.1
±0.8 (8)
11.8
±0.6 (10)
8.9
±0.5 (10)
Compliance Values for the
in parentheses)
6
I + SOX
11.2
±0.7 (11)
8.8
±0.7 (11)
Beagles (ml/cm
7
NOL + N02H
9.8
±0.6 (6)
7.1
± 0.5 (6)
H*0 ± 1 SE;
8
NOH + N02L
11.3
±0.7 (10)
9.0
±0.7 (10)
number of dogs
Groups
CM
OLE
Ccwi
CCWE
1
Control
39.6
± 4.0 (12)
65.7
± 8.0 (12)
154.9
±15.0 (12)
253.0
± 27.0 (12)
2
R
44.3
± 4.0
74.7
± 8.0
156.6
±20.0
150.0
±19.0
3
I
34.6
(9) ± 2.0 (5)
88.8
(9) ± 6.0 (5)
141.0
(9) ±15.0 (5)
144.6
(9) ± 19.0 (5)
4 5
SOx R + SOx
44.2
± 2.0 (8)
78.6
± 5.0 (8)
175.5
± 12.0 (8)
166.5
± 19.0 (8)
33.5
± 2.0 (10)
54.2
± 3.0 (10)
153.7
±18.0 (10)
232.5
±23.0 (10)
6
l + SOx
37.2
± 2.0 (11)
60.6
± 4.0 (11)
152.3
±13.0 (11)
158.7
±12.0 (11)
7
NOL + N02H
42.2
± 4.0 (6)
74.7
± 8.0 (6)
127.5
± 19.0 (6)
128.8
± 10.0 (6)
8
NOH + N02L
38.1
± 2.0 (10)
62.9
± 2.0 (10)
151.8
± 18.0 (10)
151.3
±16.0 (10)
-------
12 34 5 678
CA R I SOx R + SOx I + SOx NOL NOH
1600
1400
1200
Lung 1000
Vol.
(ml) 800
600
400
200
n
+ NO2H + NO2L
»
i
s
*
*
£
I*
i
*
3
i
E
*
z
E
4
i
— t—
i
^
*
i
*
*
i
3E
£
Figure 2
Mean values with standard errors for total lung capacity, functional residual volume, and
residual volume (top to bottom of each bar) for each group of beagles (* = significant^
C dyn
ml/cmHoO
60
50
40
30
20
10
10
20
30
50
frequency
(Cycles/mm)
Figure 3
Mean dynamic compliance values (Cdyn) at different breathing frequencies for each group of
beagles identified on the left of each Cjyn/f plot
137
-------
Table 16. Lung Volumes of the Beagles in the Control Group at the Termination of Exposure
(TE) and 2 Years After Termination of Exposure (2YR) Compared with Values from a Similar
Group of Beagles Previously Studied at DC-Da vis (External Controls).
Volume (ml)8
TE
2YR
External controls
TLC
VC
FRCN2
RV
979
819
297
163
1095
924
311
178
1028
932
373
209
aTLC = total lung capacity; VC = vital capacity; FRCN2 = functional reserve capacity
measured with nitrogen; RV = residual volume.
Table 17. Compliances of Lung (CLI&E) and of Chest Wall (Ccw) of the Beagles in the
Control Group at the Termination of Exposure (TE) and 2 Years after Termination of
Exposure (2YR) Compared with Values from a Similar Group of Beagles Previously Studied
at UC-Davis (External Controls).
Value
Units
TE
2YR
External controls
CLI
CLE
Ccw
ml/cm HbO
%TLC/cm H2O
ml/cm HaO
38.8
—
—
39.0
6.0
249
—
5.0
240
Table 18. Diffusing Capacity (DLCO). Dico/Total Lung Volume Ratio (Dico/TLC) and Total
Pulmonary Resistance (Rpui) of the Beagles in the Control Group at the Termination of
Exposure (TE) and 2 Years After the Termination of Exposure (2YR) Compared with Values
from a Similar Group of Beagles Previously Studied at UC-Davis (External Controls).
Value
Units
TE
2YR
External controls
DLCO
DLco/TLC(x1
Rpui
ml/min/mm Hg
O3) ml/min/mm Hg/ml
cm hteO/l/min
8.6
8.0
2.94
12.3
11.3
2.64
12.2
10.6
3.59
volumes, DLCO and CL with their 2YR values and analyzed each group's change with the
Student's t-test. The RV and inspiratory CL did not change in the control group but RV
increased (P<0.035 to P<0.0001) in all exposed groups except group 3 (I) (Figures 4 and 5).
TLC, VC, FRC and 1C increased (P<0.03 to P<0.0001) in all groups except group 3, but
increased the greatest amount in groups SOX, I + SOX, N02-high and NC^-low. DLCO and
increased (P<0.03 to P<0.001) in all groups except I and R + SOX.
Table 20 shows the measured and predicted TLC values for each group. The TLC predicted
TLC values were calculated with the equation TLC = 111.5 (B.W. kg), from the work of
Robinson and co-workers (7). It shows that the TLC's of all groups except the control, I and I
+ SOX increased 100 ml or more than predicted at 2YR. The dogs lost weight during the
post-exposure period, probably due to greater activity because of less restrictive caging. The
SOX, NC-L + N02H, and R groups had a large increase in TLC despite the decrease in body
weight
138
-------
Table 19. The Mean Pulmonary Function Values of the Beagles Immediately Following Termination of Exposure (TE) and 2 Years After Termina-
tion (2YR).a (Values that tend to be different between TE and 2YR are underlined. Standard errors where available are in parentheses)
Groups
1
Control
Function TE 2YR
CM 38.8
(ml/cm H2O)
Rpui 2.94
(cm H2O/1/sec)
DLCO 8.6
(ml/min/mm Hg)
DLCO/TLC 8.0
(ml/min/mm Hg/ml)
(X1000)
FRC N2 347
(ml)
ERV 142
(ml)
1C 727
(ml)
RV 232
(ml)
VC 922
(ml)
TLC 1102
(ml)
39.0
(0.4)
2.64
(0.3)
12.3
(0.59)
11.0
(0.7)
339.6
(15.3)
174
(21.1)
755.9
(45.2)
185.8
(14.0)
930.9
(56.5)
1095
(64.2)
2
R
TE 2YR
43.9 44.0
(0.4)
2.87 3.36
(0.1)
10.4 12.4
(0.73)
9.0 9.1
(0.6)
414 412.7
(17.6)
152 167.7
(19-5)
775 968.3
(52.5)
265 238.4
(7.2)
1000 1136.1
(64)
1188 1374.5
(69.2)
3
I
TE 2YR
45.0 34.0
(0.2)
3.14 3.06
(0.5)
9.36 10.5
(0.71)
9.0 8.0
(0.9)
369 396.2
(24.8)
149 181.2
(31.2)
719 838.6
(55.2)
220 228.6
(71.6)
926 1019.8
(75.6)
1087 1239.8
(90.1)
4
SOx
TE 2YR
41.5
2.85
9.3
8.0
370
159
773
212
966
1144
44.0
(0-2)
2.28
(0.2)
13.0
(0.99)
9.0
(0.8)
439.1
(19.9)
175.7
(27.6)
1011.0
(27.9)
263.4
(21-4)
1186.6
(46.4)
1450
(51.1)
5
R + SOx
TE 2YR
48.9
2.48
9.7
9.0
418
138
734
280
943
1139
33.0
(0.2)
4.50
(0.4)
11.8
(0.80)
9.0
(0.5)
401.4
(17.8)
162.7
(13.4)
836.6
(657.8)
238.3
(13.2)
1009.7
(67.8)
1248.1
JZL31
6
I + SOX
TE 2YR
47.7
2.53
8.7
7.0
376
137
790
239
999
1166
37.0
(0.2)
3.06
(0.2)
11.0
(0.68)
8.0
(0.2)
384
(15.5)
185
(18.1)
883.5
(42.4)
203.3
(8.6)
1068.9
(51.1)
1272.2
(55.1)
7 8
NC-L + NO2H NOH + NO2L
TE 2YR TE 2YR
48.3 42.0
(0.2)
2.72 1.92
(0.2)
7.7 9.8
(0.60)
7.0 7.0
(0.5)
402 429.0
(22.9)
167 174.0
(29.9)
782 905.1
(59.3)
235 280.0
(19.4)
969 1064.1
(91-8)
1183 1342.3
(97.0)
41.9 38.0
(0.2)
2.73 2.46
(0.1)
8.9 11.3
(0.67)
8.0 9.0
(0.7)
408 383.7
(11.0)
149 158.3
(18.4)
767 833.8
(43.6)
258 219.8
(24.1)
962 1042.1
(40.5)
1175 1262.5
(50.8)
aLewis et al. (6).
bCi_i = lung compliance; Rpui = total pulmonary resistance; DLCO = diffusing capacity;
Di_co'TLC = diffusing capacity/total lung volume; FRCN2 = functional reserve capacity; ERV = expiratory reserve volume;
1C = inspiratory capacity; RV = residual volume; VC = vital capacity; TLC = total lung capacity.
-------
350
300
250
200
Control
TE 2YR TE
RV
R+SOX* NOH + NO2L
2YR IE 2YR TE 2YR
ml
350
300
250
200
150
100
50
NOL+NO2H
l + SOv
R +
Figure 4
Changes in each dog's residual volume (RV) between termination of exposure (TE) and 2
years after termination of exposure (2YR). Bars joining x's represent mean change between
TE and 2YR for each group. Groups with * are those with significant increases in RV/TLC.
Those with + have significant increases in TLC.
140
-------
Inspiratory Lung Compliance (ml/cm HjO)
Control SOX I + SOX NOL+NO2H
TE 2YR TE 2YR TE 2YR TE 2YR
60
40
20
PX).5
P<0.001
P<0.035
P<0.025
Figure 5
Changes in each dog's inspiratory lung compliance (CO between termination of exposure
(TE) and 2 years after termination of exposure (2YR). Bars joining x's represent mean change
between TE and 2YR for each group.
141
-------
Table 20. Mean Total Lung Capacity (ml) of Each Group of Beagles at End of Exposure
(TE, column 1), 2 Years After End of Exposure (2YR, column 2), Change in Volume
Between TE and 2YR (column 3), Predicted Value from External Controls (column 4)
and the Difference Between Predicted and 2YR (column 5).
Groups
Control
R
I
sox
R + SOx
I + SOX
NOL+N02H
NOH + N02L
1
TE
979
1188
1087
1144
1139
1166
1183
1175
2
2YR
1095
1374
1235
1450
1248
1272
1342
1262
3
Change
116
186
148
306
109
106
159
87
4
Predicted
1028
1156
1168
1168
1096
1191
1108
1132
5
Difference
67
218
67
282
152
81
234
130
Discussion
Cardiovascular function: The cardiovascular values during and following exposure did not
show any clear-cut effects of the air pollutants. The small number of animals with evidence of
cardiovascular dysfunction by EGG, vectorcardiography and phonocardiography; the nonspe-
cific nature of the dysfunction; and the random distribution between groups makes the
conclusion of Bloch and co-workers (4) that there was an apparent association between air
pollutant exposure and positive diagnosis of cardiovascular disease equivocal The incidence
of vectorcardiographic (VCG) abnormalities (15.1%) in the exposed dogs compared to 0%
incidence in controls may suggest air-pollutant effects. However, the abnormalities were not
consistent nor were they seen with other functional or structural analyses.
Bloch and co-workers (4) showed that at the time of exposure to auto exhaust, the beagles had
high carboxyhemoglobin and small increases in hematocrit. Lewis and co-workers (6) proba-
bly correctly associated the increased pulmonary capillary blood volume in the auto exhaust-
exposed dogs with the high carboxyhemoglobin in this blood.
The cardiovascular function of all the groups of beagles studied 36 months after termination
of exposure was similar and also closely related to those values of healthy beagles reported in
the literature (20-23).
There appears to be no evidence of progressive cardiovascular disease in these beagles which
can be unequivocally associated with the air pollutants. In retrospect, complete cardiovascular
and pulmonary function measures on all beagles prior to inclusion in the study and more
careful studies on each individual dog at shorter intervals might have more clearly
demonstrated changes in cardiovascular function. This might have been particularly informa-
tive for those animals with demonstrable cardiovascular disease. For example, did any
cardiovascular dysfunction have its onset during exposure? Did the cardiovascular dysfunc-
tion worsen during the course of the exposure? Morphological studies might have confirmed
the functional evidence of myocardial lesions in individual, exposed dogs.
142
-------
Pulmonary function: These studies support the hypotheses that: 1) exposure to low levels of
specific air pollutants produces pulmonary injury and loss of pulmonary function, and 2) the
functional loss continues following termination of the exposure. We evaluated our results in
these ways: 1) comparison of internal control values to those of healthy beagles (external
controls) of similar size and age, 2) comparison of each group's values 2 years after
termination of exposure to their values immediately after termination, 3) comparison of each
group's values to all others, and 4) comparison of each individual's values 2 years after
termination of exposure to their values immediately after termination.
Comments and conclusions on group 1: The control (group 1) pulmonary function values
appeared to remain nearly the same during the 2-year post-exposure period and were similar
to those of other healthy beagles previously studied in our laboratory (7, 8, 9). There is
evidence that many pulmonary function values of dogs relate in a predictable way to body
weight (7,24,25) and/or within breeds to age (8, 9). There were no differences in breed, body
weight, or age among the eight groups of dogs, and we concluded that these factors were not
responsible for the differences in pulmonary function measured between control and exposed
groups. There were no consistent or widespread pathological lesions in the lungs of the
control group (26, 27). We concluded that group 1 was an acceptable representation of the
healthy beagle population, and was a satisfactory control for study of the effects of air
pollutants on our seven exposure groups.
General comments on exposed groups: All groups exposed to air pollutants (groups 2-8) had
differences between their 2-year post-exposure pulmonary function values and their values
immediately after exposure, and many of those of the controls (group \). We did not find a
clear pattern of pulmonary functional loss in the exposed groups (#2-8), although some trends
were evident. In general, automobile exhaust (R or I) exposure appeared to injure airways and
parenchyma, whereas SOX, NO, and N02 caused injury to the parenchyma. Combinations of
auto exhaust and SOX did not appear to augment specific functional losses caused by each
single species of pollutants. At 2YR there were only five dogs in group 3 (I) and this sample
size was unfortunately too small for meaningful comparison to other groups.
The functional abnormalities correlate well in most instances with structural changes in the
exposed groups (#2-8) (26, 27). The groups exposed to raw automobile exhaust, R (group 2)
and R + SOX (group 5), showed substantial atypical epithelial hyperplasia in their small
airways. Although less extensive, these same lesions were seen in the other exposed groups.
The groups exposed to higher concentrations of N02 (group 7) and SOX (group 4) had the
greatest airspace size and parenchymal damage as shown by several Quantitative morphomet-
ric measures. The effects of the air pollutants upon the lungs of the exposed groups are the
likely cause of the differences in pulmonary function values between exposed and control
groups.
Because of the uncertainty of the effects of anaesthesia upon the ventilation of our beagles, it
is difficult to assess the differences measured in PacCb pH of arterial blood (pHa), and VD. All
the beagles were given about the same dosage of anaesthesia, and all were in light surgical
anaesthesia as judged by their weak pedal pain response (21, 22). Since anaesthetic levels
appeared comparable in all groups, and the control ventilatory values are at one extreme of
those of all groups, the ventilatory differences in the exposed groups from control are
143
-------
probably the result of lung damage resulting from exposure to air pollutants. This view is
supported by morphological changes in the exposed lungs and by pulmonary volume and
mechanic values which differed in the exposed from those of controls and which probably are
not affected by anaesthesia.
Comments and conclusions on beagles exposed to auto exhaust: Groups R and R + SOX had
similar pulmonary function response with abnormalities in ventilatory, resistance, blood
gas-exchange, and lung volume values. Group 5 (R + SOx) had the highest PacCtei % VD, Rrs,
Rcw, and Rpul of all groups, which suggests these dogs had the greatest airway change. This
group's Cdyn/f slope was not different from that of the controls. For technical reasons, we
were unable to measure Cdyn at f s greater than 50 cycles/min. This may explain why we did
not measure a greater change in Cdyn with increased f in this group.
The ventilatory values (PacCfe, pHa, %Vo, Cdyn/f, and VD) of the R group were similar to
those of R -t- SOX group, and their Rpui increased from 2.87 to 3.36 cm H20/l/sec and 2.48 to
4.50 cm H20/l/sec, respectively, during the 2-year post-exposure period while that of other
groups remained unchanged. This suggests there was some progressive airway disease in
these groups.
In all groups except 1 (control) and 3 (I), there was functional evidence of air-trapping. In
groups 2 and 4-8 the RV's increased between TE and 2YR and in groups 4 (SOx), 5 (R 4- SOx)
and 7 (N02-high) RV/TLC's increased in the post-exposure period. The TLC increased over
150 ml during the 2-year post-exposure period in groups R, SOX, and N0£-high. Most of the
R + SOX -exposed dogs had a high RVfTLC ratio at the end of the exposure period, and 6 of
the 10 dogs in this group had RV7TLC values greater than mean control (0.17 ± 0.02 SD)i
The air-trapping in the R and R + SOX groups probably is the result of airway damage with
little or no parenchyma! injury; that in the SOX and N02-high is the result of parenchyma!
injury.
The I + SOX group's and, to a lesser extent, I group's pulmonary function values were similar
to those of R and R + SOX. The CLI increased in the 2-year post-exposure period in
individuals in groups exposed to SOX, N 62 -high and I + SOX, probably signifying a change
in the lung parenchyma of these dogs.
The DLCQ values for the control beagles at 2YR and the external control beagles were the
same and the 2YR DLCQ'S for all beagles except those in group I were greater than TE. This
apparent increase in DLCO between TE and 2YR is probably due to differences in technique
at Cincinnati (TE) and UC-Davis (2YR). The DLCO /TLC ratio increased between TE and 2YR
in all beagles except those in groups I, SOX and R + SOX. Despite the probable increase in
DLCQ values due to experimental differences between TE and 2YR, the increase in DI^Q in
groups SOX and R + SOX did not keep pace with the increase in TLC, suggesting
parenchymal damage and a real decrease in pulmonary diffusing capacity and/or increased
mismatching of ventilation and perfusion.
Literature review on auto exhaust exposure: We know of no other comparable studies in
which the structural and functional effects of auto exhaust alone or with SOX have been
studied during or following long-term exposure. Murphy and co-workers (28, 29) studies were
on rodents exposed for brief periods (2 to 6 hr) at high levels of auto exhaust from which they
reported increased total pulmonary resistance, increased mortality in the Antu-injected mice,
144
-------
and decreased activity during exposure. Other investigators have studied the effects of urban
smog, a large portion of which is associated with auto exhaust One of these studies by Reif
and Cohen (3) showed significantly more radiological evidence of lung disease in urban dogs
of 4 years and older than rural dogs of the same age. They concluded that the differences
were probably due to air pollutants. Their findings in dogs exposed to urban air pollutants for
a relatively long period lend support to our experimental results upon dogs exposed for 5
years. Swann and Balchum (31) studied the effects of Los Angeles smog upon total expiratory
resistance of guinea pigs and reported an increase when the oxidant levels were high. They
did not measure other respiratory functions, nor did their study address the long-term effects
of exposure. Kagawa and Toyama (32) concluded from their studies on children exposed to
Tokyo air pollution during a 6-month period that ozone, hydrocarbon, or S02 may affect
upper airways, NO and the lower airways, and temperature may affect both upper and lower
airways. They do not specify what they mean by "upper" or "lower" airways, nor would their
studies specifically detect parenchymal damage.
Comments and conclusions on SOX, NOL + N02H, and NOn + N02L exposure: The SOX
group had higher TLC, RV, 1C, VD, and Cdyn If values and a lower DLCO /TLC value compared
to those of controls. In addition, this group's lung volumes and RVATLC increased while
DLCO /TLC remained unchanged between TE and 2YR. This group had the most exaggerated
lung volume increase (TLC and RV), which correlated well with morphologic findings of
enlarged parenchymal airspaces (26, 27). SOX and NOL + N02H groups had substantial
increases in their lung volumes during the post-exposure period. The effect of long-term
exposure to SOX seemed to be upon the parenchyma, although we detected only a very slight
increase in CLI during the post-exposure period (41.5 to 44.0 ml/cm r^O): The SOX, NOL +
N02H and NOn + N02L groups had the greatest Cdyn/f values, and in general all of their
volume and ventilatory values were similar. Their resistance values were like those of the
controls, and their most remarkable abnormalities were in lung volumes and DLc0/TLC
measurements.
Like the SOX group, NOL + N02H and NOn + N02L groups showed the most remarkable
functional and structural changes in the parenchyma (26, 27). Their structural abnormalities
— hyperinflation, increased number and size of alveolar pores, and tissue destruction — are
similar to the naturally occurring emphysematous lesion that Gillespie and Tyler reported in
horses (33).
Literature review on SO*, NOL + N02H and NOn + N02L exposure: The correlation of our
results from SOx, NO, and N02 exposures and those of others is not clear-cut due in part to
differences in species studied; level, method and duration of exposure; and methods of
evaluation. Others have not reported evidence of parenchymal damage like that in our
SOx-exposed dogs. Frank and Speizer (34) reviewed S02 exposure effects in dogs, guinea pigs
and human subjects and reported increased nasal and total pulmonary resistance at S02
levels of about 25 ppm for 20 minutes in their dog experiments. Most other studies reviewed
(35-39) were for short periods using S02 levels (> Ippm) higher than those in our study, and
some showed increased total pulmonary resistance during SO 2 exposure. Alarie and co-
workers (40) exposed monkeys and guinea pigs for 78 and 52 weeks respectively to a variety of
combinations of S02, H2S04, and fly ash. They reported the greatest change in pulmonary
structure and function in those animals exposed to H2S04 in concentrations as low as 0.1
mg/m3. There was considerable variation in their pulmonary function values, and only the
pulmonary flow resistance appeared to have changed in their monkeys as a result of H2S04
145
-------
exposure. Thomas and co-workers (41) exposed guinea pigs to 4 mglm6 H2S04 aerosol for
periods of up to 140 days. He used three particle sizes, 0.6 mm, 0.9 mm, and 9 mm. The
largest particles affected only the upper respiratory tract. The 0.9-mm particles produced the
greatest amount of damage — slight edema, desquamation of epithelium in minor bronchi,
and reduced amounts of mucus along the bronchial tree.
Freeman and co-workers (42) reviewed the studies on the effects of N02 and described the
parenchymal damage that they and others have seen in animals following exposure. Our dogs
were exposed to comparatively low levels of NO and N02, but appear to have lung damage
similar to that reported by others (42, 43, 44).
Pulmonary function studies upon our dogs immediately after the 68-month exposure period
demonstrated modest pulmonary function changes. When these dogs were studied 2 years
after the termination of exposure, many of their pulmonary function values showed an even
greater difference from those of controls. The controls appeared to have stable pulmonary
function values similar to those of other healthy beagles. Since these dogs were handled the
same except for exposure and were of the same age and breed, we have concluded the
differences in function and structure measured are due to the effects of the experimental
exposure regimes.
We find the evidence for continued loss of function after termination of pollutant exposure in
our dogs rather convincing since it was consistent in all groups exposed to pollutants and
absent in our controls. If true, this finding should be taken into account in the design of air
pollutant exposure experiments, since one may miss the effects of exposure if the animals are
killed and studied soon after the termination of the exposure period. If changes resulting
from exposure do require time to develop, and for that reason have been missed by
investigators, this may explain in part the differences reported in the epidemiological studies
of natural air pollution disasters and those from experimental exposure.
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pulmonary function studies in normal children, ages 4-13. Am. J. Dis. Child 110: 144.
20. Ettinger, SJ., and RE Suter. 1970. Pp. 126-186 in Canine cardiology. W.B. Saunders Co.,
Philadelphia.
21. Steffey, E.P., J.R. Gillespie, J.D. Berry, E.I. Eger, and E.A. Rhode. 1975. Circulatory
effects of halothane and halothane-nitrous oxide anaesthesia in the dog: Spontaneous
ventilation. Am. J. Vet. Res. 36: 197.
22. Steffey, E.P., J.R. Gillespie, J.D. Berry, E.I. Eger, and E.A. Rhode. 1974. Circulatory
effects of halothane and halothane-nitrous oxide anaesthesia in the dog: Controlled
ventilation. Am. J. Vet Res. 35: 1289.
23. Whipple, H.E., and RE. Van Rege. 1965. Comparative cardiology. Ann. N.Y. Acad. Sci.:
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24. Spells, K.E. 1969. Comparative studies on lung mechanics based on a survey of literature
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Morphometric evaluation of lungs using automated image analysis. In Proceedings of 6th
Conference on Environmental Toxicology, Dayton, Ohio.
27. Hyde, D., A. Wiggins, D. Dungworth, W. Tyler, and J. Orthoefer. 1976. Automated
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28. Murphy, S.D., J.K. Leng, C.E. Ulrich, and H.V. Davis. 1963. Effects on animals of
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29. Murphy, S.D. 1964. A review of effects on animals of exposure to auto exhaust and some
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30. Reif, I.S., and D. Cohen. 1970. Retrospective radiographic analysis of pulmonary disease
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31. Swann, H.E., and OJ. Balchum. 1966. Biological effects of urban air pollution. Arch.
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32. Kagawa, J., and T. Toyama. 1975. Photochemical air pollution, Its effects on respiratory
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33. Gillespie, J.R., and W.S. Tyler. 1967. Capillary and cellular changes in alveolar walls of
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34. Frank, N.R., and EE. Speizer. 1965. S02 effects on the respiratory system in dogs. Arch.
Environ. Health 11:624.
35. Balchum, O.J., J. Dybicki, and G.R. Meneely. 1960. Pulmonary resistance and compliance
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36. Yokoyama, E., and K. Ishikawa. 1962. The effects of sulfur dioxide upon mechanical
properties of the lungs in dogs. Jap. J. Indust. Health 4: 22.
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38. Amdur, M.O. 1959. The physiological response of guinea pigs to atmospheric pollutants.
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39. Hirsch, J.A., E.W. Swenson, and A. Wanner. 1975. Tracheal mucous transport in beagles
after long-term exposure to 1 ppm sulfur dioxide. Arch. Environ. Health 30: 249.
40. Alarie, Y.C., A.A. Krumm, W.M. Busey, C.E. Ulrich, and RJ. Kantz. 1975. Long-term
exposure to sulfur dioxide, sulfuric acid mist, fly ash, and their mixtures. Arch. Environ.
Health 30: 254.
41. Thomas, M.D., R.H. Hendricks, ED. Gunn, and J. Critchlow. 1958. Prolonged exposure
of guinea pigs to sulfuric acid aerosol. Arch. Indust. Health 17: 70.
42. Freeman, G., S.C. Crane, RJ. Stephens, and NJ. Furiosi. 1968. Pathogenesis of the
nitrogen dioxide-induced lesion in the rat lung: A review and presentation of new
observations. Am. Rev. Resp. Dis. 98: 429.
43. Freeman, G., S.C. Crane, NJ. Furiosi, RJ. Stephens, MJ. Evans, and W.D. Moore. 1972.
Covert reduction in ventilatory surface in rats during prolonged exposure to subacute
nitrogen dioxide. Am. Rev. Resp. Dis. 106: 563.
44. Haydon, G.B., G. Greeman, and N J. Furiosi. 1965. Covert pathogenesis of N02 induced
emphysema in the rat. Arch. Environ. Health 11: 776.
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Asilomar Conference Discussion
Gillespie: The difficulty I see in Bloch's studies, and I have some tables that will show you
this, is that although there were individuals in various groups that had some suggestion of
abnormality in cardiovascular function, they were not consistent. The highest number of
repeats was two, in which they consistently found abnormalities, and the third test often
would be back to normal on individuals that were previously abnormal. The other problem I
have accepting that the cardiovascular function can be related to air pollution was that they
were not uniform-type abnormalities in the five affected. One finds, for example, a variety of
abnormalities in a particular group, and the highest number of individuals was four in the
raw automobile exhaust and there were three that had similar function abnormalities. But
again, the abnormalities were not substantiated by other tests. Earlier there was some
question about whether the pulmonary artery pressure or wedge pressure was high; they were
not, in the same individuals. They lacked back-up data on many of these individuals to show
that they were indeed abnormal and remained abnormal while in the exposure chamber.
Stara Obviously some of the observations by Bloch may be subjective to a degree. But in a
population of beagles, particularly the inbred beagle at that age, would you say that such
cardiovascular abnormalities as reported by Bloch et aL would occur normally?
Gillespie: The 15% which they report from adding up all beagles with abnormalities in the
study is higher than you would expect to see in the beagle colony at Davis or anywhere else.
However, it is difficult for me to compare his abnormal values with those of others because 1)
he includes very mild abnormalities, 2) repeat tests were not consistently abnormal, and 3)
confirmation of abnormalities by the same or other tests were not reported. The problem with
that is most of the data that you see, for example Detweiler's data on abnormalities in dogs,
identify a specific lesion and give a probability in that group, so, I'm not sure that if one
added up all the cardiac abnormalities that are possible, the total percentage might approach
15%.
Gillespie: . .. Expiratory compliance matched reasonably well with our external controls, and
the chest wall compliance, again, matched remarkably well with our external controls.
Diffusing capacity — Dr. Lewis has already mentioned that our values are consistently above
his. As I remember, you mechanically inflated the lungs with negative pressure around the
dog's body. I'm not sure that I would agree that would necessarily decrease diffusing
capacity; I think that would increase pulmonary capillary blood volume and diffusing
capacity. But for whatever reason, and I believe it probably is technical, we have different
values than Cincinnati.
Brain: Theirs were done with negative pressure and yours were done with positive pressure?
Gillespie: 'Yes, that's right.
Lewis: When we did it with positive pressure at that point in time we got comparable results.
Positive pressure opens up airways and what-have-you that may normally be collapsed.
Brain: Given the same transpulmonary pressures, I don't think the lungs will behave very
differently. I see some possible differences in the pulmonary vascular bed.
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Lewis: Pressures are distributed, I think, more uniformly across the lung when you do it
passively than when you do it by positive pressure because you have a head pressure driving
it through when you inflate the lung.
Brain: I don't think there's much evidence for that. If you plot lung volume versus
transpulmonary pressure, the curves are not very different whether they are obtained with
positive or negative pressure. There are differences in the pulmonary circulation, and I would
suspect that's what might be involved.
Gillespie: I would think it would be the opposite direction. I have expected their values to be
greater than ours, based upon predicted effects of their negative inflation technique. I suspect
there probably was something else in different techniques. Certainly we couldn't ask for a
better matching of diffusing capacity values for our external and internal controls. And again,
the ratios of diffusing capability and TLC are greater at Davis, because diffusing capacities
were greater.
Gillespie: Three groups — SOX, I + SOX, and N02-high — had increased compliance
following the 2-year post-exposure period. All three groups had significant parenchyma
lesions.
Thurlbeck: At what proportion of TLC did you measure compliance?
Gillespie: Compliance was measured at just above FRC. So, yes, it would be more like 40% of
TLC.
Brain: After coming down from TLC?
Gillespie: That's right We feel that the animals that were exposed to SOX and NO 2 had
the most substantial change functionally at the parenchyma! level Those exposed to automo-
bile exhaust had evidence of damage, both in the airways and the parenchyma. And the thing
that we also found remarkable, and I guess maybe I should just read my conclusion, is that
the abnormalities continued to occur and progressed outside the exposure chamber. And we
feel that these are pretty well substantiated in that the controls' pulmonary function values
were not abnormal and every exposed group showed some evidence of continued functional
loss in the pulmonary system post-exposure.
Albert: Very good. I really didn't quite get clear which exposure pattern had the most
damaging effect on the lung?
Gillespie: Well, I suppose the two groups I think had the most damaging effect on the lung
are the N02-high and the SO x, from our data. It is unfortunate that our I group, the
irradiated exhaust, ended up being so small. The next two groups in order would be R + SO:*,
then SOX, as far as damage. But all exposure groups had some functional abnormality.
Lewis: In your paper you talk about fast lung volumes. I don't see those data in the paper, nor
did I see the data on the slides in your presentation. Is this not a small airway-type or airway
diagnostic tool?
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Gillespie: The fast lung volume that we were interested in was resistance. In other words, you
have to have the equipment to measure fast changes. The slow ones are, of course, something
like compliance, where you inflate and deflate slowly, and all of our lung volume measure-
ments were made with a slow procedure, so you could use a box and spirometer for those. For
measurement of rapid volume changes, we used a tuned flow box. We did not measure
maximum expiratory flow, for example, or peak flows, because I frankly don't think you can
measure them unless you can do a tracheostomy and put a tube in the tracer within the chest.
One can't do these adequately with endotracheal tubes because one will only measure the
peak flow of that endotracheal tube. It's been shown that the maximum flow you can get
through a 9-mm endotracheal tube is about 1 to 2 liters per second. If you do a tracheostomy,
put a copper tube in the airway, you will find that a dog of beagle size will have a peak flow of
approximately 6 liters per second. So we couldn't do tests on these dogs because they had to
survive other tests.
Brain: Do you have data for the entire volume-pressure curve?
Gillespie: Yes.
Brain: You haven't said anything about them. I'm wondering, is there anything to be learned
from them? For example, do they tell you about changes in alveolar stability? If you compare
the volumes at 25 and 5 cm of water or between 25 and 2V6 are those ratios any different? Is
there any suggestion that stability has been affected?
Gillespie: That's a good idea. We didn't look at that, and we certainly could. We took the
residual volumes and we identified functional residual capacity positions on the curve. And
we have looked at the transpulmonary pressure, which is getting close to what you want, at
functional residual capacity, and there was no difference between the two.
Brain: It might also be fun to plot the deflation volume/pressure curves as a percent of the
TLC in order to look critically at the shape.
Gillespie: I did that with some of the groups because, again, that's the way we reported the
normal beagle data in order to get the comparisons. Again, there were no differences.
Brain: What was done to the excised lobe?
Stara: That will be reported later.
Stephens: Hindsight is always better than foresight, as everyone recognizes, but I think it's
unfortunate that you didn't do the physiology on the dogs that did have the pneumonectomy
involved. It might have been valuable. At SRI, Dr. Freeman did some pathology on those
lobes that were excised, and I did some ultrastructural work and light microscopy as well. We
did not do the pathology on the recovery animals. I think Dr. Dungworth and Dr. Hyde
worked on those, both on the scanning and light microscopy levels. In reading the abstracts
and parts of the papers last night, I can see we use different tools, but it will be interesting to
compare the data.
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Gillespie: The problem I had is that it's awfully hard to take the same amount of lung each
time you do a pneumonectomy, plus a lot of the mechanical studies we wanted to do depend
upon there being an intact or normal pleural space which you're not likely to have following
pneumonectomy. In other words, there's almost sure to be some adhesion.
Kleinerman: You only studied the dogs once?
Gillespie: Right.
Kleinerman: Is it possible, though, since you have some comparison between your studies and
the studies of Trent, that you could extrapolate back to when the first reliable changes
occurred in pulmonary function, either in compliance or in DL<-O, in these animals?
Gillespie: Well, the biggest change seems to have occurred between the termination and the
2-year post-exposure tests.
Lewis: The problem is we didn't have enough parameters at 18 months to make the diagnosis
that we could at 36 months. It's a very difficult thing to do, if not impossible. We only have
three parameters — diffusion, resistance, and compliance at 18 months — that we could
compare over the whole time frame, but even then, we don't have a pre-exposure value.
Kleinerman: Well, we're not talking about pre-exposure. We are talking about the changes
during the period of exposure. But you have those three parameters. Is there any evidence
that they change when you look at the data?
Albert: You said that at 36 months, 5/12 of the N02-high animals had a low diffusion capacity.
Lewis: That was the one and only thing that we could demonstrate at that time.
Kleinerman: But is that consistent in subsequent findings?
Lewis: Yes, at the 36- and 61-month time periods. But it would have been nice if we had had
data on TLC for instance at 18 months to see whether changes had occurred at that point, but
we didn't have this TLC data. I did want to bring out something that Dr. Stara asked me to
talk about, and I skipped over it in trying to wrap things up. Let us dwell a little more on the
case of the oxides of sulfur and perhaps their role in the etiology of cardiovascular disease, I
have to somewhat agree with Dr. Gillespie on the cardiology data. I think Dr. Bloch went to
great lengths to perhaps demonstrate that there was an effect of air pollutants on the
cardiovascular system. But many of the criticisms that you have expressed I have weighed
heavily over the years. Yet, some of the cardiac symptomatology has been confirmed, one
which was validated both by enzymes and vectorcardiology and one which was diagnosed only
by electrocardiography. These occurred one in R + SOX and one in I + SOX. Some years ago I
did a study of sulfuric acid alone. The level we used was the federal industrial air standard of
1 mg/m3. It didn't mimic the 40-hour work week, but it did mimic continuous exposure.
Although no cardiology was done, one of the significant findings that we found was both a
smaller lung size, mass, volume, etc., and also a smaller mass of the heart which was
significantly different in dogs exposed to sulfuric acid mist.
Brain: Is that an absolute organ weight, or an organ/body weight ratio?
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Lewis: That's an absolute organ weight. There were no differences in body weight.
Brain: So there was a decrease in organ weight?
Lewis: Right There was an atrophy apparently of both the lung and heart I don't know if it's
a pharmacologic response to a sulfate or not I'm not building a case that oxides of sulfur
cause cardiac disease. I just say that there's evidence within my own camp to suggest it.
Albert: Did those dogs show confirmatory evidence on necropsy of having had myocardial
infarction?
Lewis: I didn't necropsy them personally, our veterinary pathology staff did. Once again, I
don't know whether it was observed or not. The answer to your question is that no myocardial
infarctions were recorded on gross autopsy.
Orthoefer: One of his myocardial infarctions was done, it was one of the trauma dogs that had
died early. Dr. Bloch said it had a myocardial infarction, and pathology never confirmed it
The animal died within hours; at that time I don't think special stains would even have shown
in infarction; however, they might now.
Lewis: I'm not sure that's the same case, because in his findings the concluding remarks for
both dogs are "the animals gradually recovered."
Orthoefer: One of the animals that died supposedly had a myocardial infarction.
Dungworth: None of the animals that were done at the time of the scheduled killings had
evidence of myocardial scars. All dogs by 9 years of age have a triad or lesions in the heart,
usually of a mild degree. These are muscular arteriosclerosis in small arteries, minimal foci of
myocardial fibrosis and the so-called mucoid degeneration of A-V values. Other than minimal
variation in those features, there was no specific evidence of previous infarction. But that
doesn't rule it out either, because the chances of finding it are slim after a considerable
length of time, particularly if it were a small infarct.
