U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
PB-251 729
EFFECTS OF OXIDANT AND SULFATE INTERACTION
ON PRODUCTION OF LUNG LESIONS
STANFORD RESEARCH INSTITUTE
PREPARED FOR
HEALTH EFFECTS RESEARCH LABORATORIES
JANUARY 1976
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EPA-600/1-76-009
January 1976
Environmental Health Effects tearcfi Series
Health Effects Researc
Office of Research
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
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1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies re-
lating to the tolerances of man for unhealthful substances or
conditions. This work is generally assessed from a medical view-
point, including physiological or psychological studies. In add-
ition to toxicology and other medical specialities, study areas
include biomedical instrumentation and health research techniques
utilizing animals - but always with intended application to human
health measures.
This document is available to the public through the National
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TECHNICAL REPORT DATA
(Please read Instructions on the reverie before completing)
1. REPORT NO.
EPA-600/1-76-009
3. RECIPIENT'S ACCESSION"NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
Effects of Oxidant anq" Sulfate Interaction on Product!01
of Lung Lesions
January 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S) ~~
Gustave Freeman and Laszlo T. Juhos
a. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Stanford Research Institute
Menlo Park, California 94025
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
EPA 68-02-1944
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Aqency
Research Tr-ianale Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final & 4th quarterly
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT . •
This line of investigation is designed primarily to determine the sub-acute and
chronic effects of sulfuroxide inhalation alone and in combination with oxidant
exposure on the respiratory system of laboratory animals. Preliminary experiments are
being conducted to determine the optimum concentration of smal 1 -particle. H'-SOv expo-
sure to use in subsequent H 'SO.-oxidant experiments. The comparative response of rats
guinea pigs and monkeys is Being determined. The effects of S02-03 mixtures will be
determined also.
The principal biologic responses being determined are the histopatholonic response,
including ultrastructural studies and autoradiographic assessment of cell turnover
rates, biochemical studies and physiologic measurements.
PRICES SUBJECT TO CHAHft
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Held/Group
Respiration
Sulfates
Oxidizers
Air Pollution
Lung
Toxicity
Laboratory animals
06T, F
06A, P
IB. •OISTRIOUT/ON STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS /7/uj Report)
UNCLASSIFIED
21. NO. OP PAGES
20. SECURITY CLASS (This page)
UNCLASSIFIED
EPA Form 2220-1 (9-71)
Ife
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EPA-600/1-76-009
January 1976
EFFECTS OF OXIDANT AND SULFATE INTERACTION
ON PRODUCTION OF LUNG LESIONS
By
Gustave Freeman, M.D., and
Laszlo T. Juhos, B.S.
Stanford Research Institute
Menlo Park, California 94025
Contract No. EPA 68-02-1944
Project Officer
Dr. Glen Fairchild
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
ib
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
ii
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CONTENTS
LIST OF ILLUSTRATIONS AND TABLES iii
INTRODUCTION 1
PROGRESS DURING THE QUARTER
Measurements of Body Weights and Respiratory Rates
in Monkeys 2
Exposure of Monkeys to Mixtures of NO2 and O3 ....... 2
Monitoring of Exposure Chambers .... ... 2
Determination of Gross Toxicity of Sulfuric Acid Mist ... 8
Examination of Monkeys Exposed to 2 or 9 ppm NO2 for
Nine Years or to 0.9 ppm 03 for Thirteen Months 11
Pulmonary Cell Renewal Rates 17
Hematological Changes in Monkeys 17
SUMMARY OF LONG-RANGE STUDIES OF MONKEYS 20
ill
-------
ILLUSTRATIONS
1 Mean Body Weights and Mean Respiration Rates
of Monkeys on 0, 2, and 9 ppm N02 3
2 Weight Curves of Growing Stump-Tailed Macaques
on 0, 2, and 9 ppm N02 4
3 Particle Size Distribution for Sulfuric Acid Mist .'.... 5
4 Mass Distribution for Sulfuric Acid in the Mist ....... 6
5 Equilibrium Concentration for Sulfuric Acid Aerosol
as a Function of Humidity 9
6 Emphysema Following Nine Years of Exposure to
About 9 ppm N02 in Monkey Cy 14
7 Response to Exposure to 2 ppm N02 for Nine Years
in Monkey Ls 16
TABLES
1 Particle-Size Distribution of Sulfuric Acid Mist 7
2 Toxicity of Sulfuric Acid Mist to Guinea Pigs 10
3 Pulmonary Cell Renewal Rates 18
iv
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INTRODUCTION
The objectives of the research during the final quarter (June 16
to October 31, 1975) of Contract No. 68-02-1944 were to:
• Evaluate in the lungs of monkeys the effects of nine years
of exposure to 2 and 9 ppm nitrogen dioxide (NO2).