Stara: The dog in question died eventually during the night, and it was found the next
morning. We couldn't do satisfactory pathology.
Orthoefer: I really don't remember that particular case.
Lewis: I will repeat the statement I made the other day, namely that we could well have
missed the animals that had heart disease because they were the ones that were most
vulnerable to fighting and trauma and death.
Orthoefer: Actually, if we had Dr. Bloch's specific data, it's possible to go back and check out
each incident We've got to know the exact dog.
Stara: I think we should have had Dr. Bloch here. I tried to reach him and could not. He was
the one who did the work, and we permitted him to take the data with him to write the papers.
It was a massive amount of raw data. At the time, it was questionable if we should copy it all.
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8. REVIEW OF RADIOLOGIC STUDIES
D. Dungworth
I'm not going to spend much time talking about the radiographic studies, because we can't
reach satisfactory conclusions from them. There were two radiographic studies performed
about 4 months apart: the first, 18 months after the dogs had arrived in Davis, that was in late
fall of 1972; and the second, 4 months later in January-February, 1973. The main value of the
radiographic studies, in fact, was to point out to us that if we were to provide any decent
morphologic conclusions or evaluation, we would have to provide better definition of the
lesions and, even more importantly, because of overlap of abnormalities found in the control
and experimental groups we realized that we would have to apply numbers to the morpholo-
gic findings to enable statistical analysis. That stimulated us to develop the morphometric
aspects, which later Dal Hyde will tell you about. The problem as far as the radiographic
studies are concerned is that there is a background noise even in control beagles, as there is
in any aging animal. This consists mostly of an increasing interstitial pattern which results
from peribronchial and perivascular fibrosis. I am not going to present illustrations because
the lesions were relatively minor, and they would not show up particularly well on the screen.
The radiologist took the radiographs (lateral and ventrodorsal views) from the second series of
examinations and in a blind study, assigned them to one of three grades of prominence of
interstitial patterns (1 + through 3+ \ We felt unable to make clear-cut interpretations from
these data because of the small number of dogs, the overlap among the treatment groups, and
the subjectiveness of the grading. The only other finding from radiology examinations was
that there was a high frequency of hip dysplasia in both control and exposed beagles.
Asilomar Conference Discussion
Question: Were these lungs inflated to TLC?
Dungworth: These were done unanaesthetized because they were small dogs and because we
didn't want to jeopardize their continued life. We told the radiologists that these were
extremely important animals. Because there had been some anaesthetic deaths, we advised
against anaesthetizing for radiography. They were done in the standard manner, at maximum
inspiration. To put the grading of pulmonary abnormality noted radiographically into
perspective, I should point out that the range of density was a small one and even the 3 + was
less severe than the increased density that is seen in the control aged beagle population at the
University of California, Davis, that lives outdoors in a somewhat dusty environment These
beagles were indoors, not in a dusty environment Even a 3 + is, in relative proportions, mild.
Question: Are those percentages?
Dungworth: Those are percentages within the various categories and these are the numbers
of beagles in each treatment group. We didn't bother getting statistical analysis on these,
because there's too much variation, there's too much operator error. If we take these at face
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value, which we can't, there is no correlation with the subsequent morphologic and physiolo-
gic findings. This group here, the R and the R + SOX, as Dr. Hyde will tell you, had the most
obvious bronchiolar cell proliferations with no emphysema. The N02-high and SOX groups
here, these, as Dr. Gillespie told you, had the most evidence of parenchymal damage
physiologically and, in fact, they were the two most severely emphysematous groups. Here's a
group that with respect both to emphysema and proliferations in small airways were relatively
close, but look at the difference in radiologic findings.
Orthoefer: It appears that probably the most severely affected was not as severely affected as
normal animals in an outside environment.
Dungworth: Well yes, with respect to interstitial fibrosis, that is. We're looking at background
noise here. The radiologists weren't picking up the lesions that subsequent morphological
examination proved to be important, and interstitial fibrosis was not among these.
MacEweru Did you say that the Dr. Morgan paper on hip dysplasia was from measurements
made on these particular animals, but that the dysplasia was not correlated with exposure
treatment?
Dungworth: Yes, it was looked at, but there was no difference between the frequency with
which it was found in control animals and in the exposed. It was also about the same
percentage frequency in the control beagles at Davis which came from a different breeder
and so on.
Kleinerman: I think emphysema or a destructive disease in man is a difficult disease to
diagnose by radiology, by the usual chest x-rays. You really can't make a diagnosis until late
in the course of the disease. But I think most radiologists who do work on the chest in humans
attempt to standardize a means by which they take a film and they have extreme quality
control about the usability of the Roentgenogram. Also, I think they have the added
advantage of having a steady state in which they take the film at full inflation. If you had both
of these things available, a quiet chest with full inflation and good quality control of the type
of radiograph that you took, would you feel that you could get more information from these
films?
Dungworth: The quality control is good, the quality of the actual plates is good. The problem
of controlled inflation we were aware of, but we did not want to anaesthetize the animals.
Because this lesion is relatively mild compared to the severe cases in man, I would surmise
that even if they had been anaesthetized and at a standardized inflation volume, we would not
have found anything definite. Unless perhaps it is argued that we could have got numerical
data by putting these films, assuming everything was standardized, on some sort of densito-
meter. We talked about this, but we felt it would not be justified. It is necessary to be aware, if
I may use a jargon phrase, of cost-benefit analysis. You know there are some things that are
going to be much more likely to produce useful information than others, and if you can't
afford to follow all avenues and don't have the time and personnel to do them all, then you go
with those having the highest probability of providing useful information. And on that basis,
we decided it wasn't worthwhile to put a tremendous amount of effort into the radiographic
studies and probably not come up with anything. That was before we saw the lungs
morphologically. I don't think in hindsight I would have changed my mind.
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Albert: Was there consistency in the x-ray findings for given dogs, e.g., if you found a 3 +
abnormality in the first x-ray, did you find the same in the second x-ray?
Dungworth: We didn't attempt to do it twice by blind grading. The radiographs taken 4
months apart were compared for each dog, and no significant change was noted.
Albert: The reproducibility of the observations of the abnormalities would give confidence in
the results, I would think.
Tyler: Basically, we have been trying to follow radiographic changes in monkeys not exposed
and it's really interesting how those radiographic changes come and go. The consistency is
the interesting part or the change in the lung. We worked hard at it for about 6 months I
would say. We just weren't able to identify with tissue methods what the radiologists were
describing. Radiologists can make some very nice descriptions based on pictures, descriptions
of those radiograms. But correlating those with the tissue in our hands has been unsuccessful
in other attempts. We thought, why push that issue at this point in time with these valuable
dogs because we were unsuccessful in the past.
Dungworth: Again, I think the effect was not recognized. Where I put the 3 -I- abnormality
was still less radiographically than in some of the controlled beagle population kept outdoors.
We are really talking about a fairly low spectrum of changes. It's not as though massive
fibrosis or translucence isn't obvious. Had they been, then I'm sure we would have gone into
it more carefully.
Thurlbeck: Are you telling us that a 3+ radiologic lesion, shown to a human radiologist,
would be considered normal?
Dungworth: I'm saying we see this sort of thing in older people. That's exactly what I meant.
It's impossible to come up with an absolute grading. It's a relative grading among these
animals.
Nettesheim: Considering these rather minor looking physiological changes, you wouldn't
expect that they could see anything in the x-rays.
Thurlbeck: It's worrisome that two of the animals have a 3 + change. That signifies to me
terrible looking x-rays, an equivocal change that I understand.
Dungworth: Right, and that's what I meant when I pointed out that it's a relative grading, so
when he put them into three piles he had three piles. The worst pile is a very mild lesion and
is commonly seen in older dogs.
Thurlbeck: Why go through the radiology exercise anyway? But of course the reason you do
it — you never know before you start what you'll find.
Question: How many piles would one have in the clean air, if he had three from each dog?
How many would your radiology department have?
Dungworth: Depends on what you mean by clean air. If they are in a dusty environment,
where we get the same amount of dust picked up by a summer wind, in a 9- to 12-year- old
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population there would be 15 to 20%. None of them would be absolutely normal, relative to
6-month-old, perfectly healthy, out-of-the-colony beagles.
Question: But how many categories does your radiology differentiate?
Hueter: 0 to 3, 0 to 10, 0 to 4.
Dungworth: No. Remember this was an arbitrary scale that he devised to look at these
particular 86 beagles. If you ask me if he took the same age animal from the control
population at Davis, how many would have been in the 3+ ? I would say 20% or more.
Question: I was wondering if it was 4 +, 5 + and whatever?
Dungworth: No, this didn't relate to the clinical appreciation of disease. It's purely an
arbitrary thing that related to the relative abnormalities seen in this particular group of
beagles. The emphasis here and throughout the study, particularly in the early phases, was
looking for differences among groups.
Albert: From a practical standpoint, the issue is whether you would recommend that periodic
x-rays be done in subsequent chronic studies knowing that the pulmonary function studies
involve anaesthesia which has an inherent risk and that x-rays may also have risk but
probably a much lower one.
Dungworth: It depends on the intensity of insult. It just so happens that in this particular
study the insult was relatively low and the radiographic technique that was used was not
sensitive enough to pick it up. You have to do it once at least and see about where you are on
the spectrum of damage according to the level of insult.
Lewis: At least not only the level of insult but the material to which a subject is exposed —
let's say if I was doing dust exposures, e.g., coal dust — I would like to have radiographic
examinations.
Thurlbeck: X-rays are not worth doing for the detection of early small airways disease or
minor degrees of emphysema. If on the other hand, part of the results that may be seen might
be due to intermittent pneumonic episodes Let us speculate that these dogs got bits of
pneumonia or interstitial disease of various sorts that came and went. Perhaps the answer is
to do the radiology more frequently. I'm not sure how difficult this is but perhaps one should
do 3-monthly x-rays as part of the physical examination of the dogs to let you know what is
going on from time to time in the chamber.
Dungworth: If you have a radiology unit with everything on hand I would agree.
Orthoefen I think there have been radiographic studies done of urban versus rural dogs.
There were differences shown. This is a lot like the dogs in this study versus radiological lab
dogs.
^Reif, L.S. and D. Cohen. 1970. II. Retrospective radiographic analysis of pulmonary disease
in rural and urban dogs. Arch. Environ. Health 12: 698.
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Gillespie: Reif and coworkers' studyl in Philadelphia shows that downtown dogs have more
lesions than country dogs.
Busch: I've had some experience with this type of data working with Dr. Lainhart when he
was with NIOSH while they were studying the textile industry and exposure to cotton dust
We had x-rays on control group and cotton dust workers, but of course there is more variation
in the appearance of these x-rays. They first tried a 5-point rating scale, + through
+ + + + + and we learned very little from it. It was a terrible test for Dr. Lainhart;
furnishing him all the x-rays in blind, and having him rank them 1 to 5 acccording to their
degree of damage. There were many ties but he did put them in order and the signs in this
case would be rankings from 1 to 96 or however many dogs there were. We then used the
rank numbers to take average ranks for each treatment group. There is a nonparametric
statistical analysis which tells you whether these rank averages are statistically different
Perhaps you realize more sensitivity that way.
Albert: It occurs to me that here at Davis, with your dusty environment, you have a built-in
system for evaluating long-term pulmonary clearance without any effort whatsoever. You have
radiographic changes occurring in these dogs which is something that you don't see in dogs
that are kept in non-dusty environments.
Dungworth: Yes, it's a very mild form of pneumoconisis but it's mild again compared to the
human. We're talking about different levels of involvement
Tyler. Dr. Orthoefer, when you looked at these lungs at autopsy, these lungs were nice
pink-looking lungs that didn't look anything like city dog lungs. They're not the kind of dog
lungs you see working in Philadelphia at AngeL These are very mild lesions. The other dogs
we autopsy at Davis still don't look that bad. Those beagles which are outside in the dusty
area just don't compare with human lungs.
Orthoefen I agree.
Kleinerman: I think you should remember that this is the era of computerized axiotomo-
graphy. If you want to find lesions by x-ray you're going to be able to find them with the
available equipment The question is, how much do you want to spend to find the lesion. The
lesion can be detected even though it may be small, if you get the kind of equipment that is
available for human studies.
Orthoefen In response to Dr. Kleinerman, there is a running battle in the New England
Journal over the use of these three-dimensional x-ray systems which are very costly. They
computerize the images and come up with a computer display of a three-dimensional cross
section of the body.
Dungworth: Obviously, the resolution runs out at some point
Brain: I won't say anything about the radiographic analysis; I think we've already said quite a
bit about that. When considering the cardiovascular findings, I felt very much the way Dr.
Gillespie did. I find it hard to take the results very seriously. I can't imagine using the data in
setting standards, for example. However, there are a few intriguing bits that may deserve a
closer look. I found Dr. Gillespie's summary of the problems with the cardiovascular data
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quite compelling. He mentioned that the abnormalities were not reproducible in given dogs
and that they were very nonspecific. The same type of abnormality was not seen consistently
and the changes seen were not substantiated by other tests. I would like to add a couple more
reservations. Unlike the pulmonary findings, there was no progression during the post-
exposure period; this is certainly one of the most compelling features of the pulmonary
function tests. There also appeared to be a random distribution of changes between the
groups. I am also concerned that there was no morphological basis for the cardiovascular
changes. On autopsy, there apparently were no important differences. Thus, I think it is hard
to make much of the effects of these pollutant conditions on the cardiovascular system.
In contrast, I find myself impressed and excited by the pulmonary function changes. I think
this is going to be a study which, when published, will be quoted by review committees
charged with making important decisions about pollutant levels. The levels selected were
reasonable and not too far from ambient conditions. The pulmonary function studies have
been done by experienced and reliable investigators. It is hard to think of many laboratories
in North America where the studies could have been better done. I am also reassured that the
data are rarely baffling and are usually internally consistent. For example, if you look at the
data, one can compare the changes in compliance with the changes in lung values. If you
draw the volume-pressure curve of the lung and also draw the volume-pressure curve of the
chest wall, you can then sum these two together to get a curve describing the total respiratory
system. The functional residual capacity (FRC) is where the lung and the chest wall have an
equal and opposite recoil. If there are changes in lung compliance, this helps predict what
changes in lung volumes one should see. For example, if the volume-pressure shifts up and to
the left, the lungs become more compliant and if the chest wall remains reasonably compliant
then a higher total lung capacity should result. If one examines the alterations in RV and
TLC and compares them to the compliance changes, few disturbing inconsistencies are
present. The measurements of compliance and lung volume fit very well together and I find
that reassuring.
There are occasional unexplained findings. For example, the change in chest wall resistance is
mysterious. In group number 5, the R + SOx group, the resistance of the chest wall (the
amount of pressure you need to develop to get the chest wall tissue to flow) is doubled. That's
hard to understand.
A very attractive aspect of these studies is the correlation between the morphological and
morphometric findings. Another good aspect of this study has been the lack of major
interactions with other causes of altered pulmonary function. We have heard about looking
for urban-rural gradients by comparing city and country dogs. Unfortunately, the problem of
frequency and type of lung infection as another contributor to changes in pulmonary function
is a major problem. This is frequently true for many human studies as well. Even when you've
identified groups that differ with respect to air pollution exposure, they may still be very
different in the frequency of infection, occupation, smoking, or health attitudes. This study,
however, seems to be relatively free of other contributing factors. It's reassuring to hear that
both in Cincinnati and Davis these dogs did not have too many serious lung infections. It
seems less likely that the changes reported are secondary consequences. If air pollution, for
example, altered defense mechanisms, then the animals in some groups might get more
frequent and serious lung infection. In turn, that could cause the pulmonary function
changes. The possibility of this mechanism seems fairly remote in these studies.
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I think it's impressive that there are many different parameters which are significantly
different, e.g., frequency dependence of compliance, airway resistance, lung volumes and the
inspiratory compliance. Many of these changes are substantial. Another major finding is the
progressive nature of the disease. Clearly, moving the animals and continuing the study past
the exposure period was the right thing to do. The differences in individual animals over the
past 2-year period is one of the most striking aspects.
There are certainly questions that need to be asked. What's responsible for that progression?
What's going on during that 2-year period? This is a major question for everyone interested
in emphysema. What processes do you initiate, what's the clock that's ticking away and why
does the progression of the disease often remain quite slow and then accelerate? What's
responsible for that acceleration in the absence of further insult? It will be intriguing to study
such animals more thoroughly by serial sacrifice or by more repeated physiological measure-
ments in an attempt to chronicle that 2-year period and beyond, ^t's tempting to ask, what
would have happened if the study had been continued 2 or 3 more years? What if the
investigators had had a chance to study the dogs once more? Did you catch them just as they
were beginning to approach the precipice and would they drop off even more rapidly? Were
things deteriorating very suddenly? There are dangers in continuing too long if one reaches
end stage situations. Then at autopsy, the morphological and morphometric data may be less
meaningful if the animals become very sick and have more respiratory infections. Perhaps
there were some virtues as well as vices in terminating the study.
Another lesson seems to be that repeated studies on the same animal can be a very valuable
and important strategy. When the data are summarized by comparing the means of groups, it
is possible to lose a great deal of information. This has been demonstrated in a number of
epidemiological studies as well. For example, in our own department we have a number of
environmental and occupational epidemiologists who are interested in the effects of various
occupational factors on the lungs. They have been carrying out studies on fire-fighting and
correlating the number of knock-downs (collapses because of smoke inhalation) or other
indices of exposure with pulmonary function. If you take the means of groups of individuals
(of firemen in various dose categories) and compare those means, you see few significant
differences. On the other hand, if you take individuals and look at their loss of pulmonary
function and average these, the results are more interesting. For example, measure their FEV
1.0 every year for 4 years and then look at the average loss in FEV 1.0. That turns out to be a
more sensitive indicator of exposure. Repeated measurements in the same individuals or
animals focusing on the rate of loss of function over long periods of time can be very valuable.
It would be important to know about the times at which function is changing most rapidly. I
think we can't conclude very much about that in this study since measurements were made
infrequently. But most of the loss of function is after the exposure. Is rate of loss linear or
does it accelerate as the end of life approaches? It's not surprising that these later years are
very important. If you consider important human diseases such as cancer, emphysema,
bronchitis, arteriosclerosis, etc. — the incidence of all these diseases increases dramatically
with advancing age.
We'll soon talk in length about the choice of species and some of the problems that are
involved there. Certainly in the design of future studies it is essential to try to get the most
results from a finite amount of money. The question will surely be discussed tomorrow about
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the relative merits of dogs versus rodents and other experimental animals. In retrospect, I
suppose it would have been productive if some of the advantages of dogs had been exploited
more, although some of my suggestions might be unrealistic. It is possible to train dogs so
that one can perform a number of pulmonary function tests repeatedly on unanaesthetized
animals. If that has been done, the measurements of frequency, tidal volume, arterial Pc>2 and
PC02 might be more meaningful since we would worry less about how anaesthesia had
affected the control of ventilation. Also the risk of anaesthesia would be avoided and it would
become practical to make frequent repeated measurements. However, to train 80 dogs is
perhaps unrealistic; the investigators who make these kinds of measurements have usually
done it on fewer numbers of animals.
Lewis: You have to do it from midnight to eight in the morning too.
Brain: That's another problem. I would also like to ask whether the chronic exposures
influenced the vulnerability of the animals to acute exposure to the pollutants. To what extent
were there alterations in sensitivity? Were there individual animals which became more
sensitive? It may be useful to focus on the most damaged animals. Is there any evidence that
some dogs were unusually sensitive? On the other hand, there is also the possibility that there
were adaptive changes and that some animals became less sensitive. This is certainly possible
for oxidant pollutants. Also the on-and-off transience of exposure might have been an
interesting area to examine in terms of pulmonary function.
Dr. Lewis listed pulmonary function tests which measure either the properties of the airways
or the parenchyma. I think it may be misleading to divide them too rigidly into those two
separate categories. It's very hard to think of tests which measure purely the properties of
airways or purely the properties of parenchyma. There are always both primary and
secondary influences. For example, you think of tests that are sometimes thought of as
measuring airway properties such as airway resistance or the maximal breathing capacity
(MBC). Consider a situation where the initial insult is exclusively to the parenchyma. In "pure
emphysema," the emphysematous lesion characterized by loss of elastic recoil and destruc-
tion of alveolar walls diminishes support for airways. Airways will then be more collapsable
and may have reduced diameters. There will then be a secondary effect on the airway
resistance and the maximum breathing capacity; the peak flow during forced expiration and
the entire flow volume curve may also be altered. This is not because of a direct effect on
airways but because of an indirect effect on parenchyma and the support they offer airways.
Similarly, if we think of tests that we ordinarily think of as measuring the properties of tissue
such as compliance or diffusing capacity, indirect effects may again be important. If one has
airway destruction or obstruction to the extent that a piece of lung cannot be ventilated, then
the remainder of the lung will appear to be stiffer and the diffusing capacity will be reduced.
It isn't because the parenchyma has been changed but simply because the parenchyma is no
longer adequately served by patent airways. There are few pulmonary function tests which are
specific. On the other hand, perhaps it's that very nonspecificity which makes them more
sensitive. The FEV 1.0 reflects changes in both airways and parenchyma. Thus, what happens
to the lung, either directly or indirectly, may alter the FEV 1.0.
Lewis: I couldn't agree with you more but I'm forced into it by the conclusion that Dr.
Gillespie drew, saying these pollutants affected parenchyma and affected airways. I had to go
back in my data and examine both mine and his and couldn't come up with a differentiation.
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Brain: The other advantage that we have here in sorting out the site of damage in this study is
the chance for morphological and morphometrical correlates. I think that's going to
strengthen the conclusions that you both have about where the damage is. I'm talking more
about the respiratory system viewed as a black box without any other knowledge of
morphological changes. I don't necessarily disagree with your specific conclusion but I just
wanted to raise indirect and direct effects as a general problem.
Lee: The transient cardiovascular effects observed by Dr. Bloch may be explained, at least
partially, by our recent observations relative to hypertrophy in rats exposed to carbon
monoxide at concentrations of 50 and 150 ppm for only 21 days.
Comment: I find that the notion that exposure to SOX only produces a parenchymal damage
is a little hard to understand. The SOX exposure was a mixture of S02 and sulfuric acid at a
mass median of 0.6 micron. S02 is very soluble and deposits in the upper respiratory tract.
Sulfuric acid particles at 0.6 micron would grow by a factor of 2 or 3 in diameter as they
entered the lung and I would expect most of those to play out on the bronchi too. Yet, the
animals that showed the airway damage were exposed to exhaust, whereas the ones that
showed the parenchymal-only damage were the ones that were exposed to the S02, at least
from the physiological measurements. I was also surprised that damage from the irradiated
exhaust coupled with SOX wasn't worse than that with the raw because the irradiated exhaust
has a lot of oxidant in it. I thought that would have produced a lot more damage than it
apparently has.
Dungworth: There is a correlation between the morphologic and physiologic findings in that
the damage caused by SOX was at the parenchymal level.
Albert: Aren't you surprised?
Dungworth: Relative to what has been said in the literature, maybe. But I don't doubt the
fact. My trouble may be interpreting it
Albert: I think the interpretation is obvious, namely that the SOX damaged the breathing
mechanisms and the dust in the air at Davis polished these animals off.
Brain: Isn't part of the answer the reversibility of the lesions? Your scenario makes sense in
terms of the immediate short-term effects. Although there may be much more upper airway
damage, still, to a certain extent, damage here may be reversible. On the other hand,
parenchymal damage seems to be less reversible and even progressive. The fact that a few
years have passed since the end of the exposure means that the upper airway lesions may
have had a chance to disappear while the parenchymal lesions may have had a chance to
progress. Isn't that part of the problem and thus, part of the answer?
Albert: It could be. I'm not doubting the observations.
Brain: Perhaps upper airway changes in response to S02 don't persist for years.
Stara: I think that one issue should not be forgotten, namely there was approximately 0.8
micron sulfuric acid mist present in the SOX.
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Albert: That's right, but once they hit the air passages at 100% humidity and a relatively high
temperature, those particles would have grown.
Brain: To what size?
Albert: By a factor of 2 or 3 in diameter.
Stara: One micron particle study was reported in guinea pigs exposed to sulfuric acid and
sulfates. Some reports suggest parenchymal deposition as well.
Bhatnagar: I think I may have an answer for why the SOX and irradiated exhaust seem to act
differently. The reason for the irradiated exhaust being toxic is possibly the ozone, which
generates all kinds of free radicals, superoxide specifically, which is the component of all
oxidant toxicity. SOX, specifically sulfur dioxide, is metabolized in the body or rather is
chemically transformed by going through the sulfite radical HSs which in its own transforma-
tions, enzymatic as well as simple chemical, goes through a superoxide mechanism. If ozone
or superoxide is present in the tissue and sulfur dioxide is also provided in that particular
tissue, it is quite possible that sulfur dioxide transformations would compete for the
superoxide free radical and reduce the concentration of the superoxide radical and thereby
reduce the damage that would be caused by the superoxide radical.
Dungworth: My speculations run along the same line as Dr. Brain. I think maybe we've been
misled by some of the earlier S02 work. You've mentioned high concentrations can corrode
mucosa and cause bronchitis, whereas here we're talking about a very low level. We're
looking for summation of subtle increments of damage over a period of time.
Tyler: Perhaps we haven't looked at the upper airways as carefully as we looked at the
parenchyma. We looked at airways but I don't think we looked at noses.
Albert: I would have expected that with the growth of the sulfuric acid particles, a fraction of
what was administered really got down to the alveoli. Probably none of the S02 got into the
alveoli. The high potency of sulfuric acid in producing peripheral lung damage is relevant to
the automobile catalyst problem because those acid particles are pretty small, in the
submicron range. I think this is really the first data on chronic exposure to sulfuric acid that
shows morphologic effects and functional effects.
Stara: I would like to ask for some comments on dogs' upper respiratory tract, such as the
length of nares in beagles and the possible scrubbing effect of these particles in upper
airways. At Davis you have done some work on this, didn't you, Dr. Dungworth?
Dungworth: We have quoted others who have done work on this with ozone but we have not
investigated it ourselves.
Lewis: In our lab Dr. Vaughn did it. Bob Frank and others have studied removal of air
pollutants from the nose to the alveoli. The quantity of material removed is dependent on
concentration, flow rates and other variables. It's very difficult to get a good handle on it. I
did want to quote from the report of the project review committee on January 1970; the three
atmospheres that they recommended be discontinued at that time were ironically the S02
atmospheres and the two NOX exposure atmospheres, the ones that were the most demonstra-
ble and most consistent.
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Stara: I think this may imply that the dog may scrub the larger particles better than humans
and therefore you may not see as a significant an effect in the upper airway; the remaining
smaller particles may deposit all the way in the parenchyma and the very small airways.
Gillespie: You have to be very careful about that because the dog is a frequent mouth
breather.
Orthoefer: That's what I was going to bring up. They stay in the chambers with their mouth
open a lot and somebody would have to determine how often they breathed through their
nose.
Lewis: Most of the deposition for particles is dependent on the very initial point of entry
where there is a point of minimal cross section and either the particles are deposited there or
else they have a clean shot at getting into the lungs. That point of maximal deposition is
probably before these particles have a chance to grow very much. The air is not completely
saturated and the residence times are very small. They may very well grow after they reach
that point. I don't think it would be surprising even if they did grow by a factor of 1, 2, or 3
that they'd penetrate and deposit in small airways as in the alveoli.
Kleinerman: You mean there is some advantage in having a longer one?
Dungworth: We did look at the surface very carefully by the scanning electron microscope
and tabulate those features of damage. They were rather more prominent (not severe) in the
raw rather than the SOX. That seems to be the case; we have to change our ideas based on the
past information.
Orthoefer: I do have cross sections of all the turbinates in these animals. I can't make much
sense out of it as far as damage is concerned. Decalcification is somewhat damaging.
Kleinerman: Did you look for the increase in goblet cells?
Dungworth: This is being planned. Qualitatively we couldn't sort it out. The next morphome-
tric task is to evaluate mucous apparatus.
Orthoefer: The dog is a frequent mouth breather but if you look at the diameter of the nares
as opposed to the air space through the turbinate it seems to me there must be somewhat of a
flow change as far as if they did breath through the nose. The nares don't seem to be nearly
as big in cross section as the turbinates.
Tyler: Once you get beyond the vestibule, in reality, that common meatus is very large in a
dog. It seems to me that it would be almost as big as a trachea.
Lewis: One thing you will notice is that this is a dose-response type phenomena. For example,
in Dr. Coffin's infectivity studies where he used a mouse, it's much easier to demonstrate a
response where you have a short airway like the mouse has from nose to alveoli than it is in
the dog or an intermediate animal. You can usually find response to gaseous irritants in
rodent species at a lower concentration than you can in a primate or the dog.
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9. EFFECTS OF AIR POLLUTANTS ON
VISUAL EVOKED BRAIN POTENTIALS IN
CHRONICALLY EXPOSED BEAGLE DOGS
B.L. Johnson, J.G. Orthoefer and J.E Stara
Abstract
The effects of raw and photochemically reacted automobile exhaust, oxides of sulfur or
nitrogen, or their combinations on visual evoked brain potentials were studied in 88
chronically exposed beagle dogs. All dogs had been exposed daily for 16 hours over a
68-month period. The study groups consisted of one control group (17 dogs) exposed to
pollution-free air and 7 treatment groups exposed to 1) raw auto exhaust, 2) irradiated auto
exhaust, 3) raw auto exhaust plus oxides of sulfur, 4) irradiated auto exhaust plus oxides of
sulfur, 5) clean air plus oxides of sulfur, 6) clean air plus oxides of nitrogen (N02-high), and 7)
clean air plus oxides of nitrogen (NO-high). Visual evoked brain potentials were recorded
from all animals using subdermal electrodes positioned in the occipital and parietal areas. All
animals were immobilized with succinylcholine chloride and artificially respirated during data
acquisition. Evoked brain potentials generated in response to a visual stimulus were recorded
and characterized in terms of component latencies, component amplitudes, and overall
amplitude. Although all seven treatment groups showed both component and overall ampli-
tudes less than those for the control group, analysis of variance (ANOVA) indicated that the
decreased amplitudes were not statistically significant (PXX05) from control values. ANOVA
also indicated no treatment effects on latencies of component waves of the visual evoked
response.
Introduction
Epidemiological studies concerning the health effects of air pollutants have been almost
exclusively concerned with the association of pollutants with pulmonary disease. Investiga-
tions concerning the effects of air pollutants on cardiac function and brain function have
been few and confined to laboratory studies. Since the lungs are the initial site of entry of air
pollutants into the body, it is understandable why the preponderance of air pollution health
effects studies have been concerned with lung disease. Unfortunately, less attention has been
given to possible consequences of air pollutants on other physiological systems; for example,
the cardiovascular and nervous systems.
It is apparent from a review of the literature that air pollution studies concerned with the
nervous system have been concerned almost exclusively with short-term, acute effects.
Investigations concerned with the acute effects of carbon monoxide on the nervous system,
and indirectly on behavior and human performance, have been reported by several investiga-
tors and have been summarized in reviews by Environmental Protection Agency (1) and the
National Institute for Occupational Safety and Health (2). Of particular relevance to the
investigation described in this report are the reports by Hosko (3) and Putz et al. (4). Hosko
reported the effects of acute CO exposures on visual evoked brain potentials. Results from his
study suggested that whenever the carboxyhemoglobin (COHb) level exceeded 20%, there
occurred a disruption of components in the visual evoked brain potential, but not at COHb
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levels below 20%. Investigations by Putz et al. also used evoked brain potentials as an
indicator of CO neurotoxicity. In this study, it was found that human auditory evoked brain
potentials that were deemed "significant" by the subjects in an auditory discrimination task
showed signs of disruption when COHb levels reached 5%.
A review of the literature concerning the toxicity of sulfur oxides indicates few investigations
concerning the effects of SOX on the nervous system. Indeed, the only reported studies are
those of Bushtueva et al. (5) who found that exposure of humans to SO 2 concentrations
ranging from 0.9 to 3 mg/m3 disrupted alpha rhythms in the electroencephalogram and
increased optical chronaxie at 1.5 mg/m3. As with the CO neurotoxicity studies, the SOX
investigations concerned with possible effects on the nervous system have been short-term,
acute studies.
The effects of chronic exposure to ozone on brain function were reported by Trams et al. (6)
who utilized neurochemical indicators of neurotoxicity and Johnson et al. (7) who employed
electroencephalographic (EEC) techniques. Both studies utilized the same beagle dogs
exposed for 19 months to ozone levels ranging up to 3 ppm (8 hours daily exposure). Trams et
aL reported a steady decline in catechol-o-methyltransferase activity with increased Oa
exposure, and Johnson et al. noted a decrease in EEC frequency content and a disruption of
visual evoked brain potentials at Os exposure levels exceeding 1 ppm (16 hours daily
exposure).
In summary, relatively few studies have been concerned with the possible neurotoxic
consequences of long-term, chronic exposure to air pollution. The purpose of the investiga-
tion described in this report was to examine in a laboratory study the effects of air pollutants
on brain function, as measured by evoked brain potentials. The reason for examining visual
evoked potentials as possible indicators of brain neurotoxicity was due to 1) the use of such
brain potentials as indicators of visual system disorders in human studies (8) and 2) the
relative ease of data acquisition in a study involving a large number of animals.
Methods and Materials
One hundred and four beagle dogs were randomly assigned to exposure chambers which
contained four dogs each as described by Hinners et al. (9). All dogs were from an inbred
colony and were approximately 6 months old at the start of the exposure. One group of 20
dogs served as control subjects and were exposed to CBR (chemical, biological, and radiologi-
cal) filtered air for the duration of the study. The remaining 84 dogs were divided equally into
seven treatment groups as follows: 1) raw (non-irradiated) laboratory-produced auto exhaust
(R), 2) irradiated auto exhaust (I), 3) filtered air plus oxides of sulfur (SOX), 4) R-t-SOx, 5)
I + SOx, 6) filtered air plus high nitrogen dioxide plus low nitric acid (N02H+NOL), and 7)
filtered air plus low nitrogen dioxide plus high nitric acid (N02L+NOH)- Each treatment
group was exposed for 68 months, at which time the dogs were tested for signs of
neurotoxicity.
The dogs were taken from their exposure chambers on a random schedule immediately prior
to recording of electroencephalographic data. All dogs had been fasted 12 hours prior to
testing. The testing of the dogs was conducted in a manner such that the principal
investigator was blind to the treatment history of each dog. Clipping of hair on the head and
weighing of each animal were performed prior to EEC recording. Hair was clipped from the
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occiput anteriorly to the supraorbital process and laterally to the base of the pinnae. Each
animal was given Sucostrin (succinylcholine chloride) intravenously at the rate of 0.5 mg per
kg of body weight. The animals were immediately intubated with a cuffed endotracheal tube
and artificially respirated by a demand type mechanical respirator using a mixture of 95% 02
and 5% C02. The respiration rate was constant, but was adjusted individually for each
animal in order to maintain a well oxygenated state, as judged by pink tongue and mucous
membranes.
Four monopolar subdermal needle electrodes were used for EEC recording from each dog.
The electrodes were approximately 0.5 inch (1.27 cm) in length. Two electrodes were placed at
parietal locations PS and ?4 (International 10-20 notation), while the other two electrodes were
placed in the occipital area of 01 and 02. Electrodes placed in each ear flap and tied together
electrically served as the reference electrode. The parietal and occipital areas were chosen for
EEC recording because of the reported effects of some air pollutants (CO, Os) on the visual
system. Symmetrical electrode placements were used in order to investigate the possibility of
asymmetric cortical damage. The EEGs were recorded in a quiet, well-lighted room with a
Sanborn (Model 350-1500) electroencephalograph and recorded on a FM analog tape
recorder (Honeywell, Model 7600). The bandwidth of the EEC amplifier-recording system was
essentially flat from 0 to approximately 300 Hz.
The EEC recordings lasted 10 minutes for each dog. The first 7 minutes of data acquisition
consisted of recording a background EEC during minimal sensory stimulation. The remain-
ing 3 minutes of data acquisition consisted of recording visual evoked potentials recorded
from PS, P4, Oi, and 02 that were generated in response to photic stimulation from a Grass
(Model PS-2) photic stimulator. The stimulator was positioned at each dog's eye level 15
inches from the animal's nose. The photic stimulator was set at minimum intensity (Intensity
= 1). Sound absorbant material was placed within the photic unit in order to minimize the
auditory click coincident with the gas tube discharge. The visual stimuli were presented at a
constant rate of 1 flash every 2 seconds.
Averaged evoked potentials were obtained through use of the Advanced Averager program
supplied with a PDP-8 computer. For each dog, the visual evoked potentials for the first 75
photic stimuli were averaged for each EEC lead by the aforementioned computer program
and saved on punched paper tape for subsequent analysis.
The averaged visual evoked responses (VER) were characterized in this study by the following
six measures:
1. Latency of the first negative peak (Ni)
2. Amplitude of NI
3. Latency of the first positive peak (Pi)
4. Amplitude of PI
5. Ratio of Pi/Ni amplitudes
6. The root mean square (RMS) value of the VER
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In order to test for treatment effects attributable to the seven groups of air pollution
exposures, a univariate analysis of variance (ANOVA) (10) was used to test each of the
aforementioned six parameters of the VER for statistical significance. Significant differences
among treatments were to be examined following ANOVA by the use of Dunnett's test (10),
which permits comparison of individual treatments with a control group.
Results
Comparison of VER data from EEC leads PS, P4, Oi, and 02 showed no differences between
EEC leads as concerns possible treatment effects. Therefore, to simplify the presentation of
group data, only VER results from EEC lead ?4 are discussed. After 68 months of exposure
the following numbers of animals comprised each treatment group: control, 17; raw auto
exhaust (R), 11; irradiated auto exhaust (IX 10; oxides of sulfur (SOX), 8; NOn + N02L, 10;
NOL + N02H, 11;R + SOX, 10;andI + SOx, 11. Table 4 of Chapter 2 contains mean pollutant
exposure levels for each treatment group. Figure 1 illustrates a typical averaged visual evoked
potential as recorded from a control dog at EEC electrode ?4. The VER is characterized by
the presence of a large positive peak, denoted by PI, followed by a negative component NI.
Figures 2 through 6 contain treatment group means and the standard error of the mean (SE)
for the latencies of NI and PI , amplitudes of NI and PI , and the root mean square (RMS)
value of the total VER. The latter amplitude value was used as a measure of overall amplitude
of the VER.
Review of Figures 2 and 3 show no treatment effects of any air pollution, singly or in
combination, on the latencies of VER components NI and PI. For NI latencies, ANOVA
yielded F(7,80) = 0.81, P = 0.59, which, of course, is not statistically significant. The
amplitude of PI was diminished in all seven treatment groups in comparison to the control
group, but ANOVA yielded F(7,80) = 1.74, P = 0.11, which is again not statistically
significant
Possible treatment effects on overall VER amplitude were examined using the RMS value of
the total VER. Treatment means as shown in Figure 6 are all less than the mean for the
control condition, but again ANOVA showed this result to be statistically nonsignificant
(F(7,79) = 1.15, P = 0.34).
In summary, although there are indications that VER amplitudes were reduced in all
treatment groups when compared to the control condition, these decreased amplitudes did
not reach statistical significance by ANOVA.
Discussion
The lack of effect of any air pollution treatment on brain function found in this study, as
measured by evoked brain potentials, could be due to l)lack of physiological damage to brain
tissue due to treatment groups, or 2) lack of sensitivity of the VER method to detect minimal
brain dysfunction. The former possibility could be true, since there is little convincing
evidence in the literature to indicate neurotoxic effects for chronic air pollution levels at
concentrations representative of those in this study. Since carbon monoxide and ozone were
two major constituents of those treatments involving auto exhaust, the reports by Lewey and
Drabkin (12), Lindenberg et al (13) and Johnson et al (7) are relevant to findings from this
study. Lewey and Drabkin exposed six dogs to 115 mg/m3 CO for 5.75 hours per day, 7 days
170
-------
SCALED AMPLITUDE
"5
O-
ffi-
er
3.
5'
-a
o
FT
a
5'
m
CO
a.
o
o —
CD-
CD
N)
O.
O
o
O'
o.
o
Ui
§•
"m
m
m
H CO m
CD -rj ;u
I- O >
m z O
> co m
D m o
>
O i-
O m
D *
R3
D DO
£D >
N
i.
-------
o
z
140-
120-
100-
80-
60 —
20-
0 -
N1 LATENCY OF VER
VS TREATMENT
[fl
*l *
<
o
x
O
CO
+
tr
0
m
+
r
O
Z
+
Figure 2
Means and standard errors for VER component N i latency versus air pollution treatment.
172
-------
100 —
80 —
P1 LATENCY OF VER
VS TREATMENT
w
o
LLJ
1-
< 40 —
T—
Q.
20 —
n
T
*
*
r^i
f*i
rJi
*
< DC ~ XXX -IT
0 ° w w ° £
" ± 2 z
+
o
Figure 3
Means and standard errors for VER component PI latency versus air pollution treatment.
173
-------
UJ
Q
D
H
_l
Q.
5
<
100 —
80 —
§
I
N1 AMPLITUDE OF VER
VS TREATMENT
20 —
i
ft
•
•
< cc
o
it
t\
^
•
ft
--
XXX,-,-
o o o £ S
O> C/D CO O O
i
O
O
Figure 4
Means and standard errors for VER component N i amplitude versus air pollution treatment.
174
-------
100—
80—
P1 AMPLITUDE OF VER
VS TREATMENT
LU
§ 60-
t
_i
0.
< 40—
20—
n
^H
<
^B
k
M
•
•
r rr -
o
T
MB
1*1
ft
rh
i
T
T
ft
x x x -J x
o o o £ $
CO W OT ^ y
or
+ +
o1 o
Figure 5
Means and standard errors for VER component PI amplitude versus air pollution treatment.
175
-------
50—
40—
OVERALL AMPLITUDE OF VER
VS TREATMENT
g 30-
D
0.
< 20~
z
LU
2 10—
n
1
ft
ft
n
ft
ft
rh
ft
O
CC
o
a:
O
z
+
o
o
Figure 6
Means and standard errors for VER overall amplitude (RMS) versus air pollution treatment.
176
-------
per week for 11 weeks and found in morphologic examination that all dogs exhibited cortical
brain damage, but no EEC abnormalities. However, Lindenberg et aL, who exposed dogs at
115 mg/m3 CO for 6 weeks, found no brain areas showing necrosis or demyelination. CO
levels used in the study (see Table 4 of Chapter 2) were approximately equal to those of the
Lewey and Drabkin and Lindenberg studies. It would therefore appear that if CO-induced
damage occurs in the cerebral cortex as a consequence of chronic exposure to CO levels of
100 mg/m3 or less, the damage will not be reflected in abnormal EEC. Johnson et al found
EEC changes characteristic of cortical depression in dogs exposed to Oa for 18 months.
However, the Os levels (2.0 mg/m3) were considerably in excess of the ozone levels employed
in the present study (0.39 mg/m3).
As mentioned, it is also possible that minimal brain damage did occur in the dogs, but the
VER method was insensitive to detecting the neurotoxic effect. Putz, Johnson and Setzer (4)
found in a laboratory study of the acute effects of CO that auditory evoked brain potentials
were disrupted by CO only if the auditory stimuli were "significant" to the subject Auditory
evoked brain potentials judged nonsignificant in an auditory discrimination task were
unaffected by CO. These results would suggest that the sensitivity of using evoked potentials
as indicators of brain neurotoxicity can be enhanced if presented to the subjects within the
context of a discrimination task. This possibility, of course, was impossible to consider for use
with all 88 dogs in this study, but might have been possible with a small subsample of dogs.
References
1. U.S. Department of Health, Education, and Welfare. 1970. Air Quality Criteria for
Carbon Monoxide. Washington, D.C.
2. U.S. Department of Health, Education, and Welfare. 1972. Occupational Exposure to
Carbon Monoxide. Washington, D.C.
3. Hosko, MJ. 1970. The effect of carbon monoxide on the visual evoked response in man.
Arch. Environ. Health 21: 174-180.
4. Putz, V.R., B.L. Johnson, and J.V. Setzer. 1976. Effects of carbon monoxide on vigilance
performance. National Institute for Occupational Safety and Health, Report 77-124.
5. Bushtueva, K.A. 1970. Referenced in Air quality criteria for sulfur oxides. U.S. Depart-
ment of Health, Education, and Welfare, Washington, D.C.
6. Trams, E.G., C.J. Lauter, E.A. Brown, et aL 1972. Cerebral cortical metabolism after
chronic exposure to ozone. Arch. Environ. Health 24: 153-159.
7. Johnson, B.L., J.G. Orthoefer, T.R. Lewis, and C. Xintaras. 1976. The effect of ozone on
brain function. In Clinical implications of air pollution research. Publishing Sciences
Group, Inc., Acton, Mass.
8. Regan, D. 1975. Evoked potentials in psychology, sensory physiology, and clinical
medicine. Halsted Press, New York, N.Y.
9. Hinners, R., J.K. Burkart, and G.L. Contner. 1966. Animal exposure chambers in air
pollution studies. Arch. Environ. Health 13: 609-613.
10. Winer, BJ. 1962. Statistical principles in experimental design. McGraw-Hill Book Co.,
New York, N.Y.
11. Lewis, T.R., W.J. Moorman, Y.Y. Yang, and J.E Stara. 1974. Long-term exposure to auto
exhaust and other pollution mixtures. Arch. Environ. Health 29: 102-107.
12. Lewey, EH., and D.L Drabkin. 1944. Experimental chronic carbon monoxide poisoning
of dogs. Amer. J. Med. Sci. 208: 502-511.
177
-------
13. Lindenberg, R. et al. 1962. An experimental investigation in animals of the functional and
morphological changes from single and repeated exposure to carbon monoxide. Amer.
Ind. Hygiene Conf., Washington, D.C., May 13-17.
Asilomar Conference Discussion
Question: What did Dr. Johnson measure?
Stara: Dr. Johnson measured the amplitude and the latency of the positive and negative
peaks. Negative peak shows both latencies slightly lower than the controls and the amplitudes
much lower than the controls; but using the analysis of variances, as Mr. Busch described,
there were no significant differences. There were trends in all the treatment groups. This
basically sums up the results.
Orthoefer: Was the Student t-test significant?
Busch: No, he didn't use it, according to what I read. He did the ANOVA test which compares
all the means for excessive variability compared to the intragroup variability. He followed that
up with a test called Dunnett's test which compared each treatment against the control. I
don't know what results he got.
Lewis: He also did Student t-test in a random nature similar to what was done by Dr. Hyde in
Davis on the morphometry and in that situation there were some values at least that were
significantly different. He was reticent to make a comment on that until he felt it was
mathematically sound.
178
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10. ULTRASTRUCTURE OF BIOPSIED TISSUE
R.J. Stephens and G. Freeman
The lung biopsies to be described here at the light and electron microscope levels were
obtained from female beagle dogs that were originally housed in Cincinnati and later at the
University of California at Davis. Lung biopsies from 16 animals were obtained on April
26-28, 1971 in Cincinnati when exposure of the animals was terminated. Six animals were
controls, five had received irradiated auto exhaust, and five had received NOL+N02H- After
about 30 months of recovery in clear air (at Davis, California) an additional sample was
obtained from all but two of the same dogs: five controls, five that had received irradiated
exhaust, and four that had been exposed to NOL+N02H- These animals were sacrificed
between December 12 and 19, 1973.
While these studies should not be regarded as exhaustive, they were substantial. At least five
blocks were sectioned and examined at both the light and electron microscope levels for each
animal from which tissue was available, both at the end of exposure and after approximately
30 months of recovery. Additional tissue was fixed in formaldehyde in Cincinnati and shipped
to Dr. Freeman at Stanford Research Institute for routine paraffin embedding and pathologi-
cal examination. In addition to hematoxylin and eosin, sections were also stained with aniline
blue and orange G for collagen, and with acid orcein for elastic tissue.
It was not feasible to attempt to distinguish between artifactual alveolar compression and
atelectasis in the tissue prepared for pathology since the biopsies, which were fixed in
formaldehyde, were not filled or inflated through the airways. There were also areas of
distended alveoli that may possibly have represented "compensatory emphysema." There
were no appreciable differences among specimens from the three groups of dogs, whether
they were designated as control, irradiated exhaust, or NOL+N02H animals.
Possible abnormalities were limited to: 1) occasional evidence of exaggerated goblet cell
activation in bronchioles; 2) occasional multiple layering of bronchiolar epithelium; and 3)
occasional evidence of perivascular fibrosis in all three groups. Tissue was not obtained for
routine pathology at SRI after the recovery period; therefore, no data are available.
Biopsies and tissues obtained at necropsy and prepared for transmission electron microscopy
were fixed in OsCU and embedded in Araldite. One-micron-thick sections were obtained and
stained with toluidine blue for observation with the light microscope. Each block was then
carefully retrimmed in specific areas of interest and sectioned for transmission electron
microscopy. Because of previous experience with deep lung irritants in rats, dogs and
monkeys, we concentrated our attention on the terminal airways and proximal alveolar
regions. Careful examination of tissue from each animal did not reveal a significant
difference among the three groups of animals, whether tissue samples were obtained
immediately after exposure or after an extended recovery period. The ultrastructure of each
cell type was within the limits of normal variability, and it was difficult to substantiate any
consistent or significant change that could be attributed to the exposures they had received.
A comparison of data obtained from this study on beagle dogs with data from an earlier study
on dogs exposed to Os and their controls indicated some interesting differences. In the
present study, both ciliated and nonciliated cells of some terminal airways contained a
179
-------
number of lipid droplets. This was true regardless of whether they were controls or exposed
animals. Lipid droplets were not observed in the same cells from the earlier study. However,
animals exposed to Os at 3 ppm for 8 hours per day for 18 months showed several
abnormalities. Squamous metaplasia was prominent, particularly in the respiratory bron-
chioles, and there was an increase in fibrosis in the peribronchiolar and periductal tissues.
The cisternae of the endoplasmic reticulum in many type 2 cells were dilated and contained a
proteinaceous material with a regular period of approximately 750 A°. In addition, the
lamellar bodies appeared to be abnormal in cells containing the periodic material.
In conclusion, it should be noted that the total amount of tissue examined in this ultrastruc-
tural study was very small and if changes occurred that were restricted in distribution, they
may have been missed.
Asilomar Conference Discussion
Stephens: ... There's no question that there has been a very definite change in the eel] type
which was most prominent and recognizable in the respiratory bronchiolar area I think, in
addition, there's been a thickening of the basement laminar or basement membrane area as
well. We've seen this in N0;>-exposed animals.
Albert Is this in the dog?
Stephens: Yes, but for earlier studies with ozone.1
Albert: How long was the exposure?
Stephens: The exposure was 18 months and these particular micrographs were taken from
animals that received 3 ppm Os, 8 hours a day, for 18 months.
Lewis: Do you see similar lesions in the 2 x 8 or 1 x 8 or 1 x 16 in any of these treatments?
That was a factorial design of 1 x 8 versus 2x8, etc. Did you see any lesions in lower CT
(concentration x time)?
Stephens: No, we did not see this type of a change. There were some other connective tissue
changes at the lower levels, but the actual epithelial changes were not seen in animals exposed
to 1 and 2 ppm ozone. In the higher magnification showing the small cytoplasmic vesicles
there are several characteristic desmosomes cut in tangential section but they are certainly
recognizable. The tonofibrils are quite evident in the cytoplasm, clearly showing the nature of
the cellular change.
^Freeman, G., et al. 1973. Changes in dogs' lungs after long-term exposure to ozone: light and
electron microscopy. Arch. Environ. Health 26: 209.
180
-------
Albert: Is the metaplasia present higher up in the respiratory tract?
Stephens: We didn't examine the tissue from the upper airways in these particular animals.
This tissue was also acquired from Cincinnati and we did not obtain the upper airways.
Kleinerman: When you describe a lesion like this, Bob, does that mean you've seen it more
than once?
Stephens: We would see this lesion on almost every slide that we would make, but it would be
a small portion of the total epithelium that would be present.
Kleinerman: Your interpretation is that it's patched.
Stephens: That's absolutely right.
Albert: I don't get any feel for how much material you looked at You remove a piece of lung;
did you examine it in a systematic pattern?
Stephens: Yes, the amount of tissue obtained was substantial It was prepared for electron
microscopy. Terminal airways were embedded longitudinally and examined systematically
from proximal to distal areas. Upper airways were not included.
Albert: You showed a slide that looked like a whopping increase in the number of goblet cells,
then you say it really isn't increased. I just wonder whether the intensity of the examination
was adequate to support the statement
Stephens: I would have to have shown a dozen slides of each area, I suppose, to convince
everyone in the room that my general appraisal was correct, but time is limited.
Nettesheim: Transmission electron microscopy is not the most convenient way of trying to
determine whether there's an increase in goblet cells, though.
Stephens: I showed the high magnification light micrograph to describe the characteristics of
the cell types and certainly you can find various areas along the airways that have a few more
goblet cells than other areas do.
Dungworth: I think this would be more fruitful after the rest of the morphology.
181
-------
-------
11. NECROPSY
J.G. Orthoefer
Procedure
Following the final pulmonary function testing and while still under anesthesia (pentobarbital
Na) the animals were exsanguinated by incising the jugular vein and brachial arteries.
Immediately thereafter the lungs were removed, trimmed of extraneous tissue and weighed.
The weight included all lobes of the lungs and the trachea up to the third trachea! ring.
The left diaphragmatic lobe of the lung was removed and the major bronchi were dissected
out. The remainder of the parenchyma! tissue from this lobe was frozen for collagen
determination. The left apical lobe was separated at the major bronchi, weighed and quick
frozen using liquid nitrogen.
The left primary bronchus was ligated using a heavy cotton string and the lungs were fixed by
the intratracheal infusion of a cacodylate buffered glutaraldehyde-formaldehyde solution at
30 cm H20 pressure. Figure 1 depicts the infusion apparatus. A systematic procedure was
followed in dissecting the entire animal and representative samples of all tissue were saved.
The weight of all organs was recorded as removed, and representative samples of some
organs, and, in cases of pair organs, entire organs were frozen for later biochemical studies.
4-liter aspiration bottle
30 cm
Figure 1
Fixation of the right lung was accomplished using a dilute Karnovsky's fixture at 30 cm H20
pressure for 16 to 18 hours.
183
-------
Table 1. Total Body Weights and Organ Weights and Standard Deviations for 70 Beagle Dogs
Controls
Total body 9.75 ±1.80
Raw
10.90±
1.80
Irradiated
9.31 ±2.18
sox
10.78 +
1.60
R + SOX
10.44 ±1.78
I + SOX
10.62 + 2.06
NOL + N02H
9.58+1.40
NOH + NO2L
9.99 ±2. 13
weight (kg)
Lungs
Liver
Heart
(gm) 102.61
±8.58
" 296.16 ±63. 30
65.40
Lt. ventricle 46.18
& sept.
"
Rt. ventricle 19.22
Kidney
right
left
Adrenal
Spleen
Thyroid
Brain
(gm)
54.56
27.91
26.27
1.91
40.75
0.72
76.29
± 10.94
±10.63
±5.21
± 10.69
±5.87
±4.48
±0.68
±13.21
±0.22
±5.79
119.81 ±
365.1 7 ±
68.85 ±
51.42±
17.93±
63.80 ±
33.37 ±
30.47 ±
1.66±
54.40±
0.75 ±
73.13±
16.85
105.86
13.01
10.92
3.04
8.59
4.32
4.72
0.37
19.15
0.22
5.12
104.90 ±25. 17
290.15 ±57. 58
65.42 ±12.23
47.24 + 9.94
18.18 + 5.03
50.64 ±1.1 .85
25.13 ±7.52
29.48+11.39
1.97 + 0.73
38.15+10.94
0.66 + 0.26
72.46 + 5.74
123.92 +
347.24 +
69.05 +
50.18 +
18.53 +
60.91 +
29.10 +
31.98±
1.78 +
40.77 ±
0.67 ±
75.26±
12.49
60.12
8.52
9.43
3.55
13.21
5.92
646
0.53
10.38
0.18
5.27
114.38 + 20.58
326.27 ±48.27
65.03 + 7.63
48.67 + 6.07
16.36 ±1.91
58.63 + 5.75
30.96 + 2.33
27.45 ±3.18
2.24+1.86
37.20+14.34
0.68 ± 0.24
73.04 ±7.71
111.92+10.85
375.00 ±83. 18
67.30 ±10.78
51. 05 ±8.95
16.25 ±3.50
63.21+8.17
32.23 ±3.48
30.95 ± 4.84
1.76 ±0.35
44.50+18.19
0.80 ± 0.42
72.83 ± 7.55
104.53+11.52
301. 36 ±72.70
59.26 + 9.63
44.58 ±5.98
14.67 ±4.46
51. 50 ±7.29
26.46 ± 3.48
26.47 + 4.70
1.78 ±0.53
44.65 ±17.28
0.74 ± 0.23
73.27 ±4.51
108.94 ±12.85
345.57 ±100.34
65.79 + 9.84
50.11 ±7.84
15.69 ±3.36
57.28 + 8.90
29.23 ± 5.45
28.28 + 4.36
1.71+0.44
35.43+11.99
0.63 ±0.28
73.04 ±6.77
-------
Portions of heart and kidney tissue were fixed in glutaraldehyde for possible electron
microscopy. All other tissues for pathological examination were preserved in 10% buffered
formalin. The tissues underwent two changes of formalin within a week of the time they were
obtained to insure that all tissues were adequately fixed. Following fixation, representative
sections of all tissues were cut, embedded in paraffin using standard techniques, sectioned
and stained with H&E for histological examination. Additionally, bone samples were either
frozen or preserved in 10% buffered formalin for later use in determining bone lead.
Results
The organ weights are given in Table 1 as percentage of body weights. Statistically, after
correction for body weight, no treatment effect was noted for any of the organ weights;
however, values for the right kidney were close to significant in the auto exhaust atmospheres
as opposed to the non-auto exhaust atmospheres. The other values were unremarkable (1).
A number of mammary tumors were present in the animals. These were predominantly mixed
cell tumors rather evenly distributed in the various treatment groups (Table 2). Fewer tumors
appeared in the auto exhaust atmospheres (Table 3). Additional work with the mammary
tumors is being done at Davis but is not expected to have any impact upon the auto exhaust
study. It is an extension of a beagle mammary tumor study.
Another common non-pulmonary finding in old dogs was the presence of mucoidal degenera-
tion of the heart valves. This appeared to be very common and usually both valves were
Table 2. The Frequency Distribution of Mammary Tumors for Beagles Exposed to
Exhaust and Non-Exhaust Atmospheres
Atmosphere
CA
R
1
sox
R + SOx
I + SOX
NOL+NO2H
NOH + NO2L
Total
With tumors
11
3
3
5
3
5
7
6
43
Without tumors
6
7
7
3
6
6
3
4
42
Total
17
10
10
8
9
11
10
10
85
Table 3. The Frequency Distribution of Mammary Tumors for the Beagle
Exposed to Different Atmospheres
Type of exhaust With tumors Without tumors Total
Auto exhaust 14 26a 40
Non-auto exhaust 29 16 45
Total 43 42 85
aP<0.05
185
-------
involved. The findings are presented in Table 4. Very severe degeneration with a moderate
amount of calcification appeared to be the cause of the pathologic heart sound heard by this
and other investigators.
Skin tumors of a non-lethal nature were noted in several animals with no predominance in
any treatment group. There were nine papillary type tumors; three were inflammatory polyps,
two were small hemangiomas, and the rest were papillomas (Table 5). Several animals were
affected with alopecia of varying degrees but none were serious. Of the animals seen with
alopecia, none had abnormal endocrine histology. Other common findings were splenic
nodules (all hyperplastic) in eight animals and unilateral thyroid enlargement in two animals;
the latter were histologically thyroid-adenomas, most likely of non-secretory type (see Chapter
3).
The major emphasis was placed upon the pulmonary tissue; fixation was accomplished by
allowing the lungs to float on fixative of 30 cm H20 pressure for 16 to 18 hours. The lungs
were carefully tied off with a heavy silk string and volume measurements by fixative
displacement were taken. Any previously recorded abnormalities were rechecked and the
lungs were placed in fresh fixative. The details of the section preparation will be given in
Chapter 12; however, the gross lesions were as follows:
1) Eight lungs failed to completely collapse to the normal extent when removed from the
chest These came from the Mowing groups: 3/R; 3/NOH + N02L; 1/NOL + N02H51/SOX.
2) Six lungs had small, discrete, marginal zones of atelectasis.
3) One lung had incomplete extra divisions.
A summary of the pulmonary histologic lesions are shown in Table 6. Further evaluation of
the lungs will be presented in Chapter 12. The lead analyses were carried out on six femurs,
three from non-exhaust and three from exhaust treated animals. The results are shown in
Table 7.
Table 4. Mucoidal Degeneration of A-V Valves
Both Left Right
27 23 1
Table 5. Skin Lesions and Papillomas
Skin lesions Skin papillomas
Papillomas 7 Papillomas 4
Hyperkeratosis 1 Hemangiomas 2
Localized alopecia 3 Inflammatory polyps 3
Hemangiomas 2
186
-------
Table 6. Lung Lesions (No grading is included)
Number of lungs with lesions
CA
NOL+ NOH +
SOx R + SOx l + SOx N02H N02L
Inflammatory foci
Focal Type II
hyperplasia
Increased thickness
of alveolar walls
and scar formation
Severe emphysema
Other lesions
4
—
1
—
2
3
—
1
1
2
1 3
— —
1 2
— 1
— 1
1
—
1
—
2
2
—
3
—
1
1
1
1
2
1
1
1
5
4
—
Table 7. Lead Analysis of Femurs from Dogs (Beagles) Exposed to
Chronic Auto Exhaust8
Average:
Average:
Exposure
sox
NOX
CA
I
l + SOx
R
Dog#
74R18
74R16
74R66
74R12
74R41
74R55
Femur cortical
fcg/gm)
2.1828
1.2822
2.2858
1.92
4.5237
2.9742
5.5346
4.34
Femur
proximal end
(pg/gm)
0.9186
2.7816
2.1843
1.96
2.9724
7.3799
4.7517
5.03
Preliminary work on bone lead levels in six dogs. This work was done in Cincinnati and is
indicative of bone lead level in exhaust versus non-exhaust animals.
Discussion
There were no unexpected non-pulmonary findings in the dogs. Dr. Hyde and I collaborated
in the work on the lungs, which at this time is complete. Therefore the discussion of the
pulmonary lesions will be left for Chapter 12.
For the last 5 years these animals have been extensively investigated and clinical disorders
were always promptly evaluated and treated within the allowable regimen (see Chapter 3).
One additional study (2) reported earlier showed a high incidence of hip displasia in these
animals.
Only a very small number of animals were tested for bone lead. A higher bone lead level was
seen in the exhaust as compared to non-exhaust treated animals (Table 7). Further data are
not available at this time; however since all animals were essentially always treated equally,
187
-------
the additional lead component must be due to the exhaust lead. The lead levels in the exhaust
atmosphere -were discussed earlier in Chapter 1, and a more detailed description has been
presented by Barkley et aL (3).
The cardiac valvular lesions are common in older dogs (4) and are usually seen in both valves
or in the tricuspid valve. In this study the valvular lesions were more prevalent in the bicuspid
valve. Only a few of these lesions were severe enough to be of clinical importance (5).
Although mammary tumors appeared to be common in these dogs and some were diagnosed
as adenocarcinomas, no animal showed any signs of tumor metastasis.
References
1. Andersen, A.C. 1970. Beagle: As an experimental dog. Iowa State University Press, Ames,
Iowa.
2. Morgan, J.P. 1964. Hip displasia in the beagle: A radiographic study. J. Am. Vet. Med.
Assoc. 164 (5): 496498.
3. Barkley, N.E, K.A. Busch, W.L. Crider, and M. Malanchuk. 1972. The concentration of
lead in automobile exposure chambers. Am. Indust Hyg. Assoc. J. 33: 678.
4. Smith, H.A., T.C. Jones and R.D. Hunt. Veterinary pathology, 4th ed. Published by Lea
and Febiger.
5. Bloch, W.N., Jr., T.R. Lewis, K.A. Busch, J.G. Orthoefer, and J.F. Stara. 1972. Cardiovascu-
lar status of female beagles exposed to air pollutants. Arch. Environ. Health 24: 342-353.
Asilomar Conference Discussion
Orthoefen There are a lot of data on the dogs, kidney, liver, and all other organs, but really
there are few lesions that are relatable back to the treatments. Practically all kidneys have
hypercellular glomeruli. The animals have a lot of age-related lesions as distinguished from
treatment-related lesions.
Dungwortk We have time for questions, but let's leave the pulmonary-related changes until
after Dallas Hyde has presented his findings, and then we can take all the pulmonary changes
from the three together. If there are questions on the non-pulmonary, let's have them now.
Orthoefen The reason I presented this mucoidal degeneration of heart valves was because I
knew there was going to be some question about the abnormal heart sounds and the
cardiovascular work. These are the heart lesions, most of which are common in older dogs.
Everybody that has listened to the heart of these dogs from about 1967 on has pointed out
abnormal heart sounds in some dogs. The only thing it can be related to is the mucoid
degeneration of the heart valves. It occurs as dogs get older. I wanted to point out that we
didn't have any real malignant tumors of the skin. There are other lesions, some enlarged
thyroids. In fact, there were two thyroid-adenomas, but the blood chemistry data did not
indicate these were secreting or functional in any way. I did not include all that because we
just can't relate it to treatment
188
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Dungworth: The cardiac lesions in aging dogs have been well documented by Patterson and
Detweiler. I don't think there is any question about them.
Albert: What about the aorta? Were there arteriosclerotic lesions in the aortas?
Orthoefen I made sections of all the aortas and I've still got them. I've looked at them, but
have not done any special stains. The unstained aortas really looked fairly normal to me.
Possibly we should do some special fat stains on whole mounts, or maybe we could contract it
out Grossly, there were no changes in the aorta.
Albert: You don't see any arteriosclerosis?
Orthoefen No.
Dungworth: You do not generally see gross lesions in dogs unless they have diabetes or
hypothyroidism.
Thurlbeck: In Table 1 that you showed of the lung lesions, would those be grade 3
emphysema?
Orthoefen Yes, they would be a grade 3.
Thurlbeck: Using this as a point of reference, speaking in general terms in grade 3, you could
say that given a lesion of this severity in man, particularly on the gross, it would be arguable
with another pathologist whether emphysema was or was not present
Dugnworth: We made that point in the discussion of our paper. It is mild in comparison to
emphysema as usually described for man.
Orthoefen Actually, in dogs, you would rarely see the human counterpart occurring unless
you had very severe pulmonary function changes.
Dungworth: You can sometimes see severe lesions in old dogs. They tend to occur in
subpleural airspaces first, however; here we are talking about comparing treatment versus
control animals.
Kleinerman: If you had to grade whole lungs in a manner such as Dr. Thurlbeck or other
pathologists looking at human lungs have described, would you have been able to detect
emphysema on the gross cross section?
Orthoefen Possibly. One thing I felt might be significant that is mentioned in the paper under
the gross lesions is that some of these lungs had a doughy consistency. I thought it was
significant in that it represented trapped air; however, it was not as consistent as I would like
to have seen it
Dungworth: I think we have to be careful in answering that question hastily. If we took a thick
section from the control lung and a thick section from one of the N02-high and looked at
them side by side, I believe one would say yes, there is a difference there, but it is only mild if
judged by human standards.
189
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Orthoefen Actually, you can tell the presence of dilation of airspaces on the slide by just
holding up the histologic slide and looking at it. I thought maybe you were speaking for the
gross lung.
Kleinerman: That means all nine histologic slides, if you just pick one up at random from
each case, you could tell it has dilation of the airspaces, or emphysema, if you compare it to a
control slide?
Orthoefer. No, not on a 0 and 3 basis, but yes if it had severe emphysema as compared to a
non-affected animal.
Kleinerman: Or you've got a slide of lung, a cross section, and you are looking at the mean
size of the airspaces throughout the lung. Can you tell if there is emphysema in the slide?
Dungworth: In the worst affected, yes, but it was still mild as compared to human emphy-
sema.
Orthoefen I think Dr. Hyde's presentation is going to be important because you can visualize
the lesions by SEM and it is more dramatic than the histologic lesion
Dungworth; This point is addressed directly in Chapter 12. We don't say the lesions are
severe, and the worst lesions we saw are mild relative to human emphysema in its usually
noted spectrum. On that, we are clear and must accept it; however, it is unequivocally
emphysema.
Kleinerman; Were there other significant findings as far as your grading scheme was
concerned?
Orthoefen There was pigment in the lungs but it's not severe. I graded the pigments on
whether they were present In fact, it was as bad in the controls as the treatment groups. One
thing that was somewhat significant was the mucous glands, and I think Dr. Tyler has plans to
pursue the quantitation study.
Dungworth: In his paper, Dr. Stephens mentioned "occasional evidence of exaggerated
goblet cell activation and evidence of perivascular fibrosis." This is an impression dial we
obtained, but we are not in a position to quantitate it at the moment We have to apply
quantitative methods and that is in the process of being submitted for funding. So we remain
sitting on the fence with regard to how much bronchial gland or mucous activity there is and
so on, which I think is appropriate until we have more information.
Bhatnagar: Dr. Stephens, you mentioned that there were increases in the endoplasmic
reticulum in many different types of cells.
Stephens: Only the type II cells.
Bhatnagar: Only in the type II cells of the exposed animals?
Stephens: Yes, but I would have to say that it occurs in a small number of type II cells found
mainly in the proximal alveoli.
190
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Bhatnagar: That would imply they are making a large amount of whatever type II cells make.
Stephens: That might be the case. That would be an assumption, of course.
Question: Did you say that you excised the lobe, weighed it, put it in fixative, and displaced
the fixative to get the specific gravity?
Orthoefen No. What I said was that the whole right lung, after it was preserved (fixed at 30
cm HgO pressure, and expanded to its maximum), was measured using the displacement
method of finding the volume of the right lung.
Question: And what did this tell you?
Orthoefen It just tells you the volume of the right lung. The right lung is approximately 59%
of the total lung volume.
Albert In the animals that had the lobectomy was there any difference in the weight of the
remaining lung?
Orthoefen I did not weigh those lungs because the lobe was missing. Actually, the weighing
started after the 14 lobectomized animals were done. Another point I would like to clarify is
there were only 14 when the sacrifice began, because one of the lobectomized animals had
died in California. Dr. Stephens mentioned six controls, and only had five. One of the animals
he had as a control was a practice animal and it was lobectomized first before any of the auto
exhaust dogs. We wanted to see if the technique would work and this was included as a
control animal This extra animal was not an auto exhaust dog. It came from the colony in
Colorado which was maintained by the AEC.
Stephens: I was aware of that
Stara: There were originally three subgroups of five controls, five N 02-high, and five raw
exhaust that were lobectomized.
Question: Why were these selected?
Stara: At the time, we felt that these three subgroups probably could be expected to
demonstrate the most distinct changes.
Albert Well, the point I was driving at was whether or not there were any data on the ability
of the remaining lung to enlarge.
Orthoefer I don't have displacement data on these lungs. I think weight is available. I'd have
to go back and look, but I know displacement values are not available.
Littlefield: Is there anything in the literature where the^effects of very low levels of carbon
monoxide have been evaluated for as long as this exposure has gone on? You've got a 5-year
exposure of carbon monoxide. Has the brain been looked at very thoroughly?
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Orthoefen I've looked at sections of all the brains. What is it you would like to see? We have
visual evoked response and spontaneous EEGs. Dr. Johnson of NIOSH and I collaborated on
several studies when he worked at our facility. But remember, we don't have CO alone. You
have to attribute changes to auto exhaust, not CO per se. The evoked potential and
spontaneous EEC study was done by putting animals down with secostrin, and maintaining
them with artificial respiration. Then we did spontaneous EEGs and the so-called visual
evoked response using a strobe light
Question: Would that be as sensitive as a pathological examination?
Orthoefer: I think probably more so, when you're talking about brain. A morphometric study
could be done where a person could count cells in the cerebrum; however, I've not been able
to dream up such a study yet
Dungworth: That would be a mammoth undertaking to find out whether there was anything
important
Orthoefer: The organ weights were compared as percent of body weight We compared each
treatment group to controls and then compared the exhaust versus non-exhaust, and found
nothing significant in regard to total organ size regarding the brain.
Littlefield: My original question was, I don't think there's anything in the literature where
they have animals that have been exposed for that long to carbon monoxide.
Orthoefer: We do have effects on blood, hematocrit, carboxyhemoglobin, etc.
Littlefield: My point is, I don't think it's ever really been examined and this may be an
opportunity.
Stara: As Dr. Orthoefer said in the beginning, some of these side studies represent an
afterthought as the exposures progressed, such as the cardiovascular studies and the
neurophysiologic evoked potential study.
Lewis: Every time we did another such project, it represented a relative hazard to the dogs, if
it involved anaesthesia. You could lose the animals.
Stara: The problem always occurred: should we or shouldn't we make another set of
observations, and perhaps lose another dog. The evoked potentials were done immediately
after removal from the atmospheres using sedation.
Lewis: Do you mean hours or days? Do you mean they had significant carboxyhemoglobin
levels when you did the evoked potentials?
Orthoefer: Do you have the date in the paper? This information could be obtained from a
combination of Dr. Johnson's paper (Chapter 9) and the blood data I presented earlier. One
could also look at a published reference about the blood rheology of these dogs.1
lBlock, W.M., Jr., S. Lassiter, J.F Stara, and T.R. Lewis. 1973. Blood rheology of dogs
chronically exposed to air pollutants. J. ToxicoL and AppL PharmacoL 25: 576.
192
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Lewis: I think it is the spontaneous data that hasn't been analyzed yet, but Dr. Johnson talked
this over with me before I left, since we were working on many things. If you use the
conventional analysis of variance, there was nothing significant, but if you have taken the
t-test comparison where you tested every treatment back to the control, there was a response,
and he couldn't identify what that was at that time.
Stara: That is exactly the problem in the treatment of some of the data. For example, the data
reported here before were analyzed using the t-test Ken Busch and You Yen Yang must help
us to evaluate all the papers from this point, and consider either both approaches or the most
appropriate method.
Hyde: Isn't the analysis of variance test of two parameters equivalent to a t-test?
Busch: Yes. The problem is that neither of those is exact in the case of a categorical response.
You don't have time for a 10-minute statistical summary. You enumerate all possible
outcomes under the assumption that controls and test groups are merely random selections
from a total group that is in fact unaffected by the exposures. An exact test would be to
enumerate all possible ways these animals could have been subdivided at random, given that
a certain number were destined to turn out in a certain way due to their inherent biological
variability. Then you calculate this difference between rank means and note all possible
values that this statistic could assume, cut off the upper 5% of the rank values, and if your
sample value appears in the upper 5% you say this can't be a random result — it's so unlikely
under a randomization. Therefore, we conclude that it wasn't random.
Orthoefer. Is that the ANOVA test?
Busch: No, that's a test I originated. This is an exact enumeration test based on hypothesis
testing and basic statistical decision theory. You can only do it if you have a big computer and
are willing to write the computer program to do it. Some of these nonparametric tests are
made up of tables for all possible combinations. If you're willing to go to the trouble, even
with a big table with nine categories, you could write a program and do it exactly.
Stara: Dr. Johnson has analyzed the data and he's responsible. I don't feel at liberty to change
anything at this point
Busch: If you find extreme significance, at the 1% or 0.1 % level, with the t-test, then that's
probably good enough. If you get approximately 5 or 10%, I wouldn't hang my hat on it, but
I would check it by doing more precise calculations.
Stara: Gentlemen, let's proceed to the next paper.
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12. MORPHOMETRIC AND MORPHOLOGIC EVALUATION OF
PULMONARY LESIONS IN BEAGLE DOGS
CHRONICALLY EXPOSED TO HIGH AMBIENT LEVELS
OF AIR POLLUTANTS*
D. Hyde, J.G. Orthoefer, D. Dungworth, W. Tyler, R. Carter and H. Lum
One hundred and four (104) beagle dogs comprising one control and seven treatment groups
were exposed 16 hours daily for 68 months to filtered air, raw or photochemically reacted
auto exhaust, oxides of sulfur or nitrogen, or their combinations. After a further 32 to 36
months in clean air, morphologic examination of lungs by light microscopy, scanning electron
microscopy, and transmission electron microscopy revealed two important exposure-related
lesions. There were enlargement of air spaces in proximal acinar regions, with and without
increases in the number and size of interalveolar pores, and hyperplasia of nonciliated
bronchiolar cells. Proximal enlargement of air spaces was most severe, both subjectively and
morphometrically, in those dogs exposed to oxides of nitrogen, oxides of sulfur, or oxides of
sulfur with photochemically reacted auto -exhaust In contrast, hyperplasia of nonciliated
bronchiolar cells was most severe in dogs exposed to raw auto exhaust alone or with oxides of
sulfur. The air space enlargement and hyperplasia of bronchiolar epithelium correlated with
functional impairment reported as occurring in these dogs. Foci of ciliary loss with and
without squamous metaplasia were occasionally observed in the trachea and bronchi.