• Continue exposure of monkeys to mixtures of the interdependent
atmospheric oxidants N02 and ozone (O3) at realistic concen-
trations .
• Continue investigations of the interaction of atmospheric
oxidants and particulate (aerosolized) sulfate in rodents.
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PROGRESS DURING THE QUARTER
Measurements of Body Weights and Respiratory Rates in Monkeys
Monitoring of the clinical conditions of monkeys in the various
exposure environments continued by measurement of body weights and respi-
ratory rates. Figure 1 shows the means of body weights and respiratory
rates of adult monkeys, and Figure 2 shows the weights of juvenile
monkeys. No alterations from previous norms are evident; also, the
body weights of juveniles that appear to have reached adult size have
become stable.
Exposure of Monkeys to Mixtures of NO2 and 03
Exposure of three young-adult female monkeys to mixtures of 2 ppm
NO2 and 0.9 ppm 03 continued. This exposure had been initiated in August
1975. We are continuing to observe these monkeys clinically.
Monitoring of Exposure Chambers
The concentrations, of NO2 and 03 alone and as mixtures in exposure
chambers were monitored as described in Quarterly Report No. 1.
Quarterly Report No. 3 contains a description of the all-glass
nebulizer, used for the generation of sulfuric acid mist and the airs team-
heater installed for the production of smaller mean particle size. The
generated, particle size and the mass distribution, which was produced
with and without the use of the airstream-heater, were analyzed in detail
during the fourth quarter. We used a Royco Model 225 particle-size ana-
lyzer of sheath-flow and forward-light-scatter design, equipped w-l,th a
Model 241 five-channel simultaneous digital analyzer with printout; the
Chemical Engineering Department of SRI let us us.e this apparatus at no
cost to t,he contract. Table 1 shows the typical particle size distribu-
tion of sulfuric acid mist generated with and without the use of the
inlet airstream-heater.
We used the information on particle size, presented in Table 1 and
Figure 3, to calculate the mass distribution as a function of particle
size, assuming unit density and spherical configuration of sulfuric acid
particles. Figure 4 presents these data. The data in Figure 3 suggest
that the diameters of 50f5 of the particles are smaller than either 0-.73
or 0.46 p, for the unheated and heated modes of generation, respectively.
AB is evident from Figure 4, these particles represent only 0.7 or 2$
of the mass of acid in the chamber. Figure 4 also shows that 5Of: of
-------
100
E 90
-------
•T t ! I ! r 1 11 I T TT~1 I I I I I I I I I I I I I I [T I I I I 1 I I < I I n I I I I I f| M I I JTT I I p IT I I r I T I I II 1 I I 1 I I I
JtfMMLItt B*Tt V tMTTM C«VlllOm«mT
HI
IOOCC. I9M >•»•«,
19 rn. IMt NOOM Mil
It KPT no >lp>Wt
irotT K70 >M»W>t
• JULY on tOOt «M
IT M.V K7I HOW Ml
10 MA ttn
c.t
In*
0.1
00 I
M«
• t
• MO
i«o. aoo ooo wx> ooo
mo IMO
FIGURE 2 ^WEIGHT CURVES OF GROWING STUMP-TAILED MACAQUES ON 0, 2 AND 9 ppm Np2.
-------
o
10
8
c
o
u
I
1
0.8
0.6
0.4
0.2
0.1
L- i i n r
NO HEAT
• 10~3 Dilution
A10~4 Dilution
INLET TEMPERATURE, 445°F —
• 10~4 Dilution
10~5 Dilution
I I I I I I I
I
I
I
I
10 20 30 40 50 60 70 80 90 99 99.9 99.99
CUMULATIVE PERCENTAGE OF NUMBER OF PARTICLES BELOW SIZE ON ORDINATE
FIGURE 3 PARTICLE SIZE DISTRIBUTION FOR SULFURIC ACID MIST
-------
10
8
6
4
P
DC
2
1
0.8
0.6
0.4
0.2
0.1
I -
MIST GENERATOR
• Unheated
• Heated
I
I
I
0.01 0.1 1 10 20 30 40 50 60 70 80 90 99 99.9. 99.99
CUMULATIVE PERCENTAGE OF MASS OF SULFURIC ACID IN MIST BELOW SIZE ON ORDINATE
FIGURE 4 MASS DISTRIBUTION FOR SULFURIC ACID IN THE MIST
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Table