The observations indicate that enlargement of proximal acinar air spaces with some loss of
interalveolar septa can develop in the absence of alveolar fenestrations. The persistent nature
of bronchiolar cell proliferations in such circumstances was also demonstrated. Two major
toxicologic implications are (1) the production of permanent lung damage by much lower
concentrations of pollutants than previously reported and (2) the apparent lack of additive or
synergistic effects between oxidant gases and sulfur oxides.
Epidemiologic studies have convincingly demonstrated that severe industrial smogs asso-
ciated with high levels of sulfur compounds and particulates cause increased morbidity and
mortality, particularly among people with chronic pulmonary or cardiac disease (7, 28, 44).
These same reviews reveal that there is less convincing epidemiologic evidence linking
increased incidence of disease with severe photochemical smog, whose principal components
are ozone and other oxidant gases.
Many toxicologic studies have been conducted on animals in attempts to define more
precisely the nature of effects likely to be caused by the common ambient air pollutants (4,14,
21). Most have been for short exposure periods and at concentrations of individual pollutants
greatly in excess of those ever encountered in urban smog. The purpose of this paper is to
describe and comment on morphometric and morphologic findings derived from a study in
which groups of beagle dogs were exposed for more than 5 years to a variety of polluted
atmospheres of varying complexity and at concentrations reached in severe episodes of air
lThis paper was first published in 1978 in Laboratory Investigation, Vol 38, No. 4, p. 455. It
is reprinted here by permission of the U.S.-Canadian Division of the International Academy
of Pathology.
195
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pollution. The pulmonary lesions present when the dogs were examined nearly 3 years after
the end of exposures correlate well with abnormalities in cardiopulmonary function described
elsewhere (references 9,34, and 50 and J.R. Gillespie, J.D. Berry, Y. Yang, L.L. White, and J.E
Stara, personal communication).
Materials and Methods
One hundred and four (104) female beagle dogs were randomly assigned to exposure
chambers which contained four dogs each (25). All of the dogs were from a single purebred
colony and were approximately 6 months of age at the beginning of the exposure. One group
of 20 control dogs (five chambers) was exposed to CBR (chemical, biologic, and radiologic)
filtered air. The remaining dogs were divided into seven groups of 12 (three chambers), each
of which was exposed 16 hours daily for 68 months to one of the following pollutants or
pollutant combinations: raw (nonirradiated) auto exhaust (R); irradiated auto exhaust (I);
oxides of sulfur (SOX); R + SOX; I + SOX; high nitrogen dioxide (N02-high) plus low nitric
oxide, and low N02 plus high nitric oxide (NO-high): Concentrations of the pollutants are
given in Table 1. Various physiologic parameters were determined before, during, and after
exposure (references 9, 34, and 50 and J.R. Gillespie et aL, personal communication). After
exposure ended, the dogs were kept indoors in clean ambient air for 32 to 36 months before
necropsy at Davis, California. In nearby Sacramento, California, the oxidant level averaged
0.05 ppm during the 3-year period (11). The dogs were maintained as an isolated colony and
were under close clinical observation during the entire period of study. At no time was there
evidence of infection or overt respiratory disease. During the 8 years of the experiment, 19
dogs died, mostly as a result of anesthetic procedures used for the cardiopulmonary function
studies or from bite wounds received during fighting. Deaths occurred in all groups, and it
was not possible to attribute direct causal relationship to the exposure atmospheres. We did
not have access to 14 additional dogs which had right middle lobes removed surgically at the
end of exposure (five dogs each from the I and NOg-high groups and four dogs from the
control group), hence 71 dogs were available at necropsy for this study (Table 1).
From 32 to 36 months after cessation of exposure, the dogs were weighed, measured, and
killed by exsanguination after deep surgical anesthesia using sodium pentobarbital. The
lungs, trachea, and attached structures were removed from the thorax in toto and trimmed of
extraneous tissue (heart and mediastinum). Subsequently, the lungs were weighed, the left
primary bronchus was ligated and the left lung was separated for biochemical analysis. The
trachea was cannulated and the right lung was fixed in the normal dorsoventral orientation by
intratracheal perfusion with cacodylate-buffered glutaraldehyde-paraformaldehyde at 30 cm.
fixative pressure for 16 to 18 hours. The volume of the right lung was then measured by
fixative displacement for subsequent morphometric evaluation. The lungs were stored in
fresh fixative until they were blocked for microscopic examination.
Blocks of tissue (2 by 3 by 1 cm) for histologic examination were taken from nine different
sites of each right lung of all dogs (Figure 1). Paraffin sections, 7 ^m thick, were cut and
routinely stained with hematoxylin and eosin.
The paraffin sections were used for stereologic evaluation. A linear correction factor for the
shrinkage of the fixed tissue due to processing (p) was calculated by dividing the square root
of the multiplied linear measurements of the sectioning face of the fixed tissue blocks by the
196
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Table 1. Measured Pollutant Concentrations to Which Dogs Were Exposed 16 Hours a Day for 68 Months8
Atmosphere
Control air (CA)
Nonirradiated auto
exhaust (R)
Irradiated auto exhaust (I)
SO2 + H2 S04 (SOx)d
Nonirradiated auto exhaust
+ SO2+H2SO4(R + SOx)d
Irradiated auto exhaust +
S02+H2SO4(l + SOx)d
Nitrogen oxides,
1{NO2-high)
Nitrogen oxides, 2 (NO-high)
No. of
dogsb
12
10
5
8
9
11
6
10
Pollutant (mg/m3)
CO HC(asCH4) N02
112.1 + 11.5 18.0±2.9 0.09±0.04
108.6 ±22.5 15.6 ±4.0 1.77+0.68
11 3.1 ±15.9 17.9 ±2.8 0.09+0.06
109.0+22.8 15.6 ±3.9 1.68 + 0.68
1.21+0.22
0.27 + 0.62
NO Ox (as Qs)
1.78 ±0.52
0.23 ±0.36 0.39 + 0.18
1.86 ±0.54
0.23 ±0.36 0.39 + 0.16
0.31 + 0.08
2.05+0.26
S02 H2SO4C
1.10 ±0.57 0.09 + 0.04
1.27 ±0.61 0.09 + 0.04
1.10±0.56 0.11 + 0.04
aExposure was from September 7,1965, to May 7,1971. Values are means ± 1 standard deviation; milligrams per cubic meter = parts per million
molecular weight - 24 at standard temperature and pressure (modified from Lewis et al. [34]).
bNumber of dogs available at necropsy.
°Optical sizing indicated >90% of particles were <0.5 pm in diameter.
dx indicates oxides of sulfuric acid.
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Figure 1
Cranial, middle, and caudal lobes of the canine right lung are depicted from the lateral view;
the accessory lobe is depicted from the caudal view. Blocks of tissue for histologic evaluation
were selected from indicated sites in transverse plane for cranial and middle lobes, sagittal
plane for caudal lobe, and frontal plane for accessory lobe.
measurements on the processed sections (51). A screen magnification of X125 was used on an
image analyzer to obtain the primary measurements (12). A guard frame of 100,000 picture
points was used to avoid inaccurate measurements at the borders of the video screen, leaving
a screen matrix of 500,000 picture points. The image analyzer, a microscope attached to a
video system which is capable of measuring structures on the basis of density, has been shown
to give stereologic values consistent with accepted manual methods in the dog lung (27). The
image analyzer was set to detect the alveolar air spaces and the detection level was
standardized by use of a preset level of detection on a specific field of a wire mesh grid.
Four random measurements were made at each of five stratified layers on each slide (47).
Fields that contained large vessels or bronchi were rejected and other fields selected. The
volumetric densities of alveolar airspace (VVa) and alveokr tissue (Vvt) (cm3/cm3) were
estimated by the fractional area of points lying on their respective components. The
198
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volumetric density of alveolar tissue was corrected for the Holmes effect, the error introduced
by finite section thickness (51). The volumetric correction coefficient (Kva = 0.7) was
calculated from an alveolar diameter of 118 ^m for the lungs of small dogs (45) and an
interalveolar septal thickness of 10 ^m. The volumetric density of alveolar airspace was
adjusted accordingly.
The surface density of alveoli (Sva, n^/cm3) was calculated using the formula (52)
Sva = 4 N./L, (1)
where Lt is the cumulative length of test lines (scanning lines on the video screen) and Na is
the number of intersections of test lines and interalveolar septa. Alveolar surface density was
corrected for the effect of shrinkage of the fixed tissue due to processing by multiplying Sva
by(p2/p3orl/p)(51)i
The internal surface area of the unfixed right lung (Sar, m2) was calculated using the formula
(51):
S.r^Sv.-^Lr-VvpPp2!2) (2)
where VLr is the displaced volume of the processed right lung (volume of fixed right lung/p3),
Vvp is the volumetric density of parenchymal tissue estimated by point count of gross lung
slices (51), p2 is a factor for converting areas of processed to fixed tissue, and f2 is a factor for
converting areas of fixed to fresh tissue. The alveolar surface density (Sva) used in this
formula is the unconnected value. The linear correction factor for shrinkage due to fixation (f)
was determined for each dog from a modification of the formula used by Thurlbeck (47):
, _ /[TLC+WL] 0.59 \ 1/3 ,,,
f= I Vu / (3)
where WL is the weight of the lung and TLC is total lung capacity determined by a body
plethysmograph (J. R. Gillespie et aL, personal communication). WL was used as an estimate
of tissue volume. The value of 0.59 is an estimate for the population average of the ratio of
the right lung capacity to total lung capacity in normal dogs (13).
The sample means and standard errors of the morphometric parameters, WB (body weightX
WL, TLC, VLr, Sva, Sar, Vyt, Vvp, f, and p, were recorded for each group (Table 2). One-way
analyses of variance (46) were used to compare the eight exposure groups for each of the
morphometric parameters above and for internal surface area normalized by body weight
(Sar/Wb). In addition to the simultaneous comparison of the eight group means, each of the
seven treatment group means was individually compared to the control group mean using
Dunnett's method of multiple comparison (46). Further analyses of the alveolar surface
density (Sva) were performed to compare the eight exposure atmospheres within each of the
nine sample regions in the right lung (Figure 1) and to compare the dorsal regions to the
ventral regions within each group.
Each sample region was also examined histologically and graded, without knowledge of
treatment group, for three types of lesions commonly observed in old dogs (Table 3) (43).
These lesions were not evaluated morphometricalry. The sample regions were examined
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Table 2. Morphometric Parameters for Dog Lungs from Control and Exposed Groups3
WB (kg)
WL(gm)
TLC (cm3)
VLr (cm3)
Sva(m2/cm3)b
Sar (m2)c
Vvt(cm3/cm3)
Vvp (cm3 /cm3)
f
P
Group: C
No. of dogs: 12
9.08
±0.24
100.5
±3.7
1095
±64
424
±25
0.0279
±0.0013
14.85
±0.81
0.1541
± 0.0078
0.888
± 0.003
1.1835
±0.014
1.2170
±0.0120
R
10
10.24
±0.47
119.9"
±5.3
1374"
±69
500
±31
0.0279
±0.0013
18.24
±1.49
0.1398
± 0.0036
0.896
± 0.004
1.2104
±0.017
1.1840
±0.0120
5
9.15
±0.83
111.6
±11.7
1234
±90
529"
±32
0.0267
± 0.0021
16.47
±2.08
0.1303
± 0.0063
0.888
± 0.007
1.1459
± 0.028
1.1700
±0.0170
SG-X
8
9.24
±0.47
121.9d
±4.7
1446d
±51
512"
±23
0.0254
±0.0012
17.29
±1.00
0.1326
± 0.0060
0.892
± 0.007
1.2207
± 0.023
1.1960
±0.0170
R + SOx
9
9.61
±0.37
114.6
±6.9
1277
±80
466
±28
0.0280
±0.0014
16.89
±1.39
0.1562
± 0.0092
0.888
± 0.006
1.2086
±0.014
1.2060
±0.0100
l + SOx
11
9.89
±0.38
112.7
±3.2
1272
±55
497
±17
0.0257
± 0.0009
15.71
±0.73
0.1331
± 0.0043
0.884
± 0.006
1.1795
±0.0128
1.2030
±0.0210
NO2 -high
6
9.06
±0.39
108.5
±5.8
1342"
±97
525d
±28
0.0229"
±0.0019
14.27
±1.30
0.1426
±0.0116
0.857"
± 0.002
1.1770
± 0.0272
1.2270
±0.0150
NO-high
10
9.40
±0.37
108.8
±4.1
1262
±51
479
±21
0.0258
± 0.0008
15.85
±0.68
0.1395
± 0.0071
0.898
± 0.005
1.1964
± 0.0200
1.2140
±0.0160
aValues are group means ± 1 standard error of the mean. See text for explanation of morphometric parameters.
bCorrected for shrinkage due to processing.
cCorrected for shrinkage due to processing and fixation.
dSignificantly different from the control group (P<0.05, Dunnett's multiple comparison test).
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independently by two investigators who reached the same conclusions. For each lesion an
average grade (grades were averaged over the nine regions) was computed for each dog. The
dog averages were then used in the Kruskal-Wallis one-way analysis of variance (46) to test for
differences between the eight groups for each lesion. Individual comparisons of the treatment
groups to the control group were made using the Mann-Whitney U test (46).
Specimens were taken from each right lung for scanning electron microscopy (SEM) and
transmission electron microscopy (TEM). Tissue blocks (10 by 10 by 5 mm) were taken from
dorsal and ventral regions of the trachea 2 to 3 cm cranial to its bifurcation, the ventrolateral
wall of the right primary bronchus between the lobar bronchi of the middle and caudal lobes,
and the dorsal and ventral regions of the right cranial and caudal lobes adjacent to those
regions depicted in Figure 1. In the dorsal region of the right caudal lobe, tissue blocks were
taken from the more caudal site. From each of those four blocks from the right cranial and
caudal lobes, two specific sample sites were selected using a dissecting microscope. One
sample contained segmental and subsegmental bronchi, while the other contained acini
(terminal bronchioles, respiratory bronchioles, and adjacent alveolar parenchyma). This gave
a total of 11 sample sites from each lung for SEM and TEM examination. One airway within
each sample was bisected longitudinally using an alcohol-cleaned razor blade. One-half of
each bisected airway and parenchyma was processed for SEM by dehydration in a graded
series of ethanol followed by amyl acetate which was exchanged for liquid C02 in a critical
point drying apparatus. The dried tissue was mounted on aluminum stubs and coated with
carbon followed by gold-palladium in a high vacuum evaporator. An ETEC autoscan SEM
was used to examine the specimens.
The complementary halves of specimens examined by SEM were processed for TEM using a
modification of the large epoxy-embedded block technique (35). Sections (1 fim) were cut,
Table 3. Grading System for Selected-Pulmonary Histologic Lesions
Lesions Grade * Explanation
Pigment and dust 0 No significant pigment.
accumulation 1 Minimal amounts of pigment, usually within
macrophages adjacent to smooth muscle of
alveolar ducts and small airways.
2 Clusters of pigment-laden macrophages in
interstitium adjacent to vessels and airways,
sometimes associated with recognizable
lymphatics.
3 Large accumulations of pigment-laden cells in
perivascular and peribronchiolar sites.
Bronchial gland activity
Calcification of bronchial
cartilage
0
1
2
3
0
1
2
Few or rare, inactive.
Few or rare, active.
Several, active.
Many, active.
No calcification.
Minimal calcification.
Calcification in most of
cartilage in section.
201
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mounted on glass slides, and stained with methylene blue, azure II, and basic fuchsia After
histologic examination of these slides, specific areas from these blocks were excised, attached
to the end of Beem capsules with epoxy resin, and sectioned for TEM. The thin sections were
stained with uranyl acetate and lead citrate. In order to enhance the correlation between light
microscopy, SEM, and TEM, the SEM blocks with significant lesions were reembedded for
light microscopy and TEM observation of the same lesions. A Zeiss 10 electron microscope
was used to examine the specimens.
Each SEM block of tissue was graded without knowledge of treatment group according to
those lesions easily evaluated by the SEM but difficult to grade histologically (Table 4).
Determination of the criteria for the grading system was based on examination of material by
two investigators; tabulation of the data was done by one of them.
For each dog the average grade of the trachea, right primary bronchus, and segmental and
subsegmental bronchi sites was recorded for lesions numbered 1 and 2. The dog averages
were used to compare the eight exposure groups by the Kruskal-Wallis one-way analysis of
variance method (46). Individual comparisons of overall, dorsal, and ventral regions of
treatment to control group regions were made using the Mann-Whitney U test (46)i Similarly,
the average grades of lesions numbered 3 and 4 of overall, dorsal, and ventral regions were
calculated in order to compare all groups and each treatment to the control group.
Table 4. Grading System for Selected Pulmonary Lesions Evaluated by SEM
Lesions
Grade
Explanation
Ciliary loss without
squamous metaplasia3'6
Ciliary loss with squamous
metaplasia3*
Nonciliated bronchiolard
cell hyperplasia
Interalveolar pores3'11
0 No ciliary loss.
1 <2 sites0 of ciliary loss.
2 2-5 sites.
3 >5 sites.
0 No squamous metaplasia.
1 <2 sites of squamous metaplasia.
2 2-5 sites.
3 >5 sites.
0 No hyperplasia.
1 Hyperplasia in terminal bronchiole only.
2 Hyperplasia in terminal bronchiole and
respiratory bronchiole.
3 Hyperplasia in terminal bronchiole, respiratory
bronchiole, and alveolar duct.
0 Few small interalveolar pores.
1 Many small interalveolar pores.
2 Many small and few large interalveolar pores.
aPer SEM tissue specimen.
"Trachea and bronchi.
°Site = area> 100/im x 50 ^m.
"Distal airways.
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Results
No significant gross lesions were observed. Eight lungs failed to collapse to the normal extent
on removal from the chest. Three of these were from the R group, three from the NO-high,
one from the N02-high, and one from the SOX group. Six lungs had small, discrete, marginal
zones of atelectasis. This had no significant relationship to specific treatment group or to
subsequently discovered histologic lesions.
Morphologic interpretations of pulmonary lesions were the result of correlative examination
of the same airways and adjacent parenchyma by light microscopy, SEM, and TEM, whereas
quantitative evaluations of relative magnitude of involvement among the various groups of
dogs were based on SEM and light microscopy. The normal histologic and ultrastructural
morphology of the dog lung has been described by several investigators (16, 22, 37) and our
observations are in general agreement with theirs. Only those anatomical structures that are
particularly relevant to the lesions we observed in this study will be described. Major
exposure-related lesions were within pulmonary acini (Figure 2a). They consisted of proximal
acinary air space enlargement, with and without increases in number and size of interalveolar
pores, and nonciliated bronchiolar cell hyperplasia. These will be described in detail.
Inconsequential histologic lesions, which were as common in lungs of control animals as they
were in exposed dogs, were rare minute foci of chronic pneumonia or small granulomas. The
latter have usually been interpreted as being caused by migration of parasitic larvae (6).
The analysis of the graded severity of three types of pulmonary histologic lesions commonly
observed in old dogs (Table 3) showed no significant differences between treatment and
control groups.
The analysis of variance for the morphometric parameters, body weight (Wu), right lung
volume (VLrX alveolar surface density (SvaX internal surface area of the right lung (SarX
internal surface area of the right lung normalized by body weight (Sar/WfiX fixation
shrinkage (f), and processing shrinkage (p), showed no significant differences between the
eight exposure groups. The analysis of lung weight (WL) and volumetric density of alveolar
tissue (Vvt) revealed nearly significant differences between the group means (P = 0.085 and
0.090, respectively). For the remaining parameters, total lung capacity (TLC) and volumetric
density of parenchyma! tissue (Vyp), the analyses of variance showed significant differences
between the exposure atmospheres (P = 0.0175 and P = 0.0003, respectively).
Dunnett's multiple comparison test revealed no significant differences between the seven
treatment group means and the control group mean for the parameters WB, Sar, Vvt, f, p,
and Sar/Wjj. For the parameters Sva and Vyp, Dunnett's test showed the N02-high group
mean to be significantly smaller than the control group mean. For the parameter WL, the R
and SOX group means were significantly larger than the control group mean.
The average TLC for the R, SOX, and N02-high groups was significantly larger than the
average TLC for the control group. For the volume of the fixed right lung the averages of the
SOX, N 02-high, and I groups were significantly larger than the control group. The results of
the analyses of variance and multiple comparisons for each of the morphometric parameters
are given in Table 5.
203
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Figure 2.
a. Pulmonary acinus from a control dog originates from a distal bronchus (b) and consists of a
terminal bronchiole (Tb) with a nonuniform branching pattern of respiratory bronchioles (Rb)
and alveolar ducts, b. Lung from the N02-high group of dogs at the same magnification
illustrates distal air space enlargement and strands of tissue (arrow). X20.
204
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Table 5. Summary of Results for Morphometric Parameters3
Group significantly different
Parameter Analysis of variance from control*5
WB
WL
TLC
VLr
Ova
Sar
Sar/WB
Vvt
Vvp
f
P
NSC
P = 0.0850
P = 0.0175
NS
NS
NS
NS
P = 0.0900
P = 0.0003
NS
NS
None
R, SOX
R, SOX, NO2-high
SOX, NO2-high, I
NO2-high
None
None
None
NO2-high
None
None
aSee text for explanation of morphometric parameters.
bGroups significantly different from the control with more severe lesions (P<0.05, Dunnett's
multiple comparison test).
°No significant differences between group means exist (P^O.10).
Further analyses of SVa showed no significant differences between control and exposure
groups for all regions of the lung, except for the N02-high group. The cranial, caudal, and
accessory lobes and the dorsal and ventral regions of all lobes were significantly decreased
from controls in the NC>2 -high group.
Air Space Enlargement
The degree of enlargement of alveolar air spaces, which was derived from alveolar surface
density, was notably different in the N02-high, SOX, I + SOX, NO-high, and I groups in
decreasing order of severity (Figure 3). In the SOX, I + SOX, NO-high, and I groups, acinar
involvement appeared to be random and its proportion and degree varied considerably
among lungs of dogs within groups. The range was from an occasionally affected acinus to
approximately a 50% involvement in any one histologic or SEM sample. Variability was also
present among lungs of dogs in the N02 -high group, but there were samples in which all
acini were affected (Figure 2b). No preferential distribution was observed in any lobar region.
Structurally, the degree of enlargement as observed by light microscopy (Figure 4) corre-
sponds well with the morphometric ranking of decreased alveolar surface density among
exposure groups (Figure 3). Air space enlargement was centered on respiratory bronchioles
and alveolar ducts. In conjunction with these lesions, there was also apparent loss of
interalveolar septa (Figures 4 and 5).
Scanning electron microscopy revealed the apparent loss of alveolar walls more clearly
(Figure 5). An important feature of the air space enlargement was that in the lungs of most
exposure groups it was not associated with fenestrations or prominent increases in number
and size of pores in interalveolar septa (Figure 5). On the other hand, considerable increases
in number and size of interalveolar pores were found in some lungs without any air space
enlargement (Figure 6). The lack of correlation between development of air space enlarge-
ment and enlargement of interalveolar pores was further emphasized by the fact that when
the latter occurred it was distributed throughout the alveolar parenchyma, rather than being
205
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DS
or
1 8'
14-
10-
n-
1
I
I
I
L
"W
I
f
JL
ft
L
I
T
™
1
1
1
0.033
as
va
0.029
m2/cm3
0.025
0.02 I
Group C R+SOX R I N0high I + SOX SOX NO2hlgh
f Dogs 12 9 10 5 ION 6 6
Figure 3
Comparison between the control and all other exposed groups by surface density (Sva,
m2/cm3) and internal surface area of the right lung (Sar, m2). The control group to the left and
all other exposed groups from left to right are plotted by decreases in Sva- Each bar
represents the group mean ± 1 standard error. Significant differences from the control
group are marked by an asterisk.
limited to proximal acinar regions as was the increase in size of air spaces. Although there
were significant increases in frequency and size of interalveolar pores in most exposure
groups as compared to the controls (Table 6), only in lungs of the most severely affected dogs
in the N02-high and SOX groups was there concurrence between air space enlargement and a
striking increase in number and size of interalveolar pores (Figure 7).
Lesions in Conducting Airways
Predominant lesions in conducting airways were epithelial hyperplasia in bronchioles and foci
of ciliary loss, with or without squamous metaplasia, in the trachea and bronchi. The relative
severity of these lesions for the various treatment groups is significantly different from the
control group as shown in Table 6.
The hyperplasia bronchiolar lesions consisted of discrete or confluent micronodular epithelial
projections. The degree of hyperplasia was most pronounced in the R + SOX and R groups of
dogs where occasionally the hyperplasia was sufficient to cause partial occlusion of bron-
chiolar hinrina (Figure 8). In least affected lungs, small hyperplasia lesions were observed at
206
-------
sssf ^Isf '-k '^
• j ^; *sf ?*$>•* *j-
'A f\ 'V/^L: " C
f*f~-t"fi' \ ~* •
y^>-r*>
as^;
' * » " / B .x^ » , '.- - *S
,. -i**.*»; ./si' '**-'H-'
Figure 4
Comparison of distal airways and parenchyma in lungs from control and exposed groups, a.
Numerous respiratory bronchioles (Rb) and alveolar ducts (Ad) are interspersed among alveoli
in a lung from the the control group, b. Slight enlargement of respiratory bronchioles (Rb)
and alveolar ducts (Ad) of a representative lung from the NO-high group, c. Mild enlargement
and loss of interalveolar septa in alveolar ducts (Ad) in a representative lung from the SOX
group, d. Mild to severe enlargement and loss of interalveolar septa characteristic of a lung
from the N02-high group. A respiratory bronchiole (Rb) is adjacent to a greatly enlarged
alveolar duct (Ad). X16.
207
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Figure 5
Comparison of distal airways in lungs from control and exposed dogs. a. Respiratory
bronchiole (Rb) branches into alveolar ducts (Ad). Numerous alveoli and a moderate number
of alveolar pores (arrow) are present in this lung of a control dog. b. Lung from the SOX group
of dogs at the same magnification illustrates alveolar duct (Ad) enlargement and loss of
interalveolar septa It is similar to the lesion in Figure 4c. c. Higher magnification in a region
of b shows short interalveolar septa (black arrow) and shallow alveoli (a). Few interalveolar
pores are seen (white arrow). Figure 5a and b, X50; c, X120.
208
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Figure 6
Respiratory bronchiole (Rb), alveolar ducts (Ad), and alveoli have the same conformation as
controls despite widespread increase in interalveolar pores (inset, arrow) in a lung from the R
group of dogs. X50; inset, X200.
the junction of the terminal bronchiole and the first-order respiratory bronchiole. Greater
involvement consisted of more frequent and taller proliferations and their extension into all
orders of respiratory bronchioles and the first-order alveolar duct.
The frequency of occurrence of hyperplastic lesions in acini was striking in the R group (65%)
and R + SOX group (42%) of dogs, whereas it was 20% or less in all other groups. Analyses
of the average grades for the degree of severity of the hyperplastic lesion showed significant
differences between exposure atmospheres (Table 6). The average grades for the R + SOX,
209
-------
Table 6. Summary of Results of Pulmonary Lesions Evaluated by SEMa
Over-all
Lesions differences Groups significantly different Groups significantly different Groups significantly different
between from control0^ from control in dorsal regiond
-------
Figure 7
Junction between a distal respiratory bronchiole (Rb) and proximal alveolar duct (Ad) shows
air space enlargement with numerous alveolar pores and fenestrations in a lung from the
N02-high group of dogs. Fenestrations and trabeculae (arrow) are common in alveolar ducts
(inset). X60; inset, X160.
R, I, N02-high, SOX, and I + SOX groups were significantly increased over the control group
as listed from greatest to least severe. In the dorsal regions only the R + SOX and R groups
were significantly increased over dorsal regions in the controls, whereas the ventral regions of
the R 4- SOX, R, I, NOa-high, and NO-high groups were significantly increased over the
ventral regions of the controls. Within each group, however, no significant difference was
observed between dorsal and ventral regions.
The hyperplastic nodules were predominantly composed of hypertrophic nonciliated bron-
chiolar cells (Figures 9 and 10). The nonciliated bronchiolar cells in exposed and control dogs
211
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T* Q
IrSar hyperplasia of nonciliated bronchiolar cells (inset) partially occludes the lumen of a
terminal bronchiole in a lung from the R group of dogs. An aggregation of inflammatory cells
(arrow) is seen in the distal region of the terminal bronchiole. X250; inset, X920.
212
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Figure 9
Comparison of terminal bronchiolar epithelium in lungs from control and exposed dogs. a.
Epithelium of a terminal bronchiole (Tb) from the lung of a control dog has a mixture of
ciliated (arrows) and nonciliated cells, b. Hyperplastic nodules in a terminal bronchiole (Tb) of
a lung from the I group of dogs show a mixture of hypertrophied ciliated and nonciliated
cells. Goblet cells (arrow) are also visible (1 /^m Epon-embedded section). X370.
213
-------
Tb
Figure 10
Hyperplastic nodule composed primarily of nonciliated cells protrudes into the lumen of a
terminal bronchiole (Tb) in a lung from the I group of dogs. The clear areas in the nonciliated
cells (NC) represent areas of fixative-leached glycogen. A macrophage (M) and segments of a
connective tissue core (arrows) are also present. An alveolar macrophage (AM) is in the airway
lumen. XI,600.
214
-------
contained krge clear areas, scattered lipid inclusions, a few dense staining mitochondria, an
inconspicuous Golgi apparatus, sparse rough endoplasmic reticulum, and occasionally, accu-
mulations of smooth endoplasmic reticulum in the apical region. In lungs of dogs (external
controls) stored in fixative for short periods of time prior to TEM processing, the cytoplasm of
nonciliated bronchiolar cells contained large amounts of glycogen Since the lungs in this
study were stored in fixative for more than a year before being processed for TEM, we believe
the clear areas in nonciliated bronchiolar cells represent regions from which glycogen was
leached during storage. In addition to the major component of hypertrophic nonciliated cells,
ciliated cells and undifferentiated cells were also present in the large proliferations (Figure
11).
The principal lesions observed in trachea and bronchi of exposed dogs were foci of ciliary
loss, with and without associated squamous metaplasia. These abnormalities were also rarely
present in airways of control dogs. On subjective evaluation, there appeared to be a definite
increase in size and frequency of these lesions over controls in at least some of the lungs of
dogs in all exposure groups. Analysis of the grading showed significantly increased ciliary loss
without squamous metaplasia in the N02-high and SOX groups, whereas ciliary loss with
squamous metaplasia was significantly increased in the R + SOX group (Table 6).
In control dogs, the epithelial lining of trachea and bronchi consisted principally of ciliated
cells and mucous-producing (goblet) cells. The cilia were shorter and less numerous on the
dorsal membranous portion of the trachea that extends between the incomplete tracheal
cartilages. In this location, ciliated cells, nonciliated cells (Figure 12), and openings of
submucosal glands were easily observed with SEM. In the epithelium of the ventral and
lateral regions of the trachea and of the bronchi, the cilia were more numerous and long
enough to conceal most of the nonciliated cells and glandular openings from view with the
SEM.
In exposed dogs, the distribution of foci of ciliary loss associated with squamous metaplasia
differed from that of foci of ciliary loss alone. The foci of ciliary loss alone that were counted
as significant were greater than 100 by 50 ^m in size and were distributed randomly in all
levels of airway. The cilia were usually completely absent from the surface epithelium, but
some lesions had cells with long microvilli and a few short cilia (Figure 13). The composite
cells were predominantly of the cuboidal mucus-secreting type. Basal cells were in their
normal position and had no unusual distinguishing characteristics.
Foci of ciliary loss associated with squamous metaplasia were observed predominantly in the
primary bronchus and the dorsal region of the trachea, but were also observed in the
intrapulmonary bronchi in the N02-high, SOX, and I + SOX groups of dogs. Foci of ciliary
loss associated with squamous metaplasia typically protruded into the airway lumen and were
up to 500 ftm in diameter (Figure 14). Sections from reembedded SEM blocks containing
these lesions confirmed the nature of the squamous metaplasia (Figures 15 and 16). Other
components observed in this type of lesion were cells with lucent to intermediate density
inclusions, surface-ciliated cells with few cilia, cells beneath the surface with no cilia but with
numerous basal bodies randomly distributed in the cytoplasm, and a variety of inflammatory
cells. Cells adjacent to the basal lamina were often columnar in shape, and their basal borders
often had cytoplasmic projections through the basal lamina into the lamina propria.
215
-------
Figure 11
In a lung from the N02-high group of dogs, a ciliated cell (C) lies deep within a bronchiolar
hyperplastic nodule, close to the basal lamina (bl). The cytoplasm has dispersed basal bodies
(bb) and cilia are in the intercellular space (arrow). Serial sections showed cilia exiting from
this cell. X6,700.
216
-------
Figure 12
In the dorsal membranous portion of the trachea, the ciliated cells with short cilia (C) and
long microvilli and the apical portion of goblet cells (G) are easily seen in a lung from a
control dog. X2,000.
Two potentially important aspects of lesions in airways have not been satisfactorily evaluated.
These are changes in the number and activity of mucous glands and goblet cells, and
peribronchiolar fibrosis. Histologic examination provided equivocal evidence that these
features were increased above controls in lungs of exposed dogs. Accurate comparisons of the
activity of the mucous apparatus among the various treatment groups will be made morpho-
metrically.
Discussion
The findings of air space enlargement and hyperplasia of nonciliated bronchiolar cells in
lungs from beagles of most treatment groups 32 to 36 months after exposure ended have
217
-------
Figure 13
Focus of ciliary loss without squamous metaplasia in a primary bronchus from a lung in the
NOs-high group of dogs. Ciliated cells are reduced in number and have fewer cilia per cell
Goblet cells do not have apical projections (G), but ciliated cells have numerous microvilli (C).
X2,350.
important implications with regard both to pathogenesis of such lesions and for the
toxicology of air pollutants.
Morphologically, both histologic and SEM observations revealed that in lungs from five of the
seven treatment groups there was appreciable enlargement of distal air spaces which was
centered on respiratory bronchioles and alveolar ducts and was associated with apparent loss
of interalveolar septa. These observations are supported by the morphometric decreases of
alveolar surface density in the same groups (Figure 3). Since the enlargement occurred
primarily in the region of respiratory bronchioles and alveolar ducts, as opposed to being
218
-------
Figure 14
Focus of ciliary loss accompanied by squamous metaplasia is seen as a proliferative lesion (P)
which protrudes into an airway lumen of a bronchus of a lung from the R group of dogs. The
surface of the lesion has numerous microvilli and surface folds (inset). X310; inset, 6,550.
219
-------
Figure 15
Section of a proliferative lesion (P) that protrudes into the lumen (L) of a distal bronchus in a
lung from the NOa -high group of dogs illustrating squamous metaplasia. At least five cells
deep, the lesion has a connective tissue core (C) containing numerous mononuclear inflamma-
tory cells (1 fjua. Epon-embedded section). X375.
distributed throughout the alveolar parenchyma, we consider it to be analogous to an
incipient stage of human proximal acinar (centrilobular) emphysema (2). Although it was of
mild degree compared to the clinically important cases of human emphysema (41), it was
sufficient to cause abnormalities in pulmonary function. The two groups with the greatest
decrease in alveolar surface density (N0£-high and SOX groups) (Figure 3) had higher than
normal dynamic compliance at lower frequencies than the control group (J.R. Gillespie el aL,
personal communication). The investigators interpreted this difference as evidence of
decreased lung elasticity in those groups (J.R. Gillespie et aL, personal communication).
Since alveolar surface area increases directly with increases in lung volume (equation 2), the
notable increases in the internal surface area of the right lung in most exposure groups as
compared to controls (Figure 2) are primarily attributed to increases in lung volume (Table 2).
The internal surface area of the right lung of dogs in the N02-high group was approximately
the same as the control group (Figure 3X even though lung volume and lung weight were
significantly increased in the NOa-high group over controls (Table 5). It seems likely that the
loss of alveolar tissue in the N02-high group (Figure 3) counterbalanced the increase in
alveolar surface area that would be expected with a simple increase in lung volume.
220
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Thin section adjacent to the area seen in Figure 15. Columnar- to spindle-shaped cells with
intracellular fibrils (F) and numerous intercellular processes connected by desmosomes (V)
(inset) were the distinguishing characteristics of squamous metaplasia. A ciliated cell (c) sends
cilia into the bronchial lumen (L). X3.150; inset, X14,250.
221
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Comparison of our control group value for internal surface area of the right lung (14.85 m2)
with that predicted by body weight from a group of 22 mongrel and beagle dogs (18.63 m2)
(45), shows a lower than predicted value for our control group. We attribute the difference to
our use of light microscopy, whereas Siegwart et aL (45) used electron microscopy. In a study
from the same laboratory, it was shown that surface density increases proportionally to the
optical resolution of the method of measurement (29). The increase in surface density by
approximately 25% using electron microscopy (29) accounts for the difference in internal
surface areas. The possibility that overinflation in some exposed groups accounted for the
marked decreases in alveolar surface density seems very unlikely since there were no
significant differences in the fixation factor (f) between control and exposed groups (Table 5).
Hence we conclude that lung inflation and shrinkage due to fixation were not significantly
different among any of the groups of beagles.
Two patterns of morphologic development of emphysema have been described. One is the
progressive enlargement and coalescence of holes (fenestrations) in the walls of respiratory
bronchioles and alveolar ducts, with progressive shortening of interalveolar septa (23). Kuhn
and Tavassoli (32) have shown by SEM that the morphogenesis of elastase-induced emphy-
sema in the hamster is of the latter pattern. It is generally believed that the increase in size of
air spaces in human proximal acinar (centrilobular) emphysema is associated with fenestra-
tion (10,32,41,42). SEM observations in this study indicate, at least in the dog, that proximal
acinar enlargement of air spaces can develop in the absence of fenestration. In fact, this was
the usual event Increases in number and size of interalveolar pores, which appear to be the
basis for large fenestrations (10, 42X when present were fairly evenly distributed throughout
acini. Only in the most severely affected lungs of dogs in the N0£-high and SOX groups was
there concurrence between enlargement of air spaces and exaggerated numbers and sizes of
pores. Here strands of tissue considered to be hallmarks of human centrilobular emphysema
were also present.
A further aspect of the alveolar lesions detected in lungs of these dogs deserves mention;
namely that pulmonary function studies showed a gradual worsening of abnormalities
associated with air space enlargement between the end of exposure and after 2 years of
recovery in clean air (J.R. Gillespie et al, personal communication). Although we cannot
comment on the extent of morphologic changes over the same time period, the finding of
increased levels of prolyl hydroxylase activity in lungs of most of the groups of dogs in
absence of increases in total collagen content (39) suggests that there is persistent increase in
collagen turnover. This accords with continued development of emphysema after a single
administration of elastase to hamsters and the suggestion that abnormal mechanical stresses
induced by the original insult might cause persistent rearrangement and hence progression of
the lesion (32, 33). In the beagles, this would be superimposed on the enlargement of air
spaces seen in control aging populations (27).
Hyperplasia of bronchiolar epithelial cells, predominantly of the nonciliated variety, was
present to some degree in all groups of exposed dogs. We have not called nonciliated
bronchiolar cells of the dog "Clara" cells because if the latter designation is to be useful it
should be limited to cells with specific ultrastructural and functional characteristics, not used
as a blanket term for all cells without cilia-lining bronchioles in all species. Since there are
definite ultrastructural differences between nonciliated bronchiolar cells of dogs and Clara
cells such as those studied in rodents (31), we prefer to use the more noncommittal term in
our study of dogs until the situation is resolved. The bronchiolar proliferations had persisted
222
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despite removal of the original inciting insult approximately 3 years previously. Because we
examined the bronchiolar lesions at one point in time, we cannot establish whether the
proliferative nodules were slowly regressing or irreversible. The lengthy period since cessation
of exposures and evidence from pulmonary function studies, particularly measurements of
pulmonary resistance in the most severely affected group of dogs (R + SOX), make it less
likely that the proliferation was regressing. There was no evidence of atypical cellular features
considered to be indicative of a premalignant change, but these proliferative lesions might
conceivably be more responsive to carcinogenic stimuli than normal epithelium (15). A similar
persistence of nodular hyperplasia of nonciliated bronchiolar (Clara) cells was reported in
mice where proliferation caused by a 120-day exposure to ozone was still present after an
additional 120 days in clean air. Other components of airway damage such as ciliary loss and
metaplasia of tracheal epithelium resolved during the 120-day recovery period (40).
The ciliary loss and squamous metaplasia observed in trachea and bronchi appeared to be of
minor importance. Speculations regarding reasons for their persistence parallel those made
for bronchiolar cell hyperplasia. Squamous metaplasia in dogs has been caused by a wide
variety of inhaled irritants such as ozone (19), sulfuric acid mist (1), sulfur dioxides (3), and
cigarette smoke (5, 17), but only in the last named instance is there evidence that it might
progress to squamous cell carcinoma (5). The R + SOX group of dogs showed a significant
increase in squamous metaplasia in comparison with controls (Table 6). Pulmonary resistance
was also highest in the R + SO* group, possibly in part due to increased airway turbulence
associated with squamous metaplasia. It is of interest that the R + SOX group showed the
greatest severity of bronchiolar hyperplasia (Table 6) but no air space enlargement The
frequency of bronchiolar hyperplasia was also approximately fourfold that of squamous
metaplasia in the R + SOX group of dogs. Although total pulmonary resistance is usually
considered to be principally an index of resistance to flow in larger airways (36), it has been
reported that the diameter of bronchioles is the major determinant of pulmonary resistance in
a study on a series of human postmortem lungs (38). If this is true in lungs of dogs it would
clearly account for our findings. It is also possible that accompanying the bronchiolar lesions
there was a degree of bronchial narrowing which we failed to appreciate.
Several important toxicologic inferences can be drawn from the results of this study, although
assessment is made more difficult by the complexity of the exposure atmospheres and the fact
that concentrations of only a few components of the raw or irradiated auto exhaust were
measured. The production of some degree of irreversible damage in the lungs of all exposure
groups at much lower pollutant concentrations than previously reported was the salient
finding.
We are cognizant of no comparable studies in which the effects of automobile exhaust alone
or with oxides of sulfur have been studied during or after long-term exposure.
The production of incipient emphysema by nitrogen oxides and irradiated exhaust, which
contains both nitrogen oxides and ozone, is not surprising in view of previous reports that
these gases can cause emphysematous lesions (8,14,18). The low concentration at which this
occurred is surprising, however, because for nitrogen dioxide alone, for instance, the
concentration required to produce moderately severe emphysema in rats is reported to be
28.75 mg/m3 continuous exposure for the natural life span of a rat (18), whereas in this study
1.21 mg/m3 of N02 and 0.31 mg/m3 of the less injurious NO for 68 months at 16 hours a day
produced what we considered to be mild but definite emphysema.
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The presence of the air space enlargement in lungs of dogs exposed to sulfur oxides was
unexpected because S02 has not been associated experimentally with its production. In the
closest comparable study in which guinea pigs and monkeys were chronically exposed to
fairly low concentrations of S02, H2S04 mist, fly ash, or their mixtures (IX the authors
concluded that the abnormalities detected were attributable to the H2S04 at an approximate
concentration of 1 mg/m3. These abnormalities were minor amounts of epithelial erosion,
goblet cell hyperplasia, squamous metaplasia in large airways, and hyperplasia of nonciliated
bronchiolar cells. In our study, air space enlargement was caused by one-tenth the concentra-
tion of H2S04 (0.09 mg/m3) together with an amount of S02 (1.10 mg/m3) comparable to
that used in the guinea pigs and monkeys (1). The production of focal areas of emphysema in
rodents and rabbits after acute experimental exposure to high levels of H2S04 mist (49)
further supports our observations.
The order of severity of hyperplasia of nonciliated bronchiolar cells for the various treatment
groups was essentially the reverse of that for air space enlargement This is to say, the
hyperplastic lesions were most severe in the groups exposed to raw automobile exhaust, with
and without addition of sulfur oxides. These groups were the only ones without some degree
of air space enlargement (Figure 3). This can probably be explained by variation in chemical
constituents of the exposure atmospheres. Raw exhaust contains polycyclic hydrocarbons
which have been shown to cause carcinomas when painted on the skin of mice (26). It is of
interest that experimental exposures of mice to ozonized gasoline (volatilized) increased the
incidence of spontaneous pulmonary tumors and produced hyperplastic and metaplastic
changes in the bronchial epithelium (30). It is probably polycyclic hydrocarbons that account
for raw exhaust stimulating the greatest degree of bronchiolar epithelial proliferation. The
lesser effect of irradiated exhaust could be accounted for by any photochemical reduction of
the concentration of the hydrocarbons, as occurred in the case of methane, the one
hydrocarbon measured. The other theoretical explanation for separation of bronchiolar
epithelial hyperplasia and air space enlargement is that the bronchiolar hyperplasia in some
fashion protected against the development of air space enlargement We do not favor this
latter hypothesis.
Since in smoggy environments exposure is to complex mixtures of pollutants, it is necessary
to be cognizant of the degree of interaction that exists among components that up to now
have usually been tested individually. Pulmonary function tests on human volunteers acutely
exposed to SOX and ozone, singly and in combination, have indicated a synergistic or
potentiating effect of S02 on the acute bronchoconstrictive effects of ozone (24). A synergistic
effect of the two gases in causing visible damage to foliage of various plants can often be
demonstrated, although the effect on plant growth more often appears to be an additive one
(48). In this study, comparison of the magnitude of chronic pulmonary damage caused by SOX
with that of irradiated auto exhaust + SOX does not indicate either additive or synergistic
effects of SOX and oxidant gaseous components (N02 and Os) present in the irradiated
exhaust A possible explanation for this is that aggregated particulate aerosols were present in
chambers containing irradiated auto exhaust and adsorption of gaseous components on
krger particles might decrease deep pulmonary penetration of the irritant gases.
A conclusion to be drawn with respect to the toxicology of air pollutants is that accurate
experimental assessment of harmful effects of relevant concentrations requires prolonged
exposures coupled with the most sensitive measurements of functional and structural distur-
bances.
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Asilomar Conference Discussion
Hyde:... Nonciliated bronchiolar cell hyperplasia again was graded by the level of severity.
There was never a case observed where there was involvement in the respiratory bronchioles
or first order alveolar duct where it did not involve the terminal bronchiole. Thus it was
possible to reliably use this type of grading scheme.
Lewis: What's the definition of ciliary loss?
Hyde: It is where no cilia are present on the cells in the conducting airways.
Lewis: How do you know it's a ciliated cell? Is it a ciliated cell without cilia?
Hyde: This will be ciliary loss defined strictly from the surface morphology.
Lewis: I'm asking what the criterion is. How do you know you're not in an area where there
aren't any cilia?
Hyde: In the normal lung, there are no areas that are not ciliated, essentially.
Lewis: Well, you must be talking of an area of a certain size. There are certainly tiny areas
where there are no cilia.
Hyde: Yes, excuse me. The area has to be 50 microns by 100 microns in size or greater.
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Lewis: I see. So if you have a zone that large with no cilia, then that's an area of ciliary loss.
Dungworth: Excluding the openings of glands.
Hyde: Another point is that some of the sites seen were larger than 2 to 3 mm in diameter, so
they were quite extensive. Since there was an increase in alveolar pores in most of the exposed
groups, we tried to give a ranking to them since it's very difficult to quantitate interalveolar
pores. I used three levels. All the controls had a few small interalveolar pores, and when there
were numerous interalveolar pores I gave the sample a grade 1. Where there were many with
some large interalveolar pores, I gave the sample a grade 2. This was just to get a quantitative
feel for if there was any possible association between interalveolar pores and the enlargement
we appreciated with the stereology.
Kleinerman: This was the number of pores, but not size of pores?
Hyde: No, both are considered. In a grade 2 sample, large pores would be the size of two or
three average size pores that might be joined together. There had to be a large open space
that would be at least 20 to 30 microns in diameter before it would be defined as a large pore.
I tried to avoid the word fenestrae.
Albert: Wouldn't it be better if you had some pictures that could serve as standards?
Hyde: That's the difficulty with presenting a table first, followed by micrographs.
Albert: No, I mean grading. If you had some reference standards, you could take a look at the
lung and say it looks most like this particular picture or that one, and thus grade the severity
of the damage to the alveoli.
Hyde: My categories are so broad that it had to be fairly well into a category before I gave it
that grade. I went for the mean, and there's no problem looking at a few interalveolar pores.
You immediately say this sample has a few interalveolar pores, or this stub has a few; this one
has many; this one has a few and a few large ones. So the delineations of the categories are
not that difficult. If they were more fine, then I'd say, yes, I need the micrographs, but I don't
feel I did.
Thurlbeck: One normally refers to centrilobular (centriacinar) emphysema as dominantly or
selectively involving respiratory bronchioles. My concept is that the lesion should be quite
proximal and the alveoli distal to the emphysematous space should be normal. I recognize
that a dog's anatomy is quite different from human anatomy, so it's very difficult to make an
analogy. This lesion really looks like elastase emphysema. I know people argue what that is,
and I'd rather just call it emphysema.
Hyde: I should state that the lesion is similar in appearance to elastase-induced emphysema
that was recently reported on by Kuhn in Laboratory Investigation. Specifically, the appear-
ance of shallow interalveolar septa in alveolar ducts is identical. However, it's not panlobular
in distribution; it's very centrally located.
Thurlbeck: Call it alveolar duct emphysema because it's involving alveolar ducts.
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Hyde: All right From here on, I'll call it alveolar duct emphysema. A dog has so many orders
of respiratory bronchioles, 3 to 4. If a dog had terminal bronchiole and no respiratory
bronchiole, similar to the rat, the emphysematous lesions would indeed be moved much more
proximally. The other lesion was a definite increase in the number of interalveolar pores. At
higher magnification, some of the pores tend to have strands of tissue in their midst, which
were by transmission microscopy proven to be composed primarily of collagen fibers and
occasionally accompanied by a capillary.
Stephens: Is the increased size of the pores common throughout the lungs? I recently read a
paper about a similar type lesion which was described in monkeys. The control animals had
that type of a lesion but it was less prevalent than in the exposed animals. This one happened
to be Dr. Bils reporting on the monkey. Also, Jan Nowell, using ozone-exposed rats, saw
similar lesions. I forgot what Dr. Bils was doing at that time.
Hyde: He was doing nitrogen dioxide in squirrel monkeys. But to go back, there's a general
increase in the number of interalveolar pores and occasionally an increase in the size. If we
move up into the first order respiratory bronchiole, some of the alveolar duct outpocketings
have a similar type of conformation of an increase in the size of interalveolar pores. In this
case, there are some strands of tissue that are collagen fibers primarily, coursing through the
airway.
Lewis: Are you giving examples, or are these different types of treatments?
Hyde: I'm trying to give a visual representation of the two types of emphysematous lesions
observed. This type of fenestrative enlargement was seen primarily in the NOL + N02H
group, but also in the SOX group and infrequently in the I + SOX and NOH + N02L groups.
Lewis: The distal airspace enlargement was seen only in the SOX?
Hyde: No, distal airspace enlargement was seen in all groups of dogs. I said that the
emphysema observed in the SOX group was the most common form.
Albert: Do you see that Swiss cheese effect only where you see the alveolar duct emphysema,
or what you call centrilobular emphysema?
Hyde: The alveolar duct emphysema? Yes. Later, when we did quantitate the lungs that
showed increases in interalveolar pores and enlargement, they proved to be the most severely
enlarged lungs. In a lung from a raw exhaust-treated animal, you can see a substantial
increase in the number of interalveolar pores. This would be a grade 2 where there are many
small interalveolar pores, and yet looking at this alveolar duct, there is no enlargement at all.
Hence, the semiquantitative grading of interalveolar pores did not reveal a causal relationship
between interalveolar pores and emphysema.
Dungworth: Well, that could be restated by saving that there were emphysematous lesions in
the absence of any noteworthy increase in pores. There were also increases in pores without
appreciable emphysema. Then the most severe emphysema was when the two occurred
together.
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Hyde: I agree.
Albert: What do those holes actually represent? Disappearance of single cells?
Hyde: No, they're communications between adjacent alveoli through an interalveolar septum.
Albert: Would they be the general size of a single cell or several cells in the alveolar wall?
Hyde: I'm not sure if anyone knows what an interalveolar pore is other than a communicating
channel between one alveolus and another. It's lined by type I epithelium. Sometimes alveolar
macrophages or type II cells are associated with it. But other than that, I don't think there's
much else known.
Question: The question was, is it the same size? If you outline a cell there, it seems to be much
bigger than a pore.
Hyde: Sometimes they're larger. They generally tend to be the size of a single cell though.
Kleinerman: Didn't Boatman and one of his associates in Washington do some transmission
scope work on these pores and point out that they were lined by epithelium?
Hyde: That's what I just stated. The pores are lined by type I epithelium. They're normal
structures. When you see an increase in interalveolar pores, you wonder about their
significance. That's why we tried to semiquantitatively rate them and get a feeling for them.
Boatman can't make any statement about them either.
Rouser: When you say increase, do you mean increased number or increased size?
Hyde: According to the grading table, the most significant increase would be an increase in
size, and a secondary increase would be an increase in number.
Dungworth: They both tend to go together.
Hyde: I don't want to speculate on that point because we have not looked at the ultrastructure
quantitatively and before I could make a conclusive statement, I'd like to look at the
basement membrane and see if it was retracted or absent in those regions where numerous
interalveolar pores are observed. In these dog lungs, the basement membrane looks normal in
all respects, as do the type I cells and intercellular junctions.
Bhatnagar: What happens in collagenase or elastase emphysema? Could you describe that?
Hyde: The fenestrations are characteristics of more severe forms of human emphysema.
Kleinerman: Yes. You see fenestrations in human emphysema, but you see them in colla-
genase as well or elastase emphysema. One of the things that has been passed down, is that if
you look at the lungs of infants you do not see alveolar pores. I don't know if this is true.
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Hyde: It's the same with dog lungs, although it doesn't come through in Boatman's study.
You look at a young dog's lungs, and they have virtually no interalveolar pores, but at 2 years
of age interalveolar pores are very prevalent. Similarly, there are many more interalveolar
pores in old dogs' lungs that are 13 or 14 years old.
Dungworth: We're comparing control 9-year-old dogs with exposed 9-year-olds.
Hyde: Yes, they're controls compared to exposed, so I believe it's a valid comparison.
Hueter: Some people are theorizing that emphysema animal models must be based on the
number of pores the animal has relative to the number a man has. You were using the rate of
mouse or hamster, which is inappropriate, when you could use something like the monkey or
horse and now apparently the dog. What is your opinion on that?
Hyde: I don't want to take the time to make a statement on that point now. In Figure 3, the
surface-to-volume ratio, or surface density, is measured on the Y-axis and the groups are
plotted according to increasing severity of loss of interalveolar tissue on the X-axis. Method 1
is the method of using chord lengths; method 2 uses interalveolar septa or number of
intersections divided by the total length of the test lines. You can see they are very
comparable in the controls, but when you get into the severely affected, the standard
deviations tend to vary more with one method than with the other. The distribution of chord
lengths shows the greater variance, because it takes approximately 14 samples per field to
reduce the variance.
Busch: You mean those limits are ± standard deviation — the brackets shown on the grids?
Not a standard error? You don't divide by the square root of the number of samples? I'm just
trying to establish what the brackets represent. Do they represent a standard deviation or a
standard error?
Hyde: I believe they represent the standard error.
Comment: The variations would have to be enormous to give you your standard error.
Hyde: That reduces the standard error.
Thurlbeck: You're dealing with reciprocals. How do you actually get the standard error of a
reciprocal? Do you use ordinary numbers for that, or do you do something mathematical?
Busch: Standard error can be defined for any kind of strange statistic.
Thurlbeck: I have a bias against this particular measurement I like to use it the other way
around, because it's a linear measurement instead of a reciprocal measurement. Finding
means for reciprocals means talking about harmonic means and things like that that I don't
understand. If one measures the standard error of a reciprocal, do you just handle that as
though it was a linear number?
Hyde: That's how I handled it.
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Busch: If you have a large sample size, no matter what the distribution of individual values
looks like, the distribution of the mean of the values will be a nearly normal symmetrical
curve. You can treat it by conventional t-tests with reasonable accuracy, independent of how
the distribution of the individual measurement looks.
Hyde: All of these morphometric parameters on Table 2 were derived by using an area-to-
diameter-cubed ratio and directly measuring the two parameters of the airspaces with the
image analyzer using an area! detection mode and a perimeter mode. From it, one can gain
that in a comparison of the control group to the NO 2-high animals, the most severely affected
group, the volumetric density of tissue and alveoli decreased significantly. You'll notice also
that down the table the numerical density of alveoli decreased and naturally it would decrease
then for the right lung.
Thurlbeck: Are these all corrected to TLC?
Hyde: Yes, they are. They're corrected to TLC for the right lung, not for the total. In this case,
this figure was the number for the right lung.
Thurlbeck: I want to know about your correction factors. You can guess at what TLC of the
right lung is because you know the lung volume. So you then correct from that lung volume to
area and linear dimensions.
Hyde: Yes. The cubed root, or the 2/3 root.
Albert: What is it you're measuring, the four significant places out there? That's tenths of a
Hyde: These are just fractional volumes, and these are microns further down the table. These
are numbers per cm2. Here, the cross-sectional area, the volumetric density of alveolar ducts
went up and the mean diameter also increased. So from these data I surmised that one logical
interpretation is that alveoli or interalveolar septa were lost within alveolar ducts, increasing
their volume and increasing their profile cross-sectional area.
Thurlbeck Your alveolar number is not necessarily an alveolus. By definition, your alveolus is
a closed space and that closed space could be the lumen of a duct. You should really call these
things Bl, B2 and B3, because they do not necessarily correspond to anatomical structures.
They're geometrically designed structures.
Question: You may convert free alveoli by getting rid of their septa into what you call a duct?
Hyde: Yes.
Thurlbeck: That's what really surprised me, because my concept of what happens in this
lesion is that the lung, as it were, reverts back to infancy. If you start at infancy, primary
saccules form the peripheral and smallest structures within the lung and they are then
subdivided by secondary crests to form alveoli and alveolar ducts. I conceive of this lesion as
the alveolar ducts expanding and expanding, and the alveoli flattening and flattening. I would
assume that during this process the alveolar ducts would simplify themselves so you then end
up with much larger single enclosed structures. Intuitively, I would have thought that your
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simplest structure had to be larger in the emphysematous lung than in the nonemphysema-
tous lung. What you're saying is that we so seldom cut through an alveolar duct in such a way
as to completely enclose it so that this doesn't occur.
Hyde: That's correct. There are some problems, of course, looking at two-dimensional
specimens and trying to make three-dimensional interpretations, especially in altered tissue
because we use the rules for normal tissue, apply them to altered tissue and they may not hold
at all.
Busch: How did you select the dogs to be included in these samples?
Hyde: In this case where I compared all of the N02-high group with the controls, I took the
controls that were the furthest away from the mean of the group so that they were the most
variable animals for the group. I used a distal airway surface-to-volume ratio measurement for
the selection. I did it on all 12 controls because I wanted to compare, in this case, 6 to 6.
Busch: Why?
Hyde: The amount of time required for the computation was much more extensive than by
other methods. I only used this method to look at a particular group. Thus, I used an equal
number of controls to compare the six exposed dogs.
Busch: You may be biasing the control means as compared to the others since you took an
extreme subset. If the distribution is asymmetrical, then by selecting from the extreme tails,
you may be biasing the mean as compared to a more complete sample of the test group. I'm
not sure if that's true. If the distribution is asymmetrical, you would introduce a bias by not
taking a completely random sample.
Hyde: The next type of lesion seen in Figure 8 in a terminal bronchiole is characterized by
discrete or confluent aggregations or micronodular proliferations of nonciliated bronchiolar
cells. You can see that occasionally they compromised the contour of the lumen, and partially
occluded the bronchiole. In this slide, you can see some migratory cells that are located in the
distal region of this airway.
Albert: Could you go back a second? What's in here?
Hyde: That structure? It looks like there's some ciliary loss and some accumulation of mucus.
Albert: What are the ridges then? Is that a shrinkage phenomenon?
Hyde: No, it isn't. When you section these SEM samples, it proves to be a hyperplasia. I
re-embedded this particular SEM stub and sectioned it through the middle of this lesion to
see if it was truly a hyperplasia. It stands up from the epithelium in certain regions such as
this example. It is an actual fold of hyperplastic epithelial cells.
Albert: But that's ciliated.
Hyde: Some of it's ciliated and some of it's not
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Albert: And those cabbage-like structures are focal hyperplasia?
Hyde: That's right, and these are more continuous folds of hyperplasia.
Albert: But quite different than that ridge-like hyperplasia. Are they the same cells?
Hyde: Yes, I'll cover that The most common cell type found in the hyperplasia was the
nonciliated bronchiolar cell which had much glycogen in it. Here, of course, there isn't any
glycogen present, and yet when you look at normal dogs that are killed, and are processed
immediately after fixation, then the glycogen is still present These dogs tended to be stored
for 1 or ll/2 years before they were processed, and I believe the glycogen was leached out
during that period.
Question: How were they fixed?
Hyde: They were fixed by intratracheal perfusion of a paraformaldehyde glutaraldehyde
cacodylate buffered fixative at 30 cm water pressure, in a fixative bath.
Albert: Is this one of those cabbage-like structures?
Hyde: Yes, it is. It's identical to it. You can see in the center there's a macrophage and there's
also a connective tissue core to it. Generally you have some type of connective tissue core
leading up into the hyperplasia. When they are larger, there is a greater amount of connective
tissue found in it, and the basal lamina, of course, goes right along with it
Albert: What was the exposure of this animal?
Hyde: This particular dog lung? These were most commonly found in the R and the R +
SOX. I'll show you on my grading scheme at the end of the talk, the method of evaluation, and
the results. This is to show that if you take a normal dog and you kill it, those cells do have
quite a bit of glycogen. Some of these hyperplasias, besides containing the nonciliated cells,
have quite a few ciliated cells. This represents a linear fold because we sectioned this in
approximately 150 microns and it was still straight up like this. Down in the basal region of
some of these hyperplasias, we saw some unusual cells that had basal bodies and cilia in the
intercellular spaces.
Albert: Why wouldn't you call those papillomas? They look like a papilloma on a stalk.
Thurlbeck: These are linear structures.
Hyde: I call them micronodular proliferations.
Orthoefen Initially on histology I did see a couple, and I did call them papillomatous-like
lesions in the bronchi at that time. And I think they've been described by Auerbach in his
smoking dogs. I think historically they're called epithelial hyperplasias.
Hyde: In mice exposed to 1 or 2 ppm ozone for a long period of time, these were the only
lesions that didn't regress over a 120-day recovery period, thus it's not surprising to see them
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in animals that have been allowed to recover from the exposure. In mice they were the Clara
cell population. This cell type that we're seeing that has glycogen in it is the counterpart of
the Clara cell in the mouse.
Albert: Did they show any evidence of a high rate of cell proliferation?
Hyde: No, they didn't There were no high mitotic indices at all. There was no sign of an
abnormal cell population.
Figure 11 shows that some of the cells near the basal lamina that have ciliary basal bodies,
have cilia projecting from them. I'm sure that's where the cilia in the intercellular spaces
originated from. You can see they're fairly close to the basal lamina and at least 5 cells
underneath the surface.
In the trachea and bronchi, there were two major exposure-related lesions observed, and these
were ciliary loss, with and without squamous metaplasia, as seen in Figures 13 and 14. Here I
call it a proliferation because you can't tell whether it's a squamous metaplasia or not. There
is ciliary loss along here and it is a proliferative type of lesion. You can see it extends in a
papillomatous-type fashion out over the epithelium. There's also a fair area of ciliary loss
around it. You can see some cilia over here. This is how you would determine whether there
was ciliary loss or not. There are just no cilia there, and here there are.
Albert: At what level on the bronchial tree was that?
Hyde: The level? They were seen primarily in the right primary bronchus. They were seen in
the dorsal and ventral regions of the trachea and in some of the groups — specifically NOL +
N02H, I + SOx, and SOX — they were found in the intrapulmonary bronchi.
Albert: Was it any more frequent at the bifurcation?
Hyde: No, in fact, if anything, it was less frecjuent. I wasn't able to attribute the frequency to
any particular structural conformation of the airway.
Dungworth: Could we ask Drs. Thurlbeck, Kleinerman, and Nettesheim to take 10 or 15
minutes to present their summaries on the pathology of the study, and then throw it open to
general discussion from there.
Thurlbeck: I must echo some points that Dr. Brain made this morning. This is really a classic
and important study, and it's a pleasure and privilege to be associated with it. I can see, just
as Dr. Brain can, that in 2 or 3 years time this is going to be a standard reference. For the first
time long-term, low-exposure experiments have shown unequivocally lesions in the lung
accompanied by functional alterations. If one or the other had been absent, we'd have had a
little problem accepting the idea, but speaking as a morphologist, I have no doubt in my own
mind accepting the morphologic changes which have been done in an outstanding way and it
is a pleasure to see.
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I would like to comment about two things that have been described morphologically, the
airway lesions, the parenchyma! lesions, and then make some comment about the relevance of
these lesions to human disease. I was initially disturbed that there appeared to be a
discrepancy between two observers essentially looking at the same material. The SRI group
wasn't very impressed by the lesions they found in airways but these were clearly described by
Dr. Hyde. I think this is a question of sampling and the amount of material available. This is a
very good justification for having a scanning microscope because I suspect if one didn't have
a scanning microscope to visualize large areas of the lung with high resolution, these lesions
would be extremely hard to identify. This has raised the question of being able to use a
different instrument to sample properly rather than using random samples of a thin slice of
tissue. I'm really quite happy in my own mind that these are real lesions in the peripheral
airways of these animals.
There are a couple of very interesting issues related to this which nobody seems to have
commented on. One is that the airway lesions appear to be irreversible both functionally and
structurally; indeed, the airway lesions may even progress in terms of function. It's been
dogma for a long while that we're looking for "early evidence" of "early airway dysfunction"
in humans so that we can detect these changes and if people stop smoking, then the patients
will return to normal. Now, we do know from tests that small airway function does in fact
often return to normal after cessation of smoking. The lesions that we are observing at low
levels of ambient pollution may be more severe than those that occur in early smokers. It is
reasonably well documented that experimentally induced peripheral airway lesions do not
reverse after removal of the irritant. We thus have to question in our own minds the easy
notion that small airway lesions, albeit mild, are always reversible. Clearly the ones we are
looking at here are not reversible.
The next question that the airway lesions raised is whether they are related to the abnormali-
ties of function that were described, and one would guess that they probably are only poorly
related. The reason for saying this is that it is difficult to believe, using conventional McGill
and Harvard School of Public Health dogma, that lesions of this sort in the peripheral airways
would produce a very large increase in airway resistance. So presumably, this gives us a
reason for wanting to be more interested in the central airways in these animals — to know
whether there are lesions there and whether there are perhaps functional abnormalities within
the central airways. Perhaps the central airways are increasingly twitchy and one may be
looking at functional abnormalities rather than anatomical abnormalities within the airways.
These again, I'd like to emphasize, appear to be permanent and progressive. What I'm
saying is that I wonder whether the peripheral airway lesions that we are looking at are
interesting but perhaps not completely relevant to the disturbance of function that we see,
and again I think is a hypothesis that should be tested.
I'm much more interested in the parenchyma! lesion and I'm going to refer to it as the
parenchymal lesion because it raises the question (a) is it emphysema, and if it is emphysema,
what sort of emphysema is it? I'd like to stress the point that Dr. Brain has already made, that
the increase in compliance is progressive even after the removal of the irritant. We know that
in the elastase emphysema model the morphologic lesion is progressive so I would guess that
these two were going along together. Now the reason I call it the parenchymal lesion is that I
wonder if this is emphysema or not. It brings up an important concept related to the
definition of emphysema as an abnormal enlargement of the gas-exchanging portion of the
lung accompanied by destructive change. This definition trips glibly off the tongue until
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someone says, what do you mean by destruction? We now talk about loss of alveolar wall
tissue, or a disorder in the orderly structure of the lung, which is the term I like to use. Now,
what we've learned from elastase and from today's presentation is that the enlargement of the
airspaces and the increase in the pores of Cohn or the appearance of fenestrae do not go hand
in hand. It would appear to me that we should look upon these as two separate processes —
enlargement of airspaces and the formation of holes within the alveolar wall. I'm going to
raise the issue as to whether the lesions that we looked at and the lesions that were measured
are in fact a true structural change or whether they represent the result of preparing the lung.
It may be finally irrelevant which of the two it is because as I developed this idea, it occurred
to me that even if the lesion doesn't really exist in the living and intact chest, there is a
functional abnormality within the lung that results in the observed abnormality.
My interpretation of the change that's occurred is that the volume proportion that is
increased most is the core of air within the alveolar ducts and alveolar sacs, central to alveoli,
that the actual alveoli themselves have not changed in volume. You make.the point that the
average alveolar diameter is unchanged in the exposed animals. I suggest that the maximum
alveolar diameter has increased and this is an old idea of C.C. Macklin who talked about a
cup and saucer. He also talked about that at the upper limits of inflation. His idea was that an
alveolus was shaped like a cup on expiration, a saucer on inspiration and most of the
expansion occurred in alveolar ducts. Now, it is conceivable that what happens in these lungs
is that the connective tissue framework, the scleroproteins, has been altered and the lungs
have become hypercompliant. As the lungs overstretch the alveoli flatten, the alveolar duct air
increases, and the average interalveolar wall distance increases. This may be due to the
disorder of compliance, and the primary abnormality is something funny that happened to
the elastic tissue. There may be no morphologic abnormalities at FRC in situ. The analogy to
this is the old lung. A very similar thing happens if you take aged human lungs and then
inflate these outside of the chest. The argument may be irrelevant. Either there is a
morphometric abnormality and the morphometric abnormality occurs because of alteration
within the scleroproteins of the lung, or there is a morphometric abnormality in life. In any
event there is a disorder. It would be nice to see if these lesions occur in situ in frozen dogs.
The final comment I want to make is related to the significance of these lesions, and the
significance relates (a) to human diseases, as Dr. Kleinerman and I see them in the
portmortem room and (b) to the significance in terms of environmental health. These lesions
resemble a very mild degree of emphysema in humans and we would give them a score of
maybe 5 or 10 on a O-to-100 scale. It is the sort of emphysema that one commonly finds in
older people, depending on how closely one looks, seldom associated with symptoms, in fact
usually not associated with symptoms. There is probably no exact analogy of the airway
lesions in the human and they don't closely resemble the Niewoehner and the Kleinerman
lesion but I suppose that's the closest human disease that it mimics. What does this mean in
terms of the environment because, basically, the lesions resemble minimal human disease that
doesn't cause dysfunction? Is this the price that one is prepared to pay for living in an urban
environment? It's not causing disturbance, not causing dysfunction, and perhaps we should
put up with it We are getting into a philosophical area, but I think the important point is the
lesion that one sees probably would not cause much dysfunction. However, the real question
is, given that lesion, are other irritants additive or synergistic? Would these dogs be far worse
off than the clear air dogs smoking cigarettes? That of course is the exact analogy that one's
always worrying about in the human.
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Stara: Are you saying, Dr. Thurlbeck, that you would describe this pathology as an
acceleration of normal degenerative disease of the lung?
Thurlbeck: The parenchymal lesion has a very striking resemblance to the exaggeration of
the normal human aging processes. What's described in the normal human aging process is a
loss of parenchymal tissue, an increase in alveolar duct air, accompanied by either a decrease
or no change in alveolar air. One doesn't normally think in terms of focal alveolar wall
distortion and destruction in the normal aging process, at least I don't However, if one looks
at aging lungs, one often finds small areas of obvious panacinar emphysema rather resem-
bling what you have seen here, and I regard that as an exaggeration of the normal aging
process because we constantly find that in older people.
Albert: How do you know that the normal aging process isn't exposure to air pollution?
Thurlbeck: When I say normal, the average aging process that one sees in the portmortem
population. In non-smokers.
Kleinerman: Of course, you can see the aging process in people who don't live in urban
environments.
Thurlbeck: The studies that have been done have been in urban areas.
Orthoefer: Would the biochemical change that we saw (i.e., an enzyme which failed to be
turned off) be an aging change? Doesn't this occur in aging animals?
Thurlbeck: I don't know what the enzyme changes are that are described in the lungs of
aging animals. Now there is a standard collagen/elastin story which is confusing because the
collagen and the elastin in major airways, and of pleura as opposed to the alveolar wall
parenchyma, have not been separated in the studies.
Gillespie: Dr. Hyde's graphs were on aging dogs, the ones that you say look like those of aging
people, which again were from the beagle colony in Davis and not exposed to urban air and
not exposed to smoking.
Hyde: They are a different population.
Thurlbeck: It would be surprising if the lungs didn't age. Dr. Albert's question is — if we had
a non-urban population versus an urban population of human lungs, how would those lungs
look? We don't know the answer to that. I would guess they would both age, and if I could
guess again, I'd guess the urban ones would age more judging from this study.
Kleinerman: You know, batting clean-up to Whitey [Dr. Thurlbeck] is like standing in the
chow line behind the Cleveland Browns. There isn't much more to do. I personally think that
the participants in this study are to be congratulated. I think this is a landmark study. But, as
a matter of fact, it really confirms some observations that have already been made, to a
certain extent. It's true that the study was done with concentrations of air pollutants which are
lower than those which had been previously used. The major concept that has been
elaborated is that there can be a development and a persistence of lesions, probably
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development is the better word, even after exposure to these pollutant materials has ceased.
That's the interesting thing, and this of course we reported in hamsters about 2 years ago at
the ATS. After we took our hamsters out of 17 months of N02 at 20 ppm, we found that the
surface-to-volume ratio increased and we reported it with trepidation because we didn't really
know what it meant, and about that time Dr. Kuhn reported that there is progression of the
lesions after papain. Now we have the ultimate in the observation indicating that there is a
probability of continuous destruction even after removal of the exposure. I have absolutely no
problem in buying these lesions; I think the care with which the study has been done
precludes any possibility that there is error or malinterpretation.
Albert: What lesions are we talking about?
Kleinerman: We're talking about the lesions of emphysema, enlargement of airspaces, and
the enlargement of the surface-to-volume ratio which was described by Dr. Hyde.
Albert: I thought that the analysis by the scanning EM there wouldn't take into account the
enlargement of the alveolar duct
Kleinerman: There are so few ducts that their contribution couldn't make a significant
difference to the values.
Albert: Isn't that what you were talking about as the aging effect?
Thurlbeck: The surface-to-volume ratio decreases, and this is in fact a reflection of the
distance from one alveolar wall to the next,
Hyde: That's correct.
Question: Was that lesion determined correctly immediately after the end of exposure? My
impression was that the time they determined that the lesion was present was 2 years later.
How do you know what was happening right at the end of exposure?
Kleinerman: You don't, that's exactly the point.
Question: You made the statement, though, that it continued to develop.
Kleinerman: Assuming from the pulmonary function tests that at one time you did not have a
change ...
Thurlbeck: Dr. Kuhn's and Dr. Kleinerman's model shows progressive increase in interal-
veolar wall distance. They extrapolate to the functional state of increasing compliance,
without this measurement.
Kleinerman: No, we did both. Sacrificed at one time and did pulmonary function and the
surface-to-volume ratio. At that time, they let them live on 3 months. Now, there's one thing I
didn't understand that Dr. Thurlbeck said and that is that you thought the airway lesions
were irreversible. I'm not sure that you indicated that they were irreversible.
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Thurlbeck: They were present
Kleinerman: What makes you think that they're irreversible?
Thurlbeck: They're still there.
Kleinerman: I wish you would say so.
Stephens: Well, there's a lot of information to reflect on in this particular study as far as I can
see, Dr. Kleinerman. After working with NC>2 for many years, we have had an opportunity to
observe these papilloma-type structures in rats that have been exposed for various periods of
time at higher levels. When animals are removed from the exposure chamber and permitted
to recover, such structures decrease in size. We have not observed them to continue to
develop in clean air. Certainly ciliary regeneration takes place, usually in a very few days.
There is a reversal of the thickening of the basement lamina and it returns to a fairly normal
looking structure in a relatively short period of time. Most of my background is from the
examination of rat tissue rather than from the dog. We have done scanning electron
microscopy in previous studies. We were not requested to nor did we undertake any scanning
electron microscopy on the biopsies from these dogs. We did not, however, observe any
papilloma-like structures in the light, or TEM preparations of the biopsies.
I was just mentioning to Dr. Hyde the possibility that the agent that caused the dermatitis on
the skin of the animals might have been at least partially responsible for the lung abnormali-
ties. He apparently had observed the same abnormalities in some of the controls.
Hyde: I didn't mean to make it sound as though those types of lesions were present in all
controls. We only saw it in one trachea in the dorsal region of one control and a small one in
the primary bronchus of another dog lung.
Kleinerman: I don't think there's any need to explain the distribution because it's been seen a
number of times. We've seen it in our hamsters, too; there are papillary proliferations. The
point I'm raising and the question I want to ask is, why does it have to be permanent or
irreversible?
Thurlbeck: The question is, if they are reversible, to what degree are they reversible? They're
either nonreversible, progressive or partially reversible, because we're sure they're not totally
reversible.
Albert: Nor delayed in appearance, like a tumor.
Thurlbeck: You're right, except that these dogs were abnormal in terms of airway function 2
years previously.
Albert: The airway resistance was present only in the R + SOX and no other group.
Thurlbeck: Let's argue this. Let's take the notion that these things appear after 2 years. Then
this requires that something has disappeared and something else has appeared. Well, okay,
it's an intuitive argument. But it seems intuitively a very unlikely event that lesions appeared,
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and disappeared, and subsequently another lesion has appeared, and therefore these present
lesions are unrelated. But here these lesions are totally irreversible. How's that for a dogmatic
statement?
Nettesheim: Why don't we just say they're still there?
Kleinerman: The objection to this concept is that it begins to lay the groundwork that the
epithelium and the changes in the bronchioles may really be a static phenomenon. Once
they're there, they're there. That's totally incorrect, because there are turnovers of epithe-
lium. You can go from Clara to ciliated cells. You can go to hypertrophies to hyperplasias.
This can happen with dispatch. And we really don't know how rapidly these changes take
place. The epithelial nodule may be a distinctively unique lesion, and certainly it's not
distributed very widely throughout these cases, as it wasn't in our lesions or in Dr. Stephens'.
The fact is that it's probably not a very significant lesion and the important concept that I
think we should be left with is that there is a considerable epithelial turnover in these organs
and it doesn't stop because an epithelial nodule occurs. It continues, and there's no reason to
think that this will go on, at least there's no evidence of progression in our studies and in
other studies, so I don't think it should be thought of as a premalignant lesion.
Thurlbeck: I want to make what I thought is an important point. Dogma claims that there is a
lesion in the peripheral airways which is reversible. We say that in goblet cells, metaplasia is a
reversible lesion.
Nettesheim: That's something else.
Kleinerman: That's another problem in the dogma that you're talking about. That's exactly
the sort of thing that you wonder whether or not you might have to challenge, you know.
Thurlbeck: Exactly. Let me finish my argument. What I'm saying is that dogma says that
these lesions are reversible. The evidence that these lesions are reversible is really very small
indeed and there should be evidence. Let's just leave it at that.
Dungworth: I would like to ask Dr. Kleinerman, of the alternatives explaining why these
lesions are seen at the time the animals are killed, which is your preference?
Kleinerman: Which alternatives are you giving me?
Dungworth: That they are essentially unchanged and may be worse than they were at the time
of cessation of exposure or on their way down.
Kleinerman: I think that they're regressing afterward, in the terminal bronchioles.
Dungworth: So this is a long delay, 3 years ...
Kleinerman: You are at a point where you can extrapolate from acute experiments, and this is
exactly from where we must depart. Our basis of information is, what happens after you
irritate. Do you get a proliferation or a reactive injury? This regresses after a period of time.
That's the kind of information we have now. We're producing a new type of experiment, a
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chronic experiment about which we have virtually no information concerning epithelial
turnover. We do have a few fragments of information. Those studies in which you have
labeling experiments done, in chronic experiments, would indicate that the proliferation
effect in an animal who has become tolerant is somewhat depressed or decreased. If you give
an animal 20 ppm of N02 as an acute exposure, you get a turnover of so many cells per 100
cells or per 1000 cells. If you give a 20 ppm exposure after he's been exposed for 60 days or
180 days, and then you measure the turnover, you find that he no longer turns over at the
same rate and the turnover process is considerably depressed. I think that our dynamics and
our thinking about the epithelium have to come from the acute process, and, to me, the big
progress that needs to be made at this conference is that we have to set up a pattern of new
disciplines in order to deal with what happens to lung tissue and to airway epithelium after
chronic injury, because clearly it's not the same ball game as it was after acute injury.
Brain: If you point us to acute models where there's just been one insult that leads to
emphysema, can you think of an instance in which the pathology regresses and the physiology
progresses? Isn't that what your hypothesis demands?
Albert: There's confusion here because the evidence of emphysema occurred predominantly
with the SOX and the N02, whereas the hyperplasia of the bronchiolar epithelium occurred in
response to exhaust exposure, so that the progression in the abnormal pulmonary function
was in response to a different set of agents than the increase in the mucosal hyperplasia. It
seems very unlikely that the abnormal pulmonary function was due to the mucosal hyperpla-
sia, or had anything to do with it at all.
Kleinerman: I thought we were talking about the epithelium.
Comment: You're saying the parenchyma is getting worse but the airways are getting better
during the 2-year period?
Kleinerman: The airways are getting better and the parenchyma is getting worse, and I think
they're two different ballgames. That's right.
Gillespie: There is some evidence that the airways may, in fact, be getting worse as well, and
that's in the R + SOX group. Their airway resistance increased following termination of
exposure.
Kleinerman: How do you know that's an airway phenomenon and not an elastic recoil
phenomenon?
Gillespie: It could be elastic recoil. My opinion would be that in order for elastic recoil to
cause changes in airway resistance there would have to be substantial changes in compliance.
There were no compliance changes present in the R and R + SO*
Kleinerman: I think that most of us have been talking about the resistance that we think of in
terms of peripheral airway resistance and not the resistance of large airways.
Gillespie: That's right. That's why I think it's probably too simplistic to relate the airway
lesions that you were seeing in the periphery with the high resistance. I think there must be
other things that happened.
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Dungworth: Prejudice put me in the camp with Dr. Thurlbeck, but we recognize that we'll
have to find out. I think that if we draw a line over a 3-year time scale and say we're looking at
the lesion after it's been regressing, it's a little bit hard for me to take. You're right, and we
have to set up the techniques and the protocols that will examine that. You have carcinogene-
sis interests, Dr. Nettesheim. How would you read this, particularly the nodules in the
bronchiolar region? A specific instance I do recall offhand is the one paper by Penha and
Werthamer (Arch. Environ. Health 29:282, 1974) on effects of ozone where they exposed mice
for 120 days, obtained these micronodular proliferations, let mice recover for 120 days in
clean air, and found essentially the same micronodular proliferations at the end of that
period. That may have some bearing on this.
Nettesheim: There was no evidence presented that would indicate to me that this nodular
lesion has any relationship to any pulmonary function abnormalities. There were no data
presented that would indicate to me that that lesion occurs frequently enough, in enough
airways, that it indeed could cause any functional change. Once those kinds of data are
presented, then I would be more open to that suggestion. I do think that these kinds of
epithelial reactions might be important in another way than has been discussed here, namely,
if we were to learn more about the proliferative rate in these types of nodules, I think the first
question that arises is, why are these nodules there? Are they there because of an increased
cell proliferation, or are they there because of an increased survival time of the cells? What is
the principal defect in terms of cell kinetics? I think that would be an important thing to
establish in order to understand, in terms of cell proliferation, what such a nodule means.
Why is it there? If one were to know more about this, namely if indeed at the time of
observation, 2 years or whatever after the stimulus has been removed, if this is indeed a
consequence of continuous increase in cell proliferation — it doesn't have to be — then a sort
of indirect relationship to cancer becomes a possibility. There is a good story going around in
carcinogenesis circles which is backed up by a number of observations in vivo as well as in
vitro that tissues with a higher proliferative rate often also have a greater susceptibility to
neoplastic transformation or, to put it in other terms, there are certain phases of the cell cycle,
namely that of late DNA synthesis, in which cell transformation seems to take place more
readily than in others. I certainly from my vantage point would be interested in these kinds of
lesions not as preoccurences of cancer — I'm not saying that, I don't think there is any
evidence for that — but let's say as a weak spot in a system where epithelial transformation by
a neoplastic agent could more readily take place. I wasn't here unfortunately for the first part
of the meeting. I would have liked to know something more about the composition of the
material that the animals were actually inhaling in terms of analytical chemistry, but I think
potentially there are quite a few agents in that auto exhaust, of course, that have transforming
activity. I really think that's about all I have to say.
Thurlbeck: Are there reversible lesions within the peripheral areas? This is your field, isn't it,
Dr. Stephens?
Stephens: I would like to reflect just a bit on the amount of information that we have gathered
over a decade of study on N02 in particular but also with ozone, in regard to the proliferative
lesion and the possibility of its regression. In the first place, when an animal is exposed, you
have essentially a 24-hour injury/destruction phase, followed by 24 hours that we refer to as
the reparative/adaptive phase. If you compare N02 response to ozone response in the
peripheral airways, it's somewhat different. The nonciliated cells in N02-exposed animals
seem to pile up and stratify to form nodules, and it happens relatively quickly. I have a
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tendency to attribute this to some of the adaptive changes or some of the metabolic changes
that involve the relationship of the cell to the basement lamina. I think that for some reason
or other the cells do not differentiate properly or do not leave the tissue in a normal manner.
The same thing does not happen in rats exposed to ozone. However, Werthamer's study
seems to say that Os produces hyperplasia in mice, if I understand him properly. Now,
whether we can extrapolate this information to the dog and expect similar changes or not, I
don't know.
Albert: Excuse me, I don't understand what you're saying. Are you saying that the hyperpla-
sia occurs in the mouse but not in the rat following exposure to ozone?
Stephens: That's correct (in the terminal bronchiole).
Dungworth: We had the same side-by-side species, rat and mouse, in the same exposure, and
the mouse has proliferation and the rat doesn't.
Stephens: And the proliferation rate does go down in rats after it reaches a peak at about 24
hours. By 7 days it is back to almost normal. About the type II cells, that proliferative rate
does not reach its peak until 48 hours and then it progresses back down to the normal level.
We've done a number of chronic studies with rats exposed to 15 ppm NOa, until the animals
were suffering severely from respiratory insufficiency and we've then taken them out and let
them recover. They gained weight. Their respiratory rate goes down and they begin to look
healthy again. This cycle has been repeated to produce a more severe emphysematous
change. If rats are exposed to 2 ppm N02, they will survive their life span without significant
nodular formation. The proliferative lesions that we're talking about here become quite
prominent in a relatively short period of time when exposure is to 15 ppm NOa I agree with
you, Dr. Kleinerman, that the nodules regress upon exposure to clean air, but don't forget
we're talking about exposure at 15 ppm, not at 1 ppm or less that the dogs received.
Albert: Is this the mouse that develops the spontaneous incidence of pulmonary adenomas?
Stephens: No, I haven't had very much experience with mice. That was work by Werthamer.
Albert: Does it increase the incidence of pulmonary adenomas in the mouse? Maybe it's all
part of the same process.
Comment: I think they picked a species that had a pretty low incidence of spontaneous
adenoma.
Albert: Does it increase the incidence?
Dungworth: They had one paper some time back in which they reported a couple of
adenomas, but it really didn't stand up to the light of day definitely. This was a long-time
exposure to ozone at the same, I think it was Swiss white mice.
Dungworth: ... a couple of animals that had what looked like possible adenomas.
Nettesheim: We found, too, that ozone increased the incidence of pulmonary adenomas in
C57 black mice.
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Albert: So it may all be part of the same process; namely, the finding of the hyperplasia of the
bronchioles does represent the very beginning of a neoplastic process.
Kleinerman: Most of the adenomas in mice that are reported are not bronchiolar adenomas.
They're adenomas of type II cells, which are alveolar cells. So I'm not sure that you can
compare the two unless you are willing to bridge this gap and say that if it happens in one
area, it may happen in the other.
Albert: I was thinking of the tumors that were induced by plutonium in the lungs in studies at
Hanford where the tumors were adenomas, and the thinking there was they seemed to arise
from bronchiolar epithelium.
Kleinerman: That was not in mice, was it?
Albert: No, dogs. Maybe what is being seen here is just a very small step on the same path. It
may be that if the animals were allowed to live longer, some of the dogs might have developed
pulmonary adenomas.
Stephens: There's a difference between the data that Dr. Hyde presented and those we were
able to acquire from the samples that we had, and I would like to discuss it briefly. This study
was well done and certainly I accept the possibility that sampling was a problem although our
biopsies were quite large. I would expect, however, that if the lesions were present at 3 years
after the biopsies were taken that they were probably there at the time of the biopsy. In future
design you would certainly want to include scanning electron microscopy. It's a very powerful
tool for the kind of study that was needed. We have used the scanning scope before also but
at the time we were requested to become involved in this study I believe there was a crisis
situation where the experiment had to be terminated. There wasn't very much planning that
went into how we would take the tissue or what we would do with it. It was very rapidly
decided that we would take biopsies from animals exposed to N02 and the irradiated auto
exhaust. I believe a substantial question still remains as to the significance of the total data
both because of the level of exposure and the conflicting data obtained from various portions
of the study.
Orthoefer: I wonder if more shouldn't be said about the fixation process because I feel this is
the one thing that really made it possible to visualize the emphysematous-type lesions with the
SEM here, and there was some sort of question placed upon the diagram of the fixation
apparatus. I think it's the only way you're really going to see these lesions.
Kleinerman: I don't think that Dr. Thurlbeck's concept is very viable under the circum-
stances. I think it's a nice thought, but I think it has to explain the peculiar localization and
the loss, the decrease in DL^, which I think you wouldn't expect if this were just an
expansion lesion, unless you're building another parameter in to say that you also lose
capillary bed when you have these expansive hypercompliant lesions. So I think it's an
interesting hypothesis, but I don't believe it at all.
Orthoefer: After I left those lungs fixed for 16 or 18 hours, I tied them off at the trachea and
did displacement studies with them to get the volume. I think if what was said is that we have
an artifact then the whole lung would have expanded and this would have been picked up
very easily just by comparing the volumes.
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Thurlbeck: We know that's the structure of the lung when it's prepared in this way. We know
the alveoli are the same size, we know the lung is increased one-third in volume. So the
increase in one-third of the volume is in that core of air in the middle. The question is, is it
that way during life? I'm trying to say that this is a trivial question. There's no way of
guessing what the lung looks like in the chest during life. The air passages may look very
different at the highest part of the lung compared to the bottom end. What I'm saying is that
one can predict that on the basis of rotting the connective tisssue of the lung and preparing
the lungs in this way, this is the sort of lung that you would end up with.
Could we just come back to a previous point for my information? What documentation is
there of reversibility of airway lesions in experimental animals? Now, the only information I
have is a one-shot experiment of my own with a high dose of N02, where you get the severe
bronchiolitis obliterans that is entirely reversible. That's a one-shot experiment. The only
other information I have concerns a Lamb and Reid experiment in which they induced goblet
cell metaplasia, which after 5 weeks didn't reverse. Now, do you have published experiments
showing the reversibility of goblet cell metaplasia or other small airway lesions?
Stephens: In details of cellular recovery, for instance, it is quite well accepted now that one of
the initial things that happens is that there is a loss of cilia and a loss of ciliated cells as a
matter of fact. If the animals are placed in clear air and permitted to recover after N02
exposure, the cilia generate very quickly. In fact, if there's a mucous layer that develops over
the top of the ciliated cells that have lost their cilia, the cilia will reestablish themselves under
this layer of mucus even with continuous exposure.
Kleinerman: But in addition to that work in which you actually can show that there is a
proliferative response within the first 24 to 48 hours, we published that in the AEG
Symposium in 1970. We did that with 100 parts, with 20 parts, and with a smaller
concentration. We plotted it over a period of 0, 24, 48, 72 hours, up to 14 days, and described
the differential response in differential areas over the respiratory tree from the alveolus to the
trachea.
Comment: Which lesion?
Nettesheim: There are many lesions.
Thurlbeck: S02, N02, or ozone exposures produce lesions within airWys. Now what experi-
ments have been done looking at reversibility of any lesions produced by these or other
airway irritants?
Albert: Well, I can tell you that in terms of clearance with cigarette smoking in the miniature
donkey, you can keep the animal at a level of severe impairment for many months by
repeated exposure on a set pattern. And as soon as you stop, it reverts, and similarly with
repeated exposures to sulfuric acid, it can build up a response in terms of slowing of clearance
and in a matter of weeks it tapers off. So that the clearance effects come and go.
Comment: Neither of those indicates reversibility of anatomical change. It may be the
changes of mucous secretion in the quality and thickness of mucus.
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Albert: It could be, sure, but that's not what he asked. In terms of carcinogens, Kushner
showed for a variety of agents that the initial response is a severe hyperplasia, a metaplasia of
the mucosa, which then reverses. The process goes back to normal for a long period of time,
and then it starts up again, and when it does, it goes on to cancer.
Thurlbeck: What were the carcinogens?
Albert: Various polycyclic organic carcinogens, and ionizing radiation.
Dungworth: If we're to bring the discussion closer to home we should limit it to what evidence
is there that these micronodular proliferations regress or not. Not that there is damage which
is repaired, and so on, because I think that is acceptable and not relevant to those
micronodular proliferations. If we explained the effect of the ozone in the rat versus the
mouse we would get a different story completely regarding the proliferations of the bron-
chiolar cells. Now maybe the dog is more like the mouse. We don't know. I think that's the
particular lesion that we should address in talking about what models are there, or what
evidence is there.
Stephens: In our experience, to respond to that specifically, along with the micronodules is a
thickening of the basement membrane, or at least a continuing of the basement membrane,
sometimes into these nodules themselves. That reverses quite rapidly after being taken away
from the exposure chambers.
Dungworth: This is in rats?
Stephens: That's in rats, that's right. I would say that we haven't made a detailed study on the
micronodules themselves, but we have done these experiments where they have been in and
out of the chamber for extended periods of time. My very strong opinion would be that the
nodules in the rat do regress in the recovery periods, and that's on successive recovery
periods. But we haven't, as I said, done a detailed study pointed toward that aspect alone.
Thurlbeck: Is this true for ozone?
Stephens: No, ozone does not create the micronodules nearly as much as N02 does, at least in
the rat.
Dungworth: Dr. Nettesheim, because of the difference between the rat and the mouse with a
response to ozone, have you come across the evidence or suspicion that in mice, bronchiolar
cells and alveolar cells too, for that matter, are more likely to proliferate in response to injury
than those of rats or other common laboratory animals, evidence that the mouse is more
prone to develop proliferative lesions in response to irritants, whether it's N02 or other
irritants— This is, I realize, a very general question.
Nettesheim: I don't have any comparative data with mice. We have done all our studies in
rats and hamsters with respect to N02 and formaldehyde, and there both species are highly
susceptible to turning on cell proliferation with things like nitrogen dioxide, ozone, formalde-
hyde, and so on, so I don't know of any data.
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Dungworth: I'm thinking of these papillary proliferations ...
Nettesheim: No, I don't have anything.
Stephens: The problem here is trying to understand the process of the establishment of the
papilloma. Rapid proliferation takes place very shortly after the initial exposure; however, the
cells continue to build up, not because of an increased cell turnover because that returns to
normal after a few days. I believe that the relationship established between the cells and the
basement membrane gives rise to the papilloma. When continuously exposed to N02,
nonciliated cells do not differentiate properly into ciliated cells but accumulate and remain
attached to the basement lamina, giving rise to the papillomas.
Albert: Can I change the subject just a minute to get a clarification, at least in my own mind,
as to what the peripheral lesion is in these animals? The data show a decreased surface/
volume relationship in the alveoli. Was that due to a breakdown of alveolar septal walls, or
was this the same spotty process that was associated with the enlargement of the alveolar
ducts?
Hyde: It was a focal lesion.
Albert: The whole thing is focal?
Hyde: Yes.
Albert: The increased numbers of pores are not?
Hyde: Not the way I graded it, no. I graded it on the entire specimen.
Dungworth: When the increase in pores was seen, was it fairly well distributed throughout the
alveolar parenchyma as opposed to being also focal around the alveolar duct?
Hyde: Usually, when I graded the entire stub, I was looking for the entire distribution of
interalveolar pores. There was the occasion where the enlargement was accompanied by an
increase in pores and fenestrations.
Albert: Therefore, the decreased surface volume effect that you demonstrated was really part
of the enlargement of the alveolar duct process, so it was a patchy affair.
Hyde: Yes.
Thurlbeck: The answer is that it's probably both. There are a lot of things going on in these
lungs. The volume-to-surface ratio increases as the surface-to-volume ratio, its reciprocal,
decreases. And that will result automatically if the lung enlarges. It's just got to. But the point
is that the volume-to-surface ratio actually increases more than the volume increases. The
volume increases one-third. The linear measurement should change to the cube root of 1.33,
which is going to be a small change. In actual fact, the linear change is about the same
change as the volume change, so something else is happening besides just isometric
stretching of everything. We know that the internal geometry of the lung is being altered. The
analogy is exactly the same as the aging human lung. Because you change the internal
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geometry of the lung, you change the linear dimensions more than you would anticipate. But
the thing that Dr. Hyde hasn't told you, but he told me as we were talking, is that in some of
these experiments, the actual absolute surface area decreases. Now the absolute surface area
can alter, again, for two reasons. One is loss of tissue, and two, the geometry can be altered.
One would guess that both of these are going on at the same time at least in some animals.
Also we know the proportion of tissue decreases from 11% to about 7%. That change can't
be accounted for by just dilatation. I think you could certainly do the calculation, but I don't
think you would get a 50% change. You would get a 20 or 30% change; thus there is loss of
alveolar wall.
Albert: If there is an increase in the volume of the lungs by one-third due to the process that
you described, that doesn't sound like a patchy affair, does it? It sounds as if it must happen
in a substantial portion of the alveolar ducts. Does that fit the observations?
Thurlbeck: That's a good point.
Tyler: It fits the morphometric analysis quite well actually, doesn't it?
Hyde: The alveolar ducts. As far as the number involved or percent involved, there is a much
higher number in the N02-high group than in the other groups. As far as the location of the
dilatation in reference to the structure that dilated, it's primarily located in the proximal
alveolar ducts.
Albert: Well, what proportion of the alveolar ducts?
Hyde: I didn't quantitate that, but the majority of them are in the N02-high group. All I was
trying to differentiate between was where the dilatation occurs, not necessarily the percentage
of alveolar ducts in which it occurred.
Albert: But it's not such an uncommon lesion.
Hyde: It's a very commonly viewed lesion in those dogs, in the N02-high group.
Comment: And functionally you've impaired gas exchange. The diffusion capacities are low.
Hyde: The diffusion capacities are down in that group. What would be interesting would be
to take individual animals and compare the data across to see how accurately the morphology
fits the pulmonary function, but we haven't done that.
Thurlbeck: We're playing word games. I would like to re-emphasize that this model to me
looks like my concept of what happens in older people. My idea is that nearly all older lungs
on which one makes morphometric measurements have consistent changes that I detailed
before. In a minority of older people, one finds localized areas where the lesion is worse, that
there's flattening of ducts, there's "panacinar" emphysema. This is what I envisage has
happened in your animals, that you have a change that involves most of the lung, but in some
parts of the lung is worse than in others. There are two aspects — in each acinus, most of the
alveolar ducts are involved at a particular point, but in some acini, some lobules, it is worse
than in others. And that's my concept of what happens in some older people.
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Comment: Well, let us not forget, it is treatment-related.
Thurlbeck: Oh, yes.
Stephens: There's no doubt that it is a result of the treatment. It's very logical when you see
the early lesion. The early lesion is at the level of terminal bronchiole and proximal alveoli
It's not patchy. Every terminal bronchiole shows the same kind of response in the first 3 or 4
days, so if there is any relationship between the first few days of exposure and the end result,
we would suspect it to be at this location.
Comment: That's at high levels.
Stephens: That's right at various levels.
Dungworth: It begins to get very spotty down at the 0.2 level, but that's another story. We
would like to know why that occurs with ozone, what leads to it.
Albert: One of the things which is naturally of interest is dose-response in terms of building up
information related to levels of risk at different levels of exposure. How would you character-
ize the difference in response, say to the N02-high and the N02-low with respect to the
damage to the lung? Can it be quantitated, say, for example, by the surface-area-to-volume
ratio?
Comment: Can it really in this particular experiment? Because you've got another component
to this system, too, that is the opposite: the high levels of NO that accompany the low N02-
It's a two-component system, NO and N02. One is high and the other is low.
Albert: I know, but is there any evidence that it does produce lung damage?
Lewis: From our pulmonary function data, the only thing we noted was an apparent elevation
of RV. We didn't see the diffusion alterations that we saw with N02-high.
Albert: Does NO alone cause lung damage?
Comment: You don't have NO alone.
Albert: No, I mean from other studies.
Comment: I've been out of air pollution for 6 years, but when I first came in here, it was very
innocuous, NO.
Comment: NO doesn't really exist very often alone. It's difficult to have it by itself.
Comment: So is NOa
Comment: I think people are getting interested in it again and want to look at it.
Stephens: Well, for some time it was thought that N;>05 was more toxic than N02, but
eventually that was worked out. If you went back and looked at Stokinger's early review
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papers. His several reviews have been excellent in trying to straighten out all of the data and
present that which is meaningful. NsOs was thought to be the culprit, and NO, I think, has
always been assumed to be innocuous.
Albert: Well, if you assume it's innocuous, then you have the basis of making a dose-response
relationship in terms of surface-to-volume relationships.
Albert: You have the first quantitative dose-response curve for chronic lung damapre. Off we
go!
Comment: That's a weak two-point curve, but it goes in the right direction.
Stara: I'm interested if you could expand on your comments, Dr. Nettesheim and Dr.
Stephens. Did you suggest that you do not believe that there are lesions?
Nettesheim: No, I never said that.
Stara: Or that the lesions were related to the treatment.
Nettesheim: No, what I was saying, is that there's no evidence that those nodules are causing
any changes in pulmonary function, because I don't think there are any data that indicate
how many of the peripheral airways show such nodules.
Comment: It wasn't measured.
Nettesheim: Right. We just don't know. Until you can say that 80% of all the peripheral
airways shows at least one of those nodules, you can't relate the change in pulmonary
function studies to the morphology. That's all I was saying.
Stara: Dr. Stephens, these lesions were transient, weren't they? These dogs have been out of
the exposure atmospheres for 3 years. They were breathing air that shouldn't have any
re-irritating effects and yet these lesions were present in greater quantities in the exposed
dogs than in the controls according to all the measurements made.
Stephens: Well, I think it's obvious that there's considerable difference in species and their
response. I think the mouse is different from the rat and the rat is different from the dog and
maybe the mouse and dog are closer together. I don't think there are enough data yet on rats,
for instance. We've never made it a focus of a study to say that the tissue recoveries and the
nodules eventually disappear. Have you presented those data, before?
Kleinerman: Which data?
Stephens: The data about whether these proliferative-type structures or nodules do disappear.
Is there a reversion in their prominence on recovery?
Kleinerman: Not these nodules. I haven't studied that.
Nettesheim: I thought you said that, in hamsters following N02 exposure and recovery, the
nodules were positioning.
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Kleinerman: Yes, that's right, some of them were still there. But we didn't quantitate the
number between the two.
Dungworth: In regard to the correlation between physiologic and morphologic studies, I
would like Dr. Gillespie to comment. It seems from the morphologic side, when he said the
physiologic parameters indicated an emphysematous-type of change, that his ranking correla-
ted beautifully with Dr. Hyde's morphometric data, and when he listed the indication of
whether it was parenchyma! damage, that he brought up to the top a mixture of those in
which there was the emphysematous lesion and those in which there was the most severe
proliferation of the small airway epithelium. You want to comment on that, Dr. Gillespie?
Gillespie: What you said is precisely so. Lung compliance is a measure mostly of the elastic
recoil of exchange area. The compliance was increased significantly in the N02-high, I + SOX
and in the SOX. Now, Dr. Brain pointed out this morning that if you had severe loss of
communication with some of your parenchyma, then you could have changes in compliance.
That wasn't so in any of the dogs. So I think it's reasonably safe to assume that the change in
compliance, the increase in compliance, probably relates to a decrease in elastic recoil. I think
the most reasonable explanation for this function change is loss of recoil tissue. In addition,
those same groups — N02-high, SOx, and I+SOX — had the greatest change in lung
volume. Again, the capacitor of the lung is the exchange area. I believe that there was either a
loss of elastic tissue or frank loss of parenchyma! tissue. I don't think it can be explained
solely on a reshaping of the terminal airways because there was too great a change in volume.
Dungworth: That correlates well with the morphometric data.
Gillespie: Yes, that's exactly right. The animals that had increasing, or at 2 years had the
highest, resistance were those that breathed R + SOX. Group I had a high resistance but not
statistically different from the controls. These two groups had the greatest amount of
morphological evidence of damage in the airways. SOX and N02 groups had the greatest
disparity in frequency-dependent compliance, which is another way of looking at the time
constants of the lung. The high values of these groups, at low frequencies, is because there is
such a high compliance.
When one measures resistance with the oscillatory technique at FRC, the resistance value is
related to the large airways. This measurement includes the resistance of small airways, but
the predominant influence is the large airways. Dr. Hyde perhaps overstated the case a bit
when he said that that doesn't measure small airways. That's not quite true, but you have to
have substantial change in the small airways before you see great changes in resistance
values.
Albert: Is it legitimate to think in terms of the possibility that the effect of exhaust is to
ameliorate the effect of either SOX or the N02? Because, in terms of the surface-to-volume
ratios, the SO* and the N02-high are at the far end of the spectrum and the irradiated and the
raw are at the other end, and when you combine them you either get an effect in the middle
or it's even less than the exhaust itself.
Thurlbeck: The pulmonary function corroborates that, too, doesn't it?
Gillespie: Yes, that's right.
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Dungworth: That was the point that I was going to raise next. There are some slightly
mystifying aspects of looking at the various mixtures, and one being that with the irradiated
exhaust or I + SOX, there is more N02 for instance than in the N02 and NO atmospheres
alone, but yet the lesion deep in the parenchyma was less. Could this be a particle factor? We
were talking this morning about the effects of particles and deposition relative to H2S04 Is it
possible that I and I + SO x lie in the middle, but if you added up all the possible components,
they should be worse, that this was a paniculate absorption phenomenon that they weren't
carried down as far?
Lewis: It's a possibility, but the N02 could have been oxidized to nitrate before it reached the
actual alveolar zone or even the bronchial zone.
Rouser: I would like to point out the possibility that if the hydrocarbons actually enter that
membrane space and compete with things like NO 2 and so on, they could cause a lot less lipid
damage. These are physical properties of these molecules.
Busch: I don't know what process you're in, whether you're drawing conclusions which start
to be supported by the data, or whether you're building hypotheses. You seem to be building
hypotheses. If you are building hypotheses, then it's important not to confuse absence of
statistical significance with absence of effect. If you take the difference between a test amine
and a clear air amine the measured value may be here. When you allow for the possible limit
of experimental error, that's a process of putting confidence limits on the results. If the
confidence limits are to abide for the true mean, then the lower limit on the difference doesn't
go below difference, so you would say you have a statistically significant difference. Now
suppose this difference is statistically significant, and then you have another test group minus
clean air where the experimental value falls there and the limit of air falls here. In that case,
there would be no statistically significant difference. But it does not follow that this first
treatment is different than the second treatment. They both may be showing the same effect,
but because of the random variations, this one fell a little bit too close to zero to be called
statistically significant. The other one happened to fall a little higher, so you were able to
pinpoint it as an effect, but just because SOX and N02-high were the only ones to show
significant effects does not mean they were the only treatments to have an effect.
Comment: They showed the most demonstrable effects.
Nettesheim: I think the point he is making is an extremely valid one, because we were indeed
jumping the gun trying to figure out why one group shows this effect and the other group
exposed to the mixture seems to go in the opposite direction. I think that's the point you're
making, that the difference may not be real because of the absence of statistical significance.
Busch: The point I'm making is that if you have an interesting hypothesis, your data may be
consistent with that hypothesis even though one of the treatments shows an effect and the
other one apparently does not, considered by itself.
Comment: Don't let the data get in the way!
Stara: I think that Ken Busch has in one sentence described our problems.
Busch: We looked at rather simple hypotheses. We compared treatments with means and
treatments with other treatments, but we didn't develop every possible hypothesis that seem
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to be now of interest to this group. It may be that the data are consistent with some of these
hypotheses, but, remember, these were not selected to be tested.
Bhatnagar: This is one of the possibilities, and I tried to bring that out earlier today. The
difference between SO* alone and SOX with irradiated exhaust, it's possibly just simply
chemical. The main component of pure irradiated exhaust, the toxic component, is possibly
ozone which leads to the formation of the superoxide radical which is supposed to be the most
important component of photochemical smog in terms of interaction as an oxidant with
biological systems. Now, when you have sulfur dioxide alone, you get bisulfite ion, and
bisulfite ion eventually ends up as sulfate. The process continues with the generation to
superoxide. So if you have sulfur dioxide alone, you actually also produce superoxide. But in
the presence of ozone, the superoxide may not need to be generated in going from bisulfite to
sulfate, and the sulfur dioxide may actually compete for the superoxide radical formed by the
interaction of ozone with tissues. Because of this both sources of superoxide arise in the
irradiated exhaust. Superoxide may be generated by sulfur dioxide alone or be dissipated by
its subsequent reactions. So you have at least one component of the toxicity missing here
simply because of the competition between these two.
Comment: If that theory were true and tenable, one would suspect that SO 2 would be more
toxic than sulfuric acid, and that's not the case.
Bhatnagar: Well, SO 2 is transformed very rapidly.
Comment: No.
Bhatnagar: Anyway, the thing that somebody pointed out there was that bisulfite is very
rapidly transformed in the biological system.
Nettesheim: There are other reasons why sulfuric acid is more toxic than S02, that have
nothing to do with the chemistry.
Bhatnagar: I'm simply pointing out that there's competition for the superoxide ion between
sulfur dioxide and the irradiated exhaust.
Comment: You were saying that sulfur dioxide would generate superoxide.
Bhatnagar: By itself, it would generate superoxide.
Comment: We've exposed dogs to 5 ppm of S02 for a long time with no impairment.
Bhatnagar: I don't know if sulfur dioxide has any toxic effects on these dogs. The irradiated
exhaust does have toxic effects, and sulfur dioxide seems to ameliorate that effect, that
particular effect. This is what I've been hearing here.
Comment: That's not sulfur dioxide.
Bhatnagar: SOX.
Comment: The next generating problem is really sulfuric, which is 80s and H2S04.
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Bhatnagar: Yes, and 80s would compete for the superoxide radical, reduce the concentration
of the superoxide radical, and therefore somehow lessen the toxic effect.
Comment: There's gaseous S02 in there, too, metered in from tanks.
Bhatnagar: SO 2 would do the same thing.
Stara: I wonder if we could take a. moment. Two or three times during the meeting, we have
had some questions or problems with some of the statistical treatment of the data, and I
wonder if you would, for the record, Dr. Busch, summarize the issue and what you think
should be done.
Busch: The serious problem I saw would be sample sizes on Dr. Hyde's data, the number of
tissue samples. There were relatively few animals represented, but large numbers of samples,
because 20 or so samples were taken per animal, I believe. There is a high correlation, a high
degree of agreement between results on replicate samples from a given animal. Depending on
the relative size of the inter-animal and the intra-animal components of variance, the sample
size needs to be adjusted. If the inter-animal variability is large compared to the intra-animal,
then the sample size should be the number of animals. You must first pre-average the
replicate samples on each animal and then consider those as single observations where the
number of animals is the sample size. That's the major error that I've found. There was the
matter of selecting the extremes as a sample size, the extreme deviates from the mean of 12 as
a sample for the tissue examination. I don't really know what property that has.
Hyde: That was just on comparing the N02-high and some of the control animals in one
method of measurement. In the others I did use all of the animals available.
Tyler: He had two studies, this study used all of the animals and then the one in which he had
two tables; just the two animals, six controls and six from one NO group.
Hyde: There were about 14 animals that I didn't look at for the SEM and TEM. That was just
a time limitation. I looked at all controls and then to see the greatest amount of variability I
just intuitively took those that were — I did not select those that were closest to the means of
the group for distal airway margins.
Comment: Your morphometric measurements in comparison included all 71 or 72 animals.
From this complete sample, sub-samples were selected for your scanning electron microscopy.
Stara: This was exactly my point. If you use all the animals available, how much difference
does it make if you take 20 samples from each animal and pool the groups together or if you
individualize?
Busch: It makes a huge difference in the standard error for the mean.
Stara: We must be able to analyze the data accurately, because we have a relatively small
sample size; if we examine many lung sections of each group, doesn't it improve the statistical
error even though we have small number of individual animals?
Busch: That's the way it is. If you believe that the animals' lungs were identical so that in fact
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result on the average, then under this model you could examine your data to see if they were
consistent with that model, to see if the samples were the same from every dog's lung. If they
were, then treat them all as one big sample from a master dog, pool dog.
Nettesheim: It still has to be treated such that it is clear from how many dogs the 40 samples
are derived.
Stara: In dealing with the dam and the offspring in teratological studies is the sample size the
dam or the newborn?
Question: When you take many samples from one animal and pool a lot of animals, does that
tend to decrease or increase the mean? If you take 5 samples from each animal as opposed to
20 samples from each animal, would this tend to decrease your standard error?
Busch: It's a matter of how you assess the variation in the mean. I think you've greatly
underestimated the variation in the mean by treating the sample size as a larger number than
it should be.
Comment: It also possibly allows a smaller difference to become significant if you have a
much larger base of numbers. When N is large, then a much smaller difference is significant.
Busch: There is one final point I have to make. I think what may have happened is that the
statistical analyses were canned and computerized. We got into the habit of running analysis
of variance, and toward the end analysis of variance may not have been applicable.
Stephens: I would like to make a comment about potential relevance to human experience
and human exposure, particularly in conjunction with cigarette smoking and the calculation
with N02 there. I guess the level of NOathat was being used in this particular experiment was
relatively low in comparison to other experiments that have been done. It appears to me that
some of the options between animal species are not open, one to another. We have never seen
stratified squamous metaplasia in rats and yet that seems to occur from Dr. Hyde's
experiences with N02 in dogs and certainly from our previous study with ozone in dogs.
Apparently the cells in rats do not have that option open to them. Look at what the human
experience might be from the data that must be available. You who work with humans might
be able to determine the level of N02 in cigarette smoke. A puff of smoke might contain as
high as 200 ppm. I guess that has been adjusted one way or another and is down in the order
of possibly 25 ppm at the present time. If there is any reality to the amount of N02 in
cigarette smoke, then the lesions that we see in the dog at these low ppm in comparison must
be available or could be appraised from autopsies from human experience from heavy
smoking. I'm not saying that the data are readily available but it's something we ought to try
and acquire additional data on.
Albert: How can you not test squamous metaplasia in the rat?
Nettesheim: The rat is the animal that more than any other tends to develop squamous
metaplasias. There is no animal that I know of that gets squamous metaplasia as easily as the
rat.
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Stephens: We have never seen squamous metaplasia in the terminal airways of the rat as a
result of NOa or ozone exposure. If it's in the trachea, that's another question, I have not
sampled that often enough.
Dungworth: So you would have to specify precisely where anatomically in the lungs?
Stephens: Right.
Question: Do you have any tissue besides the lung? What tissue did they keep in the
necropsy?
Orthoefer: Everything.
Stephens: The material that we have already worked on is still in block form. There may be
some tissue from the biopsies but I think that Dr. Freeman still has that
Orthoefer: You're referring to the biopsy because the rest of the lung is available. All the right
side of the lung js still available except for small pieces that have been taken out for
pathology. Lobectomy was done on the apical lobe of the left side so the rest of the lung is still
available. Scanning electron microscopy could be done with the same size sections and
compared with some of the other animals. It seems to me that you got a disproportionately
large number of the dogs that had high NO 2.
Stephens: We've got controls plus two exposures, the irradiated automobile exhaust and the
N02-high which had lobectomies.
Comment: You've got five or six N02-high. Five of the eleven were left.
Comment: I don't think it should be limited to the lung. We've gone to a great deal of
expense and time to conduct the study. I don't see why not, since they can come up with the
money but don't, do detailed histopathology on the brain. We talked about this earlier. There
is a theory that there is permanent brain damage at various epidemiological levels. It's never
been proven. You've got the opportunity here to look at it, and you've got all the tissues.
There are potentially other target organs. How about the blood vessels? CO could have an
effect on them.
Comment: Somebody already said they did look at the arteries. There was no atherosclerosis,
is that correct?
Comment: Not with any morphometry.
Dungworth: You have to be realistic because each of the studies that we talk about is going to
take somebody a long long time.
Comment: First you take some sampling from each group. You've got the tissue and you
spent a lot of money to get it. It has never been available before.
Dungworth: All we are saying is that observation is fine. It doesn't show anything that is
convincingly different. This means that you are going to have to get into some quantitative
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sampling and once you talk about sampling quantitatively you're into the whole morphome-
tric analysis. It's a large undertaking.
Comment: With all of these different compounds you are using you should potentially have a
whole bunch of different target organs.
Tyler: Morphometric analysis of the brain is tough to do. It's not simple and I'm not sure we
even know what tests to use on it. Are we really talking about total neuron disappearance or
are we talking about disappearance of buttons? Certainly we see both processes quantitatively
happening in certain disease processes or in certain behavioral patterns in some cases other
than disease. Both are possible. Doing the stereology there is one hell of a lot tougher than
doing it in the lung. It would be a tremendous undertaking. I am impressed by pathologists
looking at nervous tissue. We autopsy a lot of monkeys at our shop and these guys came up
with this PML lesion which was hard to find and so it was really a sideline with these
pathologists. At this point in time, unless there is going to be a tremendous amount of effort
expended, I'm willing to take the pathologist's opinion that there were no obvious lesions in
the brain. I'm willing to take his opinion that in the tissues that he evaluated, the major
vessels were within a normal range for animals of that age. We could measure those till hell
freezes over and I don't think we would necessarily come up with anything"more.
Stephens: Could you increase your sample size in the statistical problem by at least taking a
look at the animals in which the lobectomies were made. It may well be that they were a
problem. I know the physiology wasn't done on them. If you use the opposite side from which
the lobectomy was taken, it may be a way of improving your data base and satisfying the
statistical problem.
Hyde: What he is objecting to is the cumulative calculation of all the animals in samples
rather than a per dog data base. It's just a method of calculation, not really the results that
much. I don't believe it will shift the results. We will change our calculations accordingly. We
didn't have a statistician advising us along these steps so it's natural to make this mistake.
Orthoefer: I feel that throwing the lobectomized in with the normal animals is going to cause
a lot of problems because they are not normal. Once an animal has been lobectomized, he has
had a lot more insult than with the auto exhaust.
Comment: You don't have to do that. There were some animals in the same groups that were
not lobectomized.
Orthoefer: We can't throw them all together.
Comment: We have some control dogs that were lobectomized too so you should be able to
see if there is some difference in those controls.
Dungworth: I think the need is not so much for morphometry but to look at the lobes that
were taken out with the methods that we have used in the more recent investigation and
compare them with pulmonary tissue from 3 years later.
Stara: Drs. Hyde and Tyler both suggested further work with the quantitative pathology of
the EM sections.
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Stara: I was very much interested to see what the expert here would say about the pathology
data and particularly the morphometry because this phase was performed only on the
histological sections. Dr. Hyde is suggesting that this work should be expanded into the EM
tissue section, right?
Hyde: Yes, I propose the use of low-level transmission electron microscopy, about a primary
magnification of 2000, in which you look at the interalveolar septae, examining the compo-
nent parts, type 1 epithelium, basement membrane and the endothelium. You can count cell
populations, measure the capillary density. Just about every measurement that would relate to
a significant difference in the pulmonary function.
Stara: The amount of funds requested to be spent is not large. What we need to know is the
validity of the work and the reasons for it. There was another phase proposed by Dr. Tyler.
Tyler: Basically we want to take the tissues, go back and look at the bronchi primarily and
somewhat at the trachea, using Spicer's histochemical methods which we have applied to
tissues fixed in this manner in the past, but have not applied to tissues fixed and stored this
long. If the storage were a problem, we would simply use Dr. Orthoefer's blocks from his
study which have been in paraffin and hence are not subject to any leaching problem; I don't
think that happens that much with the mucopolysaccharides present. Then apply morphome-
tric methods for the evaluation of numbers of units and relative numbers of units of each
type.
Albert: Is there anything that can be done to further elucidate the primary lesion in the lungs?
Thurlbeck: Certainly the simplest thing to do is just to look at major and minor airways. It
might be useful if you want to do simple things and do things cheap to look at the SOX group
that had the high airway resistance as against the control group. It is easy enough to count
and measure dimensions in small airways in paraffin sections. You won't get too much joy out
of the major airways, but you can measure all those things pretty easily and quickly. You can
measure mucous glands, you can point count, you can do muscle. It's very easy if you're
talking of small numbers of animals as a pilot.
Stara: Would it be worthwhile at all after all this discussion of cardiovascular lesions to look at
the heart and maybe at arteriosclerotic lesions in the vessels?
Lee: What is the chance of getting Tappel's group to look at fluorescent pigments to examine
the acceleration of aging process?
Tyler: This is an extraction procedure; he homogenizes tissue and extracts it, as I recall, and
then measures the fluorescent pigments spectrophotometrically. I don't think it can be done
on fixed tissue.
Kleinerman: No, it cannot.
Tyler: It can be done on the frozen tissue. I agree this is an elegant method for some tissues
but is it really the right one for pigment in lung?
Lee: Rather than the lung, I think the heart samples would be more valuable.
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Rouser: Yes, heart and brain.
Tyler. The fluorescent stuff you've seen has got to be lysosomes, doesn't it? He's quantitating
in an homogenate the secondary lysosomes which you see in EM.
Stephens: I haven't done that type of cytochemistry for a long time but it seems to me that it
is a possibility that it could be looked at. Certainly the numbers of lysosomes in some of those
epithelial cells in particular. It might be as valid to do it cytochemically or biochemically if the
tissue only permits that.
Lewis: There is evidence of lead toxicity in these studies; therefore, it wouldn't be worth
doing. The only question is the biological availability of irradiated and raw exhaust Is the site
of deposition within the bone different? However, it may only be an absorption phenomenon
because we are dealing with soluble and insoluble lead.
Hueter: We have only a very poor measurement of lead in the exposure.
Orthoefer. What about the pilot lead study? Don't you think that shows an increase in bone
lead of animals exposed to auto exhaust?
Hueter: Yes, but the question is what would it tell us. We couldn't use the information for
anything productive.
Nettesheim: I would also say that while there are some interesting things that can still be
followed up with the materials still available, it would probably be more productive to use the
money and manpower to address the questions that have been caused by this experiment.
Albert: There is just one point that occurred to me and that is you showed some damage to
the cilia in the trachea. Was that in the region where the intratracheal tubes were put, and did
they have an inflatable cuff?
Hyde: My samples were taken 2 cm cranial to bifurcation of the trachea and that's quite a
distance away from the larynx where the cuff would go.
Lewis: All dogs were tubed and it was only found in N02-high. It would be very coincidental
that in one of eight treatments we could see effects, when they were all handled the same
throughout the study.
Albert: Would you always put the cuff in the same place in the trachea?
Orthoefen Essentially it was placed in accordance with the length of the tube and by
palpations along the trachea.
Albert: I wondered if the injury was in the area where the cuff was placed?
Hyde: It would be much lower than that.
Lewis: While we were reporting on it, swabs of the trachea were taken to look for infectious
organisms. Essentially everything was negative.
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Albert: A cuff like that does produce a lot of damage to the mucosa?
Hyde: I'm saying that where we sampled from was far distal to where the endotracheal cuff
was placed.
Stara: We have reviewed the possibilities and we can take it from there. Perhaps a discussion
of how this study may help in planning and funding other chronic low level projects would be
useful.
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13. CRITICAL REVIEW.
IMPLICATIONS AND PLANS FOR THE FUTURE
W. Tyler, D. Dungworth and J.E Stara
This chapter consists of a critical review by a panel of experts of the data presented in the
previous chapters. The panel members were: R. Albert (New York University Medical Center),
J. Brain (Harvard School of Public Health), G. Kleinerman (St. Luke's Hospital), J. MacEwen
(University of California Aerospace Medical Laboratory), W. Thurlbeck (University of Mani-
toba Medical SchoolX E Nettesheim (Energy Research and Development AgencyX B. McLees
(National Institutes of Health), K. Busch (National Institute of Occupational Safety and
Health), N. Littlefield (National Center for Toxicologic Research) and G. Hueter (U.S.
Environmental Protection Agency). Hosting this review session were: Dr. Jerry Stara (U.S.
Environmental Protection Agency), Dr. Walter Tyler (University of California) and Dr.
Donald Dungworth (University of California)
Tyler: Today's sessions will address some longer range aspects of this and future studies.
Perhaps we should look to future research plans first, as our discussions about the toxicology
of this study and about the implications on air quality will be long and a consensus may not
be possible. While the experimental plan of our present study and the limitations of the work
that we have done are fresh in our minds, we ought to consider first the next long-range study.
From our discussions over the last 2 days, we see that there has been some value to this study
and that more long-range studies are needed. Perhaps our panel of experts would address
themselves first to the plans for future long-term low-level studies in "higher mammals."
Brain: The most intriguing and powerful aspects of the current study are the morphological
and physiological correlates. Another aspect of the morphological/physiological correlations
which hasn't been done yet, but might, is more attempts to correlate data from individual
animals. I'm sure you both, Drs. Gillespie and Hyde, have the numbers of individual animals.
I think it would be useful to look at the correktion coefficients between various pairs of
morphometric, morphological, and physiological correlates. For example, if, on an individual
animal basis, one plotted the changes in TLC relative to the controls versus alterations in
compliance, one could calculate the correlation coefficient, "r". By looking at r2, one would
then have an estimate of what fraction of the variability in altered TLC could be attributable
to the changes in compliance or to some particular morphometric measurement. One can
think of various pairs of parameters that could be correlated on the basis of individual
animals: e.g., changes in TLC compared to reduction in surface-volume ratio, frequency
dependence of compliance correlated with some measurement of small airway obstruction. Is
it possible to quantify the degree of small airway obstruction? Could one quantify the total
cross section of small airways? One could try to correlate the total resistance with large airway
changes or cross section. Residual Volume could be correlated with small airway changes or
with changes in compliance. One could try to develop specific hypotheses about possible
mechanisms and then see whether the correlation coefficient supports the hypothesis. It also
seems that it might resolve some other questions. In the current studies, it is hard to interpret
dead space, PAQ2 and Pc02 values because of the anesthesia problem. However, if one
correlated those variables with morphometric variables and it turned out that r2 was a
reasonable number, that might give you more courage to say yes, the reductions in PAQ2 or
the elevations in Pccfe have some relation to the exposure conditions.
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Tyler: I think you have raised a very interesting point. Yesterday we talked about what should
be done in the future relative to these animals, and one of the things we did not talk about
specifically was what priorities should be given to the individual animal correlation using
multiple methods of evaluation. May we have further comments on this aspect?
Nettesheim: Personally, I think that to try to correlate the function and morphometric data on
an individual basis would be one of the most exciting things to do and I would give that a very
high priority, alongside the completion of some of the morphometric studies as we discussed
yesterday.
Thurlbeck: I agree with that I think we should also try to look at the problem of reversibility
since you can work with the lobes from the animals and the lungs that were removed 2 years
later from the same animals, and from other dogs in the treatment group. I recognize there
will be difficulties, but that appears to be a key.
Littlefield: A correlation should be tested in situations where there were two samplings at
different time intervals on a specific animal; that is, the reversibility or progression of a
specified parameter or lesion needs to be tested. Progression and regression studies are of
interest to the regulatory agencies and are relied on heavily for determination of what
regulations and restrictions need to be imposed. In instances with this study all that is needed
is to take the different parameters that had two samplings or more at different time intervals.
A problem may be encountered with the specific sample sizes.
MacEwerc We had a lot of discussion yesterday on whether these morphological changes
either progressed or regressed, and there was really no experimental evidence. I was hoping
perhaps that some of the tissues that were collected might be in a usable fashion to be
recompared. That is, not the second sampling from the animals that were lobectomized but
the lobe that was removed earlier. Is that tissue in any kind of decent condition or was it
properly prepared in a way that can be compared to tissues that were taken subsequently, at
Davis?
Stephens: I think there may well be some small portions of that original lobe that were fixed
in a way that they could be looked at I do not think it was inflated so it may be quite a
difficult task. We will only know by taking a look at what still exists.
Nettesheim: I would like to say something about the reversal of lesions before we go on to
something different, because it might save you some trouble. We recently did a "reversibil-
ity" study after carcinogen exposure. The term reversal can be dangerous and one can get
caught in a trap. We induced rapidly appearing hyperplasias and metaplasias with carcino-
gens and, under various conditions, these disappeared, so we were tempted to use the term
reversal When we took a closer look, indeed the metaplasias had disappeared, but something
else had appeared instead which was much less conspicuous. I think the term reversal implies
reversal to normal, rather than entering another phase of the disease.
Kleinerman: The important issue to me is not so much the correlation between animals and
not the definition of the emphysema, because I think it is unquestionably there. Rather it is
what the mechanism of this lesion is. If we are to investigate the mechanism of the lesion
starting with whatever tissues are left over, you have to look at the ultrastructure because that
is what you have left. Such a study may not be a quantitative ultrastructural study, although
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that can add some information. The approach should be to ask a question or make a
hypothesis about what has happened to the tissue and then specifically look for the effects.
For instance, if you think that there is increased lysosomal activity in all the cells, can you find
it? Can you really find this change in the interstitial cells or the fibroblasts? Instead of doing a
blind study of quantitating everything that may be present in the remaining tissue and
coming up with numbers, address yourself to specific questions about what may have
happened and try to provide the answers.
Brain: What bothers me about that approach is that the smoking gun is long gone. Even
though the lesion may have been initiated by release of lysosomal enzymes or fibroblasts may
have been importantly included, they may have done it years ago. The kind of approach that
you have described may make a lot more sense if it could be done on a longitudinal basis.
Kleinerman: We have no proof that the lesions really have stopped. There is no evidence that
they have stopped, no evidence as to when these lesions began, if they have stopped or are
going to progress further. I think that's a hypothesis that could be explored in addition.
Albert: It seems to me that the major point is that this study demonstrates the usefulness of
long-term studies in animals. It points up the need for a systematic long-range program
particularly to define dose-response relationships and interactions of important components
of atmospheric pollution. I think if this study will help to develop a program of this sort, it will
have made a major contribution aside from the enlightenment it provides.
Tyler: I think we need to concentrate on what should be our next approach to these animals.
There appear to be three main aspects. One is that individual animal data should be
correlated. The second is progression versus reversal of the lesion. The third is studies of
mechanisms responsible for observed effects. May we have comments on this.
Brain: It seems inconceivable not to carry out those studies. The additional amount of money
needed to gather that additional information is small in comparison to what has already been
invested. I would recommend it with great enthusiasm as money very well spent to do those
three or four things you've just described.
Tyler: I have not heard much discussion about the Reid index. It was very popular in the late
50's and early 60's. Dr. Kleinerman, would you want to comment on that?
Kleinerman: I will comment on it only in a general way. I'm not sure I agree with Dr. Brain
about the philosophy of wanting to study everything available. From the point of view of
realism in terms of time and money, you've got to ask specific questions rather than try to
cover the universe. You can do the Reid index or point count goblet cells or enough things to
fill one man's lifetime if you are to study everything about these tissues. You have to get down
to asking a specific question or two that may help in the understanding of the things thought
to be crucial. If you think that studying the Reid index is a crucial index, which I do not
happen to believe, then do it. The crucial question has to be defined and from it the
appropriate experimental approach taken.
Nettesheim: I tried to point that out yesterday, too. I do not believe that it is useful in these
studies to try the "hospital approach": admit the patient, send him through the laboratory
mill and get any and all data you can possibly get. I do not think this approach has ever made
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any significant contribution to our knowledge of pathophysiological processes. I agree with
what Dr. Kleinerman says. We should focus on what this study has unearthed as the major
finding and try to pin it down further and go from there in designing the next set of
experiments. We need to ask what are the crucial questions that are raised by this study
rather than to try to determine which out of an endless number of biochemical or physiologi-
cal parameters might have been changed by the exposures.
Thurlbeck: The Reid index in beagle dogs may be pointless because the glands are so small
and they are in the trachea mostly. After SOX exposure, the glands get big and distended but
not hypertrophied and hyperplastic. I don't know what the best procedure would be for those
dogs with a high airway resistance comparing them to the normal. I would probably
quantitate everything in sight in the major airways and look particularly at muscle. Goblet
cells should be quantitated.
Gillespie: I think we need to look at the surrounding tissue in the large airway to try to
account for the correlation.
Orthoefer: We have more information on these glands in larger airways. They will have to be
quantitated. I have done it on a grading system and it was nearly significant in certain groups
of dogs.
Littlefield: It would appear that what is done with this tissue in addition to what has already
been done would be guided by what you plan on doing in the future. If you plan on doing a
carcinogenesis study, then hyperplasia might be interesting to look into. I agree with what
Paul says, that is, you can't analyze everything, however, the studies you may want to conduct
in the future should dictate to a large extent what you concentrate on in the analysis of this
study.
MacEwen: I would like to return to one point, the need to take the available data we have on
the pulmonary function studies, try to put them into common terminology, ensure that they
have been given exactly the same statistical treatment, and then find out whether we can plot
a dose-response curve for the exposure group with various levels of NOX.
Brain: Another critical aspect of this study is this: to what extent are the atmospheres used a
good model for air pollution. I know you've got piles of data about the atmospheres, but I still
haven't been told whether the atmospheres generated are typical of urban atmospheres. Can
we dismiss the trace metals? Can we dismiss the other compounds that weren't analyzed?
When we look at a physiological change and correlate it to high N02, is there anything else
that might be causing the observed changes? Are there people here who can summarize the
situation and say "Yes, this really is an accurate model," or "No, it's unusual to the extent
that..." and so on? Another important question is to what extent is the dog a good model?
To what extent is the dog more or less sensitive than man? The pattern of airway branching is
more "pine tree" than man and there is also more collateral ventilation. Furthermore, the
ability of the upper airways to scrub soluble gases may be superior to man and/or some other
animals. The other concern is whether we have adequately defined the dose. We have defined
the dose in terms of exposure, not in terms of the actual dose delivered. Each dog's dose is a
function of his size, metabolism, and level of activity. I wonder whether these factors have
been considered. It is true that different animals breathing the same particles do not always
receive the same dose in terms of particles per gram of lung. There are significant species
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differences. I would be interested in any comments that any of you have as to what extent that
the dog is similar to other animals and man.
Albert: I would like to urge a better characterization of the actual dose of sulfuric acid
particles given to beagles. Another point is that it might be a good idea to take a systematic
look at the vessels to see whether there is arteriosclerosis. It would be a shame to lose that
data.
Lee: That was one of the suggestions I was going to make. Look at the aorta biochemically
and morphologically.
Orthoefer: There are slides of the aorta; also one-half of it is still available in 10% buffered
formalin. The other half of a lengthwise split was given to the biochemists.
Nettesheim: I would like to hear what the rationale is for doing things like this. I don't see
what would prompt you to want to look at the aorta.
Albert: What about carbon monoxide?
Question: Does that cause vascular disease?
Brain: That assertion has been made. On the basis of what we have heard here there is
nothing to suggest doing it. But the assertions that others have made and the magnitude of
the problem may make it worth doing.
Tyler: Mr. MacEwen, I think you have done as much CO work as anybody. You've looked at
the effects of CO.
MacEwen: We did not see any evidence of change in the aorta or in the heart, but these
exposures of dogs were considerably longer than our exposures and I think they are well
worth looking at. There have been a considerable number of comments made by epidemiolo-
gists, including John Goldsmith of the State Health Department here in California who has
claimed that lower CO levels than the dogs in these experiments received have caused
disease.
Nettesheim: I would like to say that if you get into this, the aorta may be the wrong place to
start You might be much better off starting with the smaller vessels, for example, the
coronary arteries and other smaller vessels. If you are willing to make a commitment to it,
that's fine. It is not something you're going to do just quickly on the side.
Stara: The cardiovascular studies were not a part of the original study design, they were
started in 1970.1 want to make a point that one-half of these dogs was exposed for 5 years to a
level of approx. 100 ppm of CO, the other half was not. I do think that the careful analysis of
these data may be useful. Several of the investigators have some of the tissues frozen if we
want to use them; one of them is Dr. Rouser.
Thurlbeck: There is important material that we haven't spoken about — the small mammals
that were exposed for a year. My understanding is that they were negative experiments, right?
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Rouser: Yes, they were negative.
Thurlbeck: Could the material be reviewed since there are new techniques available? One has
to think about whether one can do a lifetime exposure in some other species, such as hamsters
or gerbils, in a shorter period of time.
Stara: Those tissues are not available; they were discarded a long time ago. There are some
data available on small rodents exposed to auto emissions. I don't think that we should start
another study to clarify further the rodent data.
Tyler: I am hearing a strong recommendation to EPA to evaluate the vessels in these dogs,
both the large vessels and the small vessels,
Thurlbeck: I think the point is that you are not very likely to get any answer that's meaningful
by saying this is a simple study that can be done easily by staining a few strips of aorta. This is
a very tough problem that has been confounding people in the vascular field for a long time.
Albert: There is a perfectly straightforward question that you can ask here. Is there any
atherosclerosis?
Kleinerman: That is not as straightforward a question as you make out. The answer that you
are apt to get may be influenced by the nature of the diet These animals do not have a diet
similar to that of the human.
Albert: Suppose there is no difference.
Kleinerman: But there is, and the dietary factors, uncontrollable as they were in this study,
are likely to have an effect which may overwhelm the effects of the CO exposure. In any event,
we would not be certain after having performed a long, detailed and expensive study how to
separate the effect of CO from that produced by diet hi the development of vascular lesions.
Tyler: Can we get back to the relative priority of correlation of physiology and morphology on
mechanisms of the lesion. Dr. Dungworth, you would probably like to talk on that for a
minute.
Dungworth: I agree with the comments that Dr. Kleinerman made. I think further ultrastruc-
tural aspects of lungs can be incorporated into the study that Dr. Hyde is planning, and
should be, so that he is looking for answers to specific questions.
Hyde: I agree with you, but it is not difficult to collect extra data when you do stereology. You
don't always know what is going to happen. I don't mean to quantitate everything and I
intend to ask specific questions. What are the structures that possibly will be changing, such
as capillary density and capillary surface areas? With CO these are specific questions that
should be asked. Lysosomal activity requires a different level of magnification but that easily
could be another step. One should ask specific questions, but it is not difficult to quantitate
other structures while one is in the process of answering the original question. There must be
a balance between what you can measure economically and measuring those structures that
you expect to change because you may not have asked the proper questions.
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Lee: Yesterday the possibility was mentioned of analyzing for fluorescent pigment in these
tissues to examine them for possible changes in the aging process.
Tyler: We'll add to our list of possible tasks fluorescent pigment evaluation. Are you
recommending lung tissues, other tissues or both?
Lee: I would pick heart tissue over lung tissue. The order of preference would be heart, brain,
and then lung.
Tyler: Other comments from our panel of experts on the fluorescent pigment evaluation?
Albert: On another subject, what about quantitating those holes of early emphysema?
Tyler: One can quantitate the holes. There are a couple of ways of doing it. The usefulness of
quantitation is for dose-response data and evaluation of pathogens.
Thurlbeck: Numbers are a lot better than descriptions.
Nettesheim: No, that's not true. I think that's a generalization that I cannot agree with. I
think numbers are often better than descriptions, but sometimes they are just a fancy way of
saving the same thing. I think you have to ask yourself, what am I going to learn if I know in
this group A versus group B versus group C that there are twice as many holes? Maybe that is
important in this case. I'm asking another question. What do you really learn beyond and
above the fact that you can say qualitatively there are many more holes in this versus the
other one? What have you learned if you can say there are 50% more?
Thurlbeck: If there are really more. The sort of experimental pathology, when you look at
pictures alone is art criticism. I suppose that there are very good art critics. I believe in
numbers. It may be that the subjective description of holes in the alveolar wall is biased. You
can't bias a machine.
Bhatnagar: We've got a very great resource here with all this tissue around and we are trying
to figure out what to do with it I think the scientific community at large should be given a
shot at it. A lot of people who are not here and do not hear from anybody from this group
would like to have some of these tissues and look at various things. Lots of times I've seen
write-ups in Science, for instance, where studies funded by National Institutes of Health or
other agencies have materials available and they invite a proposal from outside to see if
anybody would be interested in taking some of the material and handling it. If you follow a
procedure like that and put a write-up in Science or something, describe what we have and
ask who is interested and who would submit proposals, then you can evaluate those proposals
and take it from there. They can find their own funding to do it.
Tyler: Can we move on then to the next major topic, which is the plans for further long-term,
low-level studies in "higher mammals." I'm not trying to bias this toward primates. Primates
are in short supply and should be used in critical studies where they truly are the animal of
choice. Dr. Brain has made some studies of distribution of particles in lungs of animals of
various sizes and we've all worried about dose to the tissue. Dr. Brain, we would like to hear a
little more of your philosophy on that
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Brain: We have looked at particle doses in five different species ranging in size from mouse to
dog. We have completed some experiments where we have these five different species
breathing the same particles at the same time. If you express the data in terms of particles per
gram lung, you do see systematic differences which in part are predictable on the basis of
different metabolic rates and different ventilation per gram body weight It is in the direction
of smaller animals getting a higher dose. There may also be some systematic differences in
collection efficiency but it depends on the particle size. The main point is to raise the general
problem that very frequently we define the dose in terms of exposure rather than in terms of
amounts deposited or absorbed in the respiratory tract. There are many other things that can
influence dose. We did an experiment where we pre-exposed mice to a cage environment for a
half-hour and then added some naive mice and let them both breathe the same radioactive
aerosol. The naive ones had 2 to 3 times as much particle deposition in their lungs as the ones
that already had been there. During the first half-hour in a new environment the mice were
very curious and active. Other factors may also be important, temperature for example. There
is a zone of thermal neutrality and if the animals are warmer or cooler than that, they tend to
breathe more. Thus temperature can influence the dose to the lung. In this study my question
is; do we know whether animals in different groups had the same ventilation? Did auto
exhaust or the other agents added affect their level of activity?
Gillespie: I agree with everything that Dr. Brain said and certainly that will have to be taken
into account when we select an animal. We're going to have to know what its neutral zone is
and what its ventilation is in the environment at any particular time. One of the things that I
think came out very clearly is that we need tests, a variety of tests to study individual animals
sequentially, knowing what they were before they go in and then watching them as they go
along. One of the problems that we have in physiological monitoring of the cardiovascular
and pulmonary system is that of size. It is true that you can study in a rather sophisticated
way the function of the pulmonary system of the rat, but it's very tedious. You have a huge
problem of measuring flow events from the lung of a rat because very soon the equipment
becomes the overwhelming resistance in the whole system. As Dr. Brain knows, Dr. Leith and
his co-workers labor endlessly trying to measure accurately the flow events from these small
mammals. The advantage of having an animal of some size, something like a beagle or larger,
is that it allows large-scale monitoring equipment with fewer technical problems. In a
practical sense, when we select an animal it is going to have to be one of size.
Stephens: We have had extensive experience with the morphological response of animals
exposed to N02. This background includes both acute and chronic exposures of a variety of
animals over a significant portion of their life span. We have also studied animals permitted
to recover after long-term exposure to 2 or 15 ppm N02. This experience leads me to the
conclusion that care should be exercised before one comes to the conclusion that the changes
reported in the conducting airway are due to exposure to N02, at such a low level as was used
in this experiment
Dr. Freeman has been exposing a number of monkeys to 2 and 9 ppm N02 for up to 10
years. Some of these have been sacrificed and preliminary observations have been made with
both the light and electron microscopes. Unfortunately, we have not examined enough
animals yet to make any statements at this time. My point is that these animals are being
exposed to comparatively high levels of the gas and have survived for substantial periods.
Question; Have you ever run a pulmonary function test?
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Stephens: Yes, and it is my understanding that the pulmonary function tests for a single
animal do not correlate well from one test to the next At least that was the case several
months ago. These tests are done in Dr. Freeman's laboratory and I am not certain of the
current data.
Dungworth: I would like to ask Dr. Brain a question concerning the relationship between an
ambient concentration of a pollutant and the dose that the pulmonary parenchyma actually
receives. This relationship is going to vary for different species of laboratory animals. I
wonder if he has any suggestions as to the one or two species of experimental animals for
which the relationship between ambient concentration and dose to pulmonary tissue would
most resemble man. Then we should not be introducing another variable in concerning
ambient concentrations for human populations and chamber concentrations for experimental
animals.
Brain: I don't suggest comparisons to man as the only guide in selecting species for
experiments. I think there are many other considerations such as cost Another problem is
that a free-ranging animal breathes with quite a different pattern and gets a different aerosol
dose than one who is breathing through a nose cone shoved in a little tiny chamber.
Littlefield: I feel we should discuss the objective of what you want to do next. We are talking
very generally at this point about animals. Unless you know the objective of the study and
what you want to do next, you really can't get down to specifics.
Nettesheim: I just want to support this statement because I think this generalized discussion
might be very helpful. Statements to use for taking ambient air contaminants are of no use
unless you can define specifically what it is you are trying to determine. Only then can you
decide what the right species is and whether the ambient air level used is the right one.
Gillespie: One objective I really feel we need is to have some spots or more sampling points
along the way so that we can extrapolate about time-dependent changes during the course of
exposure. I think they need to be functional, morphological, and biochemical.
Tyler I think we really need some U.S. Environmental Protection Agency (EPA) advice as to
what kind of objective they want achieved in future studies.
Stara: We have with us Dr. Albert who has the knowledge and who was during the past year
deeply involved in planning and developing objectives for the EPA Office of Research and
Development to solve the problems that EPA is facing from the standpoint of required
regulatory actions. We invited also representatives from three other federal agencies that may
contribute to the question what it is that their organizations need in terms of long-term
studies. On that basis we can develop future programs. I don't think that we can just say, let's
study NO* I would like to ask Dr. Albert if he would give his views on EPA needs.
Albert: The things that are being talked about are: a fine paniculate standard, a short-term
N02 standard, an emission standard for sulfuric acid from automobile catalysts. That's about
it, I think. It is difficult to talk about what goes through the minds of people at the regulatory
end. The biochemical aspects of the problem are not necessarily the dominant issue. It
depends on the protocol considerations.
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Tyler: I think what Dr. Albert is saying is that to survive in the higher echelons of the EPA,
you have to be a politician and not a biologist.
Albert: Not exactly. There is an essential need for biomedical data. They prefer to know what
levels won't produce an effect. All they really want to know is what level won't cause any
harm. When you tell them that you can't really say, they find that difficult to live with. We
know what the ground problems are. We know that there needs to be work done to define the
nature of the pollution effects, both qualitative and quantitative. What, in all the mixture
these animals have been exposed to, is producing nasty effects and what is the nature of the
dose-response relationships? There was a serious question raised yesterday as to whether the
lesions observed in dogs are comparable to those seen in humans.
Stara: There are five ambient air pollutants for which a standard was set: carbon monoxide,
hydrocarbons, nitrogen oxides, sulfur oxides, and particulates. The reviews of the present
standards are to be done every 5 years based on new information that may determine if a
revision is needed. There are no ambient standards for a great number of air pollutants; but
with the new Toxic Substances Bill, it is obvious that some regulatory action on many of these
pollutants will have to be taken. Do we need to study in more depth the regulated ambient air
pollutants, or do we need to study the compounds that we know less about? Where is the
greatest need? For example, the EPA is facing a court decision regarding 65 compounds in
water; more information on all these materials is needed. I was hoping that in the discussion
of proposed future studies this group would address some of these issues.
Hueter: I think the people in the research arm of EPA, and I am not sorry to be there, are
continually harassed by trying to fight the pollutant-of-the-month approach. Although EPA
has been charged to produce documents relative to control of 65 substances in water, they're
not going to do that based on any research that's been done, to my knowledge, within the
EPA. There is no time to do that.
Albert: That's water.
Hueter: I don't care if it's water or air. If they had 65 air pollutants to address, they wouldn't
base that on any research that we were going to do at this point. It would be based on the
literature available now. As far as any chronic study that is to be done, people like yourselves
and other people in the country and the world should identify now those areas in which a
chronic study has a chance for producing meaningful data for application 10 years from now.
It might be carcinogens or fine particulates, whatever that means. The EPA has not decided
how they are going to approach the problem of respiratory particulates. NOX is still a major
problem because the standard which was set in 1970 was based upon one study that was done
in one place, which has been subsequently criticized. It should never have been set.
Stara: Initially, that was also true of CO.
Hueter: We lucked out on CO because subsequent work more or less supported the standard.
Kleinerman: It's obvious that you've got to give us a little more guideline, a little more focus
on the objectives. Are we supposed to help in defining experiments that will determine the
agent or the combination of agents which are the initiators and in what concentrations? Is it
more important to focus on defining experiments which give you something about the
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mechanisms of action of these agents? Is it also important for us to define safe limits, because
to design experiments with regards to safe limits of these agents would be different than
talking about a long-term chronic experiment. Do we have the money and time to initiate
another 10-year study to answer these questions, or are you more interested in finding a
model which will give us some of these answers in a year or two or less if we go to a different
kind of species? The kind of answers you are going to get from addressing questions to us will
depend in large measure on the guidelines you give us to some of these questions. I would
like to reverse the role and ask you, can you answer some of these questions? If you do, I
think you will get a more succinct and definitive response from all of us.
Albert: The concern is with public health and the magnitude and nature of the impact. The
issue of mechanisms comes into play in giving guidance to the interpretation of the
quantitative data. This gets particularly important at low level responses where the actual
observed data have so much uncertainty that the way you draw the curve depends on what
you think is the underlying process. For example, if you believe that you are dealing with a
one-hit curve, you tend to draw a straight line through a scatter of data. Mechanisms are the
basis for understanding the nature and the magnitude of the problem, but per se don't figure
in at the level where the decisions are made about regulatory action. Looking at the SOX data,
from the analysis, you get an effect in the production of emphysema. Maybe the major health
effects from a regulatory standpoint are not those on airway resistance but the induction of
chronic lung damage.
Gillespie: I think it's impossible to get into the framework unless you say, here we are, here
are our skills, and here are our backgrounds. Just as you pointed out, we have come this far
and we've shown these things, some of them not as definitive as somebody that's in the
regulatory field may wish them to be, but they're still going to be more useful than a lot of
other things that you try to do or try to anticipate doing. I think we're just absolutely going to
go round and round in circles if we're going to try to do what we think will please EPA. First
of all, I think it will be wrong, and second of all, it will very likely be outside the sphere of the
best skills that we have. The exciting things that I see coming out of this last study really
weren't those that are going to set levels. That may be a side product. But the exciting thing, I
think, is that we have shown under these conditions a very interesting dysfunctional and
structural lesion associated with reasonably well defined pollutants or irritants, and you can
look at that as being a response of the lung, a reaction of the lung, or you can look at it as air
pollution, whichever pleases you.
Albert: I would just like to raise the issue of asking what actually has been observed? And I
think it's not at all clear. If you look at your data, the biggest effect of course is the N02-high,
and the next one is the SO* But if you look at the irradiated exhaust, the irradiated exhaust
has more N02 than the N02-high. The N02-high has an average of 0.64 ppm NCh; the
irradiated exhaust has 0.94 ppm. It's one-third more NOa You put the irradiated exhaust
together with the SOX, and you get a smaller response than with the N02-high. Now, SOX
produces a big response, N02 produces a big response. You take irradiated exhaust, which
has more N02 than the N02-high, and you put it together with the SOX, and you get a smaller
response. The problem is, what is going on?
Kleinerman: That's still the first question we asked. Do we determine which agents or
combinations of agents are the initiators, or do we determine mechanism?
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Nettesheim: We need to make that decision. I think we should forget about the regulatory
agency. You have done the experiment and I think what should come out of this now is to
decide what the most important question is that has been raised by this set of experiments.
That's what we should agree on. The decisions which the EPA or somebody else finally
makes, that's their problem, not ours.
Stephens: The point I wish to make is in conjunction with the design of any future experiment
using chronic exposures.
The first large scale chronic exposure study on lung that I am aware of was done in the Los
Angeles area over 15 years ago and the resulting data were not very useful. The reason of
course, recognized early in the experiment, was that the level of exposure (ambient air) was
too low and inconsistent to be accurately interpreted. The data obtained were equivocal,
giving rise to speculative conclusions that subsequent investigations have had to reinterpret.
In regard to the present investigation, I believe Dr. Albert has a point in suggesting that there
are significant questions about the importance of the current findings. He points out the
inconsistency in the data based on the level of N02 exposures.
In any case, during the past decade there have been a substantial amount of data obtained
from animals exposed to somewhat higher levels of various pollutants.
These experiments have included acute and chronic exposures with the pollutant being
administered either continuously, or intermittently. With this extensive information to draw
on, we are probably now at a point where a useful long-term study can be designed.
The data should be individualized so that the progression of the disease could be assessed
more accurately. Two or more levels of exposure would be necessary, one at a level
approaching that found in severe air pollution and at least one at a significantly higher level.
With our current understanding of the response of tissues to single pollutants, perhaps a
simple mixture of gases and particles would be most productive. Animals with both short (2 to
4 years) and long (5 to 20 years) life spans should be included.
In brief, I believe there is ample information now available to construct a major chronic study
that would give unequivocal answers to at least some of the major health concerns related to
the exposure of lungs to toxic gases and particulates but much time and effort should be
devoted to the design because of the expense involved.
Stara: I wonder if Mr. Malanchuk would comment on this. Is it possible that the cycling of the
engine may have influenced the emissions to produce variations in the NOX levels? It
certainly does affect it, and I wonder if the data you are talking about might be misleading
because of that.
Brain: I think that's an interesting question. It relates to air pollution in general and relates to
flexible fuel strategies for power plants specifically. Is it occasional and infrequent peaks that
initiate and cause disease, or is it the long-term average level. I think that's an interesting
question and, to my knowledge, it hasn't been addressed in any chronic study.
Stara: Intermittent level exposure versus constant level exposure?
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Hueter: This isn't a question of intermittent exposure, though. It's a question of do you have
to cycle the engines in order to be able to get the pollutants that we're interested in. For
instance, N02 is generated when the engine is under a certain phase of the cycle, so these
might be average concentrations, but they certainly did vary throughout the day.
Brain: It sounds like, at least in the irradiated exhaust, those chambers are large enough to
damp out any cyclical changes even though what's coming out of the tailpipe may be going
up and down. That should be smoothed out by the time the animals breathe it.
McLees: I would like to know if anybody knows the chemical species involved in any of these
mixtures. Has anybody looked at free radical concentrations in an enormously transient
species that can be very, very devastating from a physiological point of view? Superoxide is a
good case in point. I think it's impossible to work out unless someone has really done very,
very detailed physical chemical studies that I know do not exist in these areas. I don't think
that apparent inconsistencies between exposures are a difficult thing to explain away, because
I think you don't know the chemical species you're working with. I don't think it really makes
the data terribly difficult to interpret. It seems to me that you have demonstrated a very
important thing. Low levels have caused unequivocal biochemical, pathologic, and physiolo-
gic changes in the lungs. To me, that is a very important piece of information.
Lewis: Everyone ought to be put in a real world situation and try to run one of these studies in
5 years, and imagine the cost and prioritizing everything you would like to be able to study.
One could spend a lifetime and billions of dollars just characterizing the atmospheres. And on
top of that, consider the economics and logistics to encompass the biological phenomena that
go with atmospheric characterization, the appropriate species, the appropriate tests. And a
very good point that Dr. Gillespie brought up, you don't have every available expertise on
hand while the animals are being exposed, and you have to match experts with what you're
trying to achieve and all the other primary considerations that are there.
Tyler: Dr. Littlefield, you had a comment?
Littlefield: I'm not sure it's applicable anymore. What I was hearing at one time is that
they're actually talking about two basic types of studies here. One is a chronic dose-response
study and the other one is a factorial type study where they study individual components and
then try to factor out the actual component that is responsible for the particular response.
Lewis: That may have been an oversimplification. There are many complications in exposure
situations such as antagonism and synergism. For instance, we know there were a large
number of nitrate particles in the chambers with irradiated exhaust which was not true for the
NOX chamber. But it's difficult to simulate the real world situation under laboratory
conditions. Animal activity and all these other variables cannot be controlled for 5 years. You
have to make certain compromises because of practical limitations in numbers of animals and
chambers. Everybody said we should have studied more irradiated auto exhaust; we couldn't
generate more irradiated auto exhaust.
Hueter: The same way, if you could control the activity and so forth, how would you apply
that to a study situation for a population of people? You wouldn't just apply it to a set level of
activity.
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Brain: I'm not suggesting that it necessarily be controlled, but it might be possible to measure
it. For example, if because the animals felt sick or irritated in one of the chambers they
exercised less and breathed half as much, that might be an important part of the discrepancy
that Dr. Albert described. Maybe the animals with the auto exhaust don't feel well, are
inactive, and they just lie around and breathe only half as much. I doubt that's true, but
perhaps it's a possibility. It's important to know in a situation like that whether the ventilation
and the dose are similar in all groups. We assume that the dose of N02 was defined by the
chamber concentrations, and I'm saying that this may not always be the case.
Lewis: As Project Officer through much of the actual exposure part, my clinical interpretation
was that there were no differences in activity among chambers, etc. The maximum activity
was at night when there was no exposure and they were going to be fed. There would be
sporadic activities when we had visitors or something like that But the greater part of the
day, they were either lying around or relatively inactive.
Brain: It sounds like an unlikely possibility, then, from what you say.
Lewis: Yes.
Hueter: We actually at one time, I believe, sent someone through and we were counting
respirations on these dogs over a period of weeks and comparing respiratory rate across
chambers and, as I recall, we couldn't detect any differences.
MacEwen: I don't think you could ever detect it in a long-term experiment, because it will
change from day to day. You could for short-term acute experiments, but not for any kind of
chronic experiment. They've got to average out pretty much the same or else it would be
obvious in their growth rates and in other clinical measurements.
Orthoefer: As far as what Dr. Lewis was saying, the people who worked in the chamber room
and took samples did not excite the dogs, but if a stranger went in there, the dogs all became
very excited and barked and climbed all over the chambers. This type of study could be very
stable if you keep strangers out and the amount of activity was programmed. The problem is
what do you allow visitors to see and not to see. If you hide the study then you are suspected
of doing something out of the ordinary. If you let people see the animals then excitement
occurs, injuries occur and problems develop. I don't know the answer, but in future studies
using larger mammals it will have to be dealt with effectively.
Lewis: Just a sidelight, the maximum excitement occurred if one dog jumped out of the
chamber and ran down past all the rest of them while they were still in the chamber - that's
when they really got excited. But that was very occasionally.
Thurlbeck: I'd like to follow up Dr. Gillespie's point. We ought to recommend that someone
look at mechanisms of disease production. We have a lesion, we know its functional effect, we
know its relevance in terms of environmental exposure, but we don't know the mechanism of
production of that lesion.
Dungworth: That's the point I would like to make, that if we asked everybody around the
room what materials and methods were necessary for even minimal effective experimental
design, we could obtain the highest common factor. But even with that, we're talking about a
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large experiment and one that is going to cost a lot of money and needs a lot of forethought,
and probably is going to need interagency funding.
Kleinerman: You mean, decide where to begin to study mechanism? Do you want ar
expression of whether the study of mechanism is the way to proceed? If we're talking aboi t
mechanism then clearly we're back to the problem of how to produce emphysema. That
problem revolves around the balance of proteolysis and antiproteolysis. One can get into the
way at any point, measuring what is happening in terms of proteolysis in the lung or serum at
various times during the period of injury and repair. One of the specific problems that needs
to be answered is the question of how chronicity can produce lesions so distinctly different
from those which are present in the acute phase.
Nettesheim: What I would like to see us do is to ask everybody present what, in his opinion,
are the most important issues that have been raised by the study and that should be proved;
not in order to generate an agreement but rather to get an idea of what the various people
with their particular expertise conceive of as the most important questions that have been
raised. I think the material that would come out of that kind of discussion could provide the
basis for formulating ideas and defining priorities for future experiments.
Tyler: We should address in the short time that remains why EPA should be interested in
chronic studies. Chronic studies do cost a lot of money. Dr. Brain, will you start please.
Brain: The mechanisms involved and the damage produced by acute exposure may be very,
very different than the lesions produced by chronic exposure. Whether the real threat of air
pollution is bronchoconstriction or whether it's the kind of progressive, irreversible lesions
that have been described is crucial to determine. The concept of a lesion which progresses
after the termination of an exposure is crucial. Perhaps one could even suggest that this study
was not prolonged enough. Another crucial question we haven't discussed is the whole idea of
impairment. To what degree were these dogs really impaired? The degree of emphysema
produced when compared to humans is not very serious. The same thing is true in terms of
the physiological evidence. The changes were not life-threatening. If you asked the dogs how
they felt, they probably would say "fine." An important unresolved question is: would that
lesion have progressed to the point where it would cause a drastic impairment? Would it even
become life-threatening? That's an unanswered question but I suspect it may. The rate of
change over the 2-year periods in some of the groups was severe. RV doubled over a 2-year
period and if one extrapolates that for another 4 or 5 years, one might reach levels which are
life-threatening.
Kleinerman: The question of why we must pursue chronic studies comes down to two basic
reasons. First, the concentrations that have been studied are realistic in terms of the
exposures to which urban populations are exposed. Secondly, we are addressing a major
public health problem to which we have no answers, in terms of understanding disease
processes.
McLees: I would like to turn it around and say, in view of the results that you have presented
over the past 2 days, how could you not do chronic studies? You have shown, from the result
of a chronic study, using low-level emission, pathologic, physiologic, and biochemical changes
in lung. You don't understand them completely. You don't even know what in the mixtures
that you've exposed these animals to is responsible for it. It doesn't make complete sense, as
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Dr. Albert pointed out. I really turn the question around. I don't see how you can get by
without doing more studies. Perhaps they need to be longer and designed somewhat
differently. I don't see how you can get around not following this up and getting some idea of
what's happening, the evolution of the disease process and what it's like.
Tyler: Thank you. Dr. Nettesheim, why don't we pick you up next, please.
Nettesheim: I will agree with everything that has been said. In addition, I would say yes, do
chronic studies. But, there are various ways of doing chronic studies, and I can conceive of
pursuing some of the Questions that have been raised by the previous study but taking a
somewhat different track. I'm not so impressed with the necessity of staying with near-
ambient air levels. I would, for example, if I were given the charge to design new experiments,
use two or three times the concentrations that have been used attempting to induce similar
kinds of lesions perhaps in a matter of 2 years rather than 5 years. Then I would address
myself to the question of reversibility of some of these lesions. I think this chronic experiment
has raised enough important questions concerning mechanisms as well as concerning which
the crucial factors in the mixtures are. These questions should be provided in a somewhat
modified experimental design which yields results within a shorter time.
Thurlbeck: I think the only thing I can do is echo Dr. McLees' comment. These experiments
show unequivocal evidence of emphysema. To me, it is self-evident that further experiments
have to be done. It is the EPA's responsibility and it is also probably the only funding
mechanism for long-term exposures. I assume that it is unlikely that other agencies are going
to get involved with this sort of work.
MacEwen: Back to the question of why do chronic studies. I think there's no question about
the need for chronic studies, for EPA or any other regulatory agency that is going to have to
define ambient limits for exposure. My only concern is whether what has come out of this
study is adequate for defining ambient limits. I'm not sure we've got enough information yet
in dose-response. We have a positive finding and yet we don't have a negative finding. We
don't have a lower limit of response. I believe that EPA should take the finding of this study
and undertake additional studies designed to give a higher level as well as lower levels to
define where the response ends and to try to quantitate the degree of impairment in terms of
concentration or dose-response curves. I think that is the more important thing to define,
both the N02 as well as SO* and I don't know how you're going to do it with irradiated
exhaust very easily because you can't dilute it very easily without changing your whole
exposure pattern in terms of CO and everything else involved in that complex mixture.
Littlefield: Well, to make it unanimous, I agree that the best way to get at this problem is the
chronic study. This is the real world. In an evaluation of a literature review, whether it's for
carcinogenesis, a pesticide, or for an environmental chemical, most of the studies are
invalidated because the exposures are too short. This is in my experience, anyway. If you try
to get an effect on an acute or subacute study, it is often necessary to raise the dose to
abnormally high levels which may cause a different physiological mechanism to be brought
into play and you begin to get a false picture. Therefore, a chronic study is the best way.
Hueter: I also believe in chronic studies, or else I wouldn't have got involved in this thing in
the first place. But I just wonder how many other federal agencies are doing chronic studies.
Is National Center for Toxicologic Research (NCTR) doing chronic studies? I really don't
know.
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Littlefield: That's essentially the prime mission of NCTR, that is, dose evaluation at the lower
end of the dose-response curve, which essentially is a chronic study.
Hueter: Really, EPA is not the only federal agency in a position to fund chronic studies. It
might perhaps be more appropriate if some of the federal agencies might consider setting up
a special group to do this kind of work. Their research people do all these quick response
studies on the lung. Yet, at the same time, experts like this group rightfully tell the agencies
they ought to be supporting chronic studies. There's no way they can do both at the same
time.
Albert: As I said before, the main contribution of the chronic exposure studies is the
demonstration of effects which are quite different and, in my opinion, much more serious
than those that are induced by acute studies. Besides which they supplement the epidemiolo-
gic studies in a very useful way. Because one thing the epidemiologic studies haven't done is
to answer the question, to what extent does air pollution really produce emphysema and
chronic bronchitis. This is aside from the issue of whether or not it aggravates pre-existing
disease. But I think one of the major gaps is whether air pollution really does play a primary
role in causing irreversible destruction of the lung.
Thurlbeck: One additional reason for chronic studies is that other than accident, suicide, and
homicide, all the important causes of death in man are chronic diseases.
Busch: I don't think it's important to simulate community atmospheres. I think it's more
important to get information about particular components of the exhausts which we breathe
which we can identify with health problems. Some components are being given attention in
the EPA regulations; SOX is one, with respect to catalytic converters. If we confound SOX with
the whole exhaust mix, we haven't perhaps gained as much information which would assist us
in making decisions about alternative exhaust control mechanisms (engineering devices), as
we would if we experimented with mixtures of SOX and CO and N02 or whatever. Perhaps we
would gain more information which could be extrapolated to arbitrary fuels or engine types, if
we experiment with more basic components. I also don't believe in dose-response studies.
Think how nice it would have been if we had had 50 animals in each group instead of 12. We
would have something really definitive. We could quantitate incidence very closely. We could
have done serial sacrifices. If we get into another experiment where we try to take a
"birdshot" approach at 8 or 10 atmospheres, the money will have to be divided correspon-
dingly, and what we really have is a very expensive mix of small studies. Each atmosphere by
itself wouldn't constitute much of an experiment, but collectively they cost a great deal of
money. So I would make it more simple and bigger. I'd use concentrations high enough so
that the effects could be accelerated, so long as it's not so high that you get into a different
mechanism for production of the effect. Retain the mechanism but accelerate the effects. I
think the reason it isn't so important to simulate community atmospheres is that we don't
regulate community atmospheres. We regulate the content of exhaust emission. There's a.
rather tenuous relationship and a very imperfect correlation between what comes out of the
exhaust pipe and what we breathe anyway, so why is it so important that we simulate a
community atmosphere? The important thing is to find out whether what comes out of the
exhaust may produce certain types of effects.
Tyler: I would like to ask our consultants once more regarding design of future studies
because we must soon bring the conference to a close.
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Brain: It is important to define dose-response curves for our major pollutants and this
requires a number of levels of exposure. I would also add that in addition to number of levels,
a future design might also want to incorporate important interactions such as infection,
smoking, occupational dusts, genetic susceptibility, very young and very old animals, and
animals with pre-existing disease. Also, perhaps species differences can be exploited. For
example, it might be useful to pick out the species which are both most sensitive and least
sensitive. Let's look at the extremes of the bell-shaped curve and ask, what is responsible for
those differences in susceptibility? Is it morphological? Is it physiological? Does it relate to
life span? One man's noise can be another man's signal. Perhaps that might be an aspect of
species differences that could be utilized in a useful way.
Kleinerman: We must be careful when we draw conclusions from studies at high concentra-
tions and apply them to lower concentrations. We must prove that the consequences of these
different exposures are similar before we can use different exposures interchangeably. I agree
that if we can study the effects of single substances such as N02 or S02 or ozone, we will be
farther ahead in understanding the mechanism of the lesions. The species of animal which we
use is very important. The choice will depend in part on what one's goal is. If our goal is to try
to understand mechanism, a small animal species may be as useful as any other provided that
the desired experimental end point results. We can then manipulate the variables and observe
the effects over a short time period. Important factors in utilizing a small animal species are
its life span and the time necessary to create the end point.
McLees: I think there are a number of important things to glean from this sort of experiment
biochemically and hemodynamically with respect to physiology and biochemical changes in
the kinds of lesions that have been presented.
Tyler: You're speaking in favor of a mechanistic approach.
McLees: Well, finding out what's happening. Right ,
Lewis: I take the opposite opinion of just about every point Mr. Busch raises. With the new
technology in automobile engines, unleaded fuel, catalytic converters, and so on, the pollutant
problems that we're dealing with are going to be different 10 years hence than they are now.
This study has some relevance, but will not answer all the questions of the toxic hazards of
automobile emissions from automobiles with catalytic mufflers and unleaded fuel. That's one
thing. The other thing that Mr. Busch said is let's take it as it comes out of the tailpipe.
Busch: I didn't say that You misunderstood me.
Lewis: Well, we need to cause a photochemical reaction to simulate the Los Angeles type of
situation. It's going to be with us forever. Auto exhaust contains thousands of components.
'aid, although this study with the internal combustion engine as we know it now relates to it
jualitatively, it may not relate to it 10 years hence quantitatively. I do believe we want to try
to approach dose-response studies, because if you don't have a "no-effect" level, the people
that are setting standards have to do it somewhat arbitrarily. That's the key point. You're not
going to be able to create synthetic atmospheres that either can be factorially removed from a
complex mixture or necessarily relate to it. I think this study proves it on a biochemical
response phenomenon, the differences between I versus N0;>-high. The known toxicologic
phenomena of synergism, antagonism, and additive effect, all come into play on both a
physical and a biological basis.
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Busch: May I rebut that? Everything you say supports what I said. You just misunderstood
me. You said that we won't know what we're going to have in the air 10 years from now, what
kind of auto engine emissions control device we'll have. That's exactly the reason I thought
we should use identifiable components, because if we can identify the toxicity of components,
then we'll be able to make some extrapolation from the data we take today to some arbitrary
or alternative engine emission control system. But if we experiment with a particular
configuration now which contains unknown components, we won't be able to extrapolate
from that to anything else. So I think what you said tends to be consistent with what I said.
MacEwen: I was a little startled at what Mr. Busch said. He is recommending the use of purer
compounds or essentially identifiable compounds rather than complex mixtures. I understood
him earlier to say he was opposed to dose-response studies, and I still definitely feel that it is
the only way we are ever going to be able to extrapolate these data to other mixtures of fuels
and to define the "no-effect" level for establishment of any kind of emission standards.
Busch: I said the "no-effect" level relates to a community atmosphere in which we breathe.
None of us breathes diluted auto exhaust or irradiated auto exhaust. We breathe that plus all
the industrial emissions and whatever comes from our home heating systems. I think we need
to consider the whole community atmosphere and not just the auto exhaust emissions.
MacEwen: But I can define my community atmosphere in terms of SOX or N02?
Busch: No, you can't But you can probably make some inferences about the toxicity of the
atmospheres if you know that certain components of it are toxic in themselves.
Malanchuk: The aerometry that was connected with this study was very considerable in spite
of what some people might consider were just a few compounds being monitored. We had to
address ourselves to doing what we did because of some limitation of instrumentation, people
and time. Mr. Busch suggested limiting it to a couple of compounds, doing a chronic study
over a long length of time, and using 50 animals instead of only 12.1 must admit if I had been
in that original committee back in 1965,1 think I would have approved of their recommenda-
tions to eliminate several exposure atmospheres. If that had been done, where would we be
today? So I think you have to spread it a little bit thin and try to avoid complete dependence
on statistics. I was hoping that we could supplement statistics with a little bit of intuitive
knowledge as to what direction it was taking.
Hueten I just have a couple of remarks. First, I firmly believe it is the scientist's responsibility
to assess the hazard of a pollutant, but it isn't his responsibility to set its risk. Times have
changed since this study was planned in 1963-65. We are in a better position to identify what
the components of these new energy technologies might be and to evaluate our screening
methodologies from the standpoint of cytotoxicity. We can now better select the components
of a mixture on which we should concentrate the toxicology and chronic studies. I really
believe that we could learn more by not looking at the mixture but using the other disciplines
that are available to us now ahead of time. So I support much of what Dr. Lewis said.
Tyler: You're supporting pure compounds?
Hueten Simple mixtures of pure compounds. And I also firmly believe in mechanistic studies
as long as they have a reasonable balance within the EPA laboratory with practical applied
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research, which the regulator needs. But for the long-range response, I think we have to study
mechanism because mechanism might predict significant disease at some future date. One
example of this is the infectivity model, which is the only mechanistic thing we've ever done,
and it's very useful now.
Nettesheim: I would like to have a clarification, because I can't get the mixtures and the
compounds straightened out I heard Dr. Lewis emphasize the mixture as it is being
produced, but I heard you emphasize a mixture of identified compounds. Is that correct? To
me, that makes a big difference, and I think the latter goes pretty much along with what Mr.
Busch said.
Hueten I personally believe that if one has the proper chemical characterization of the
effluent of interest, then, at the present state of lexicological knowledge and by using experts
assembled to assess those identified compounds, you get further faster by looking at
individual compounds that are given toxicity priority or simple mixtures of those compounds
where you feel an interacting effect probably is going to occur, than you are by putting such
an expensive facility to use with a mixture which can give you all kinds of interpretative
problems.
Nettesheim: Yes, I think that's clear.
Tyler: Now I'm going to call for brief comments from people who were involved in the
original study.
Lee: My support for chronic studies has been expressed by all previously, particularly by a
fellow biochemist, Dr. McLees, so I'm not going to elaborate any further on the same point
But, I want to say a word about the support for the whole emission studies and single
pollutant studies plus various combinations of single pollutants. I know this is probably going
to sound like talking from both sides of the mouth, but without doing the whole mixture
study, I don't think you can see the effects of interactions of certain compounds with the
particulates and with other chemicals. So I think the whole mixture study is really necessary,
and if I had one at this point, offhand, I would start with emissions from fossil fuel
combustion because most of the sulfate, some 96%, is coming from stack emissions rather
than auto emissions. For future studies, this is something that has not been brought up in this
conference. I would also like to look at the nutritional influences. In many experiments,
animals are overfed and overprotected, and in some cases, as Dr. Rouser mentioned, they
may be deficient in certain essential nutrients. Another important point that I would like to
mention is the provision of stable working atmosphere along with a stable funding mecha-
nism rather than allowing some administrators or politicians to attack the study for its
nonproductivity before its conclusion.
One other point that we should pay attention to is so-called positive results vs. negative
results. Positive results in the conventional sense of basic research are important, however,
negative results from well-conceived and well-run experiments could be just as significant in
terms of public health.
Stara: What did this study show? Did it show that certain air pollutant mixtures produced
from automobile exhaust caused definite emphysematous lesions? Is that what we can expect
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to occur in the human population? And if so, can these results be shown using short-term
studies or even long-term studies of single pollutants? I believe that certain mixtures or major
components of auto emissions together prove to be harmful and must be studied further, and
perhaps the individual exhaust components as well. But without at least one group of animals
being exposed to the actual total emissions, we couldn't develop an airtight case and obtain
the results that we need. I also know that the auto industry, the power industry, and coal
industry have projections into the future: they are now developing future technology and
fields and so we could possibly predict at least some of the pollutants that may be associated
with these processes. In some respects, we know what will occur in future years; therefore we
could now plan the research on the effects of some pollutants that will be pertinent in the
future. Hence if we started such a study now, we would have the data ready when they are
needed. It would be a sort of "prospective" toxicologic research.
Tyler: Dr. Hueter, I'm going to call on you next. Then I'll call for comments from the people
who were involved in the study but not with EPA, and then our consultants can make
comments.
Hueter: I just had a couple of other comments. Dr. Lee talked about most of the sulfate
coming from stacks and that's true, but that's probably a different kind of sulfate. That's aged
sulfate aerosol. The catalyst is fresh, perhaps ultrafine sulfate, sulfuric acid, which is a little
different problem. We're looking into that. Secondly, I think we've been a little short-sighted
in that we're concentrating on just animal toxicity, and as far as EPA is concerned that isn't
the only tool we have to look at health effects. We combine epidemiology, clinical human
exposures, and animal toxicology. And when you're using all three disciplines to attack a
problem, I think that puts a different perspective on the type of exposure that you're giving
animals in a lexicological situation, because you have a possibility of verifying it in some
instances with human exposures under controlled conditions and in other instances with
correlative epidemiological findings.
Tyler: Thank you. I'm going to start with Dr. Dungworth, because he was the major
contractor on the West Coast, as I understood it
Dungworth: The only point I want to make is that it disturbs me to hear people talking about
exposing animals to an ill-defined mixture of pollutants because it is impossible to sort out
and analyze interactions within it. If we talk about defined mixtures, that is reasonable. If we
want to know the interaction between particulates and oxidants and reductants we must use
well-characterized exposures. Unless we know what the animals are breathing, and this is in
the air, not necessarily the tissue dose, we are incapable of sorting it out. We have an oxide
mixture that we have been discussing the last 2 days, the raw and irradiated automobile
gasoline. And we have admitted that we can't sort out why apparently there was less damage
in the raw and irradiated gasoline than there was with the purer compounds.
Bhatnagar: One of the things I'm concerned about is the fact that the study did not take into
account a number of factors. For instance, human beings, when they are being exposed to
this kind of exhaust, are not protected the way our dogs were. There were no social
interactions, there was no possibility of catching a viral pneumonia which very likely would
have been exacerbated simply by the fact of having been exposed. Then there were other
factors such as nutrition which Dr. Lee has pointed out. We only used female dogs which were
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never allowed to be mated. I think these conditions should be taken into account in any kind
of long-term study. Now, we've talked a great deal here about the realities of doing factorial
types of experiments, whether we should look at a mixture or whether we should look at
individual pollutants, and I really don't think this kind of thing can be solved very easily. We
can't do a very large number of animals; obviously it's a very expensive operation. So we've
got to find simpler, cheaper ways, and I think we have to think in terms of doing experiments
using tissue cultures, organ cultures and other in vitro models.
Rouser: I would like to just add one thing which I discussed earlier. I think there is a very
serious omission that doesn't have anything to do with biochemistry. It has to do with the
developing organism. I think that development studies should start day one to try to assess
what the effects are of agents both on the in utero development and the immediate postnatal
period, where so much change takes place. I think that's something you should give some
careful thought to.
Busch: I want to temper what I said before. I didn't mean to imply that we should use simple
gases. Dr. Lee brought up the important point that it may be necessary for these gases to
exert their effects to have particulates present, and if it's necessary to generate the kinds of
particulates that are typical of the freeway and the city by using an engine, then that's a good
reason to use an engine. But if you can get them in some better, simpler way so that they're
more identifiable, then I would think you should. The best reason to use an engine is to be
able to generate the types of identifiable components we want to examine.
Bhatnagar: I got the feeling that we as biochemists failed to provide useful information and
useful input in this study. We got a very limited amount of data which do not explain the
morphological or physiological changes. The reason for that perhaps is in the fact that our
involvement was at the very terminal state whereas as a biochemist I know that the initial
response may be the most important thing. For instance, we should have looked at the
animals after the initial exposure to see if there was inflammation, if there were any kinds of
changes in the whole biochemistry of the lung, or the whole organism. What kind of
metabolic changes are occurring in different organs? What kinds of changes occurred in the
tissues themselves? Lysosomes might have been mobilized to cause emphysema. So, in any
future chronic study, I would like to recommend very strongly that a biochemical component
should be included.
Stephens: I would like to take a brief look at the history of concern about health and air
pollution. Originally (during the 1950's) much of the concern grew out of industrial problems
but by the late 1950's epidemiologic studies had indicated that the air pollution and smoking
were related to chronic diseases as well as cancer of the lung. A major criticism of early
studies that exposed animals to whole cigarette smoke and smog was the lack of identity of
the specific toxic agents within these complex gaseous and particulate mixtures. The major
question was: what specific elements were harmful?
In the following decade or so there has been a marked increase in funding from NIH to study
the problem and numerous studies on specific pollutants, such as sulfur dioxide, nitrogen
dioxide, ozone, cadmium, and the like have been undertaken. A volume of data is now
available to design and conduct a reasonable chronic investigation. The problem, as Dr.
Dungworth put it, is what kind of a defined mixture can we handle and interpret There are
many people who can, and should, contribute to the design of any future chronic study
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because it will be extremely costly, and to date, chronic studies using low-level exposures do
not have an impressive track record. It can probably be assumed, however, that regardless of
the exposure regime agreed upon, there will be substantial criticism. If an exhaustive effort is
made, however, to design the experiment well, fully utilizing the available data, then that is
the best we can do.
There are only certain kinds of questions that can be answered with chronic studies, however
they are important to the general public. I think the public needs to clearly understand the
health risk involved in exposure to smog and other pollution. Chronic studies are tedious and
it may be difficult to acquire the needed commitment on the part of qualified scientists to
continue with such a protracted study.
Current information supports the usefulness of shorter exposures at higher levels and the
reasonable extrapolation of such data to levels that currently exist in the environment In
addition, there is strong evidence that intermittent exposures evoke the same response as
continuous exposures. Obviously there is a level of exposure to each toxic element that will
overwhelm the animal and it will die after a brief period of exposure, usually under 48 hours.
However, at only modestly lower levels the animal will adapt and survive for much longer
periods giving the tissue time to respond.
In the case of ozone, it has become clear that levels between 0.2 and 1.0 ppm are useful for
chronic studies of modest length (up to 1 year). Exposures to nitrogen dioxide from 1.0 ppm
to perhaps as high as 20.0 ppm appear to be useful in terms of permitting various species to
survive so that associated cellular and tissue responses can be studied and evaluated in terms
of their health effects.
Mechanisms are obviously complex and involve biochemical, structural and physiological
alterations that are progressive. While there are many things happening during the first few
days, the insidious progression of the disease takes much longer. Protracted studies of several
decades may be required to understand the details of various mechanisms involved and the
use of simplified systems, such as protein gels for the study of free radical production
resulting from nitrogen dioxide or ozone may be useful as demonstrated by Dr. Pryor.
For the past 2-1/2 years we have been working with postnatal animals, and their response was
surprising in that they are remarkably resistant to both nitrogen dioxide and ozone. They
provide a most interesting model.
MacEwen: There is one point I would like to make. It refers to the early set of cardiovascular
studies that were done on these animals when they were still on the experiment. I think there
is a report in the package concerning the measurements and electrocardiograms of the whole
series of dogs which implies that there were some treatment effects, particularly in terms of
right heart hypertrophy, some infarcts, and also widening of the QRS complex. I think that
that should be repudiated at this point. I don't believe there were any changes. I don't think
there is any evidence to substantiate it. Electrocardiographic changes, infarcts of any
magnitude that would show up on randomly taken or periodically taken electrocardiograms,
should have been of a sufficient magnitude to have been seen clinically or to have been
observed at the necropsy. At least some scarring should have been visible if it was at that
magnitude to have persisted over any period of time. However, none of these effects were
seen twice in the same dog, as I understand. I don't think there is sufficient incidence of
cardiovascular changes.
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Littlefield: As far as the present study is concerned, it appears that the bulk of the tasks left
are largely statistical in nature. What is needed to conduct a thorough statistical analysis of
each end point?
Albert: I think this study provides a basis for thinking that there's real usefulness in a
balanced, long-term program for chronic inhalation studies. It's obvious there needs to be a
balanced mixture of dose-response studies, mechanism studies, single pollutant effects,
interactions of different pollutants, species differences, etc. One thing that hasn't been
mentioned here which I think is an important part is the intercomparison of human and
animal populations. For example, the same quantitative morphologic studies could well be
done on autopsy material on people living in cities with different levels of pollution. I think
the immediate thing to do regarding this study is to finish the quantitative morphologic
analyses on the rest of the dogs and on the excised lobes and get an overview paper written
which would describe the characterization of the atmospheres and the functional and
morphological effects and the interrelationships between the morphology and function. I
think this would be very useful toward getting a long-term federal commitment toward the
support of centers for chronic exposure studies.
Nettesheim: I agree with everything that has been said and I disagree with everything that
has been said, which means that there is some justification for the various points of view that
have been raised. I would like to turn that into a recommendation to the EPA. The thing that
has kept me away from the EPA is the fact that they tend to avoid mechanism studies. I can
see the need for the kind of study that has been done here, but I think in the long run, without
support for basic research starting, we will run out of the tools and the knowledge needed to
deal with the practical problems like the ones stemming from pollutarfts. I would say that any
agency that expects the scientists to deal with the day-to-day problems must support basic
research also. Therefore, I think it is wrong to permit a polarization between basic and
applied research. We have to create a balance. The agencies must see to it that that is being
done. Also, I do believe that what is being developed in carcinogenesis research is badly
needed in toxicology. Namely, short-term assays and early indications. This may not always
provide us with the complete answers but it will give us important leads. These assays may
have biochemical end points or provide cellular end points in tissue culture, because anything
that causes emphysema must have a cytotoxic effect at some time; if you find an agent is
cytotoxic, it doesn't necessarily mean that it is an emphysematogenic agent, but if it is not
cytotoxic it is not going to cause emphysema. I think the need for short-term assays is an
important one and I think there needs to be a considerable amount of investment into the
development of such assays. That's not a quick and easy thing to come up with. The National
Cancer Institute has been supporting the development of in vitro carcinogenesis tests for the
last several years, and it is anticipated that another few years are needed before we have the
tests that are really needed. Lastly, I would like to say that I congratulate you on this
conference. I think I have not been involved in anything like this before. I think it is a great
idea to get all the people together that have been involved in such a complex study, plus a few
others, to evaluate what has been accomplished at the end of a long and large experiment.
Tyler: Often the first indication that something has gone wrong in the health of the
population is going to show up at the autopsy table. While I'm calling on Dr. Thurlbeck last,
indeed many times pathology at autopsy is going to be the first indication.
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Thurlbeck: Dr. Brain made a point which is critically important to the human pathologist,
namely that one is often looking at interactions. The concept of modifying a long-term
experiment to examine interactions with infectious and other agents is a critical one. I have
no feeling about mixtures versus pure compounds other than that pure compounds sound
much more logical. Sulfates and sulfuric acid seem to me a lot more important than I had
previously recognized.
We have talked about advantages of using small animals because we can get quicker results.
This is worth following. I'm suggesting that one might duplicate this experiment using small
mammals and see if anything happens at the end of a couple of years. We may, however, be
fooling ourselves in regarding chronicity in terms of lifetime of an animal. Lifetime and
duration may turn out to be two different things. If lifetime and duration exposures are two
different things, the human situation is very interesting. We have to think in terms of 50-, 60-,
and 70-year exposures and extrapolate backwards to the longest possible exposure rather than
the lifetime of an animal.
Two observations have surprised me. The chronic destructive lesion within the lung appears
to be progressive after cessation of exposure, whereas intuitively it should be static. We are
unclear as to whether the airway lesions are reversible, partially reversible, or progressive.
They certainly cannot be totally reversible and I would have originally guessed that airway
lesions would be reversible after cessation of exposure. This particular variable can be
examined. I'd like to stress the point about the importance of mechanisms. Mechanisms are
finally what it's ah1 about If we knew what the mechanisms were, then as changing
technologies occur, as changing environments occur, one could then find rational solutions
rather than make empirical decisions. Every applied agency has to recognize that it has to be
looking at mechanisms at the same time.
I'd like to pick up the point about what we can find in the autopsy room. The available
studies really have not addressed themselves properly to this problem. The reason is that the
studies are extremely difficult. It would be useful to try to identify people who have lived in a
non-urban environment for a long period of time and look at a whole variety of morphometric
and functional variables on excised lungs. You can get a lot of good information out of
excised lungs, and then compare these findings to the lungs of people who have lived in cities.
The previous questions that have been asked are crude ones — these have been questions
like, are there big holes in the lung? Are there big holes more frequently in the lungs of
smokers than nonsmokers? Are these big holes more frequent in people who have lived in
Montreal compared to Malmo in Sweden? These questions are crude ones, and we should be
asking much more subtle ones like, is there loss of recoil in the lungs? Or are there subtle
morphometric changes in dimensions of the lung in subjects who have lived in an urban
environment? We should be asking, I think, whether there is evidence that lungs are
prematurely and excessively aged because of environmental exposure.
Tyler: Thank you. I'm going to summarize the conference by saying that the experiment was
much better planned than I had realized. It was conducted at a high level, and I think that it
has shown significant results. I'd like to thank all of you for participating. With that, I'll turn
it over to Dr. Stara.
Stara: I want to thank all of you for your input and interest; particularly for your willingness
to work the long, early and late hours. I think this was an extremely hard-working and
successful conference.
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TECHNICAL REPORT DATA
ff lease read Instructions on the reverse before completing)
EPA 600/8-80-014
3 RECIPIENT'S ACCESSION NO.
Long Term Effects of Air Pollutants:
In Canine Species
5 REPORT DATE
July 1980
6. PERFORMING ORGANIZATION CODE
J.F. Stara, J.G. Orthofer, D.L. Dungworth,
W.S. Tyler
8. PERFORMING ORGANIZATION REPORT NO
10. PROGRAM ELEMENT NO
A1LH1A
SAME AS BELOW
11 CONTRACT/GRANT NO
SPONSORING AGENCY NAME AND ADDRESS
Environmental Criteria and Assessment Office
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
Series 8 - (1970-1980)
4. SPONSORING AGENCY CODE
Research and Conference Proceedings Being Published Together
The Clean Air Act of 1970 as amended in 1977 requires that a comprehensive
data base be established to assess human health effects caused by air pollu-
tion from mobile sources. The spectrum of potential toxic effects can be
viewed from two perspectives: The first is the identification of toxic ef-
fects from combined low-level effects of the individual major ambient air pol-
lutants, which are combustion by-products of automotive exhaust. Ideally, the
major components of a data base used to develop health risk assessments are
well-designed epidemiologioal studies and long-term, low-level animal studies.
The 9-year study presented in this monograph reviews the effects following
exposure of dogs for 68 months to automotive exhaust, simulated smog, oxides
of nitrogen, oxides of sulfur, and their combinations. Studies using canine
species over extended periods of time have proven useful in the evaluation of
risk to humans, especially when combined with epidemiological studies and
human clinical investigations.
All of the data were reviewed at a conference held at Asilomar, California
by invited expert scientists. Their evaluations and judgments form a signifi-
cant segment of this monograph.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c COSATI Field/Gro
19 SECURITY CLASS (This Report)
21 NO. OF PAGES
304
!0 SECURITY CLASS (Thispage)
n 22200 (9-73)
288
ft US GOVERNMENT PRINTING OFFICE 1980-660-934
U.S. Environmental Protection Agency
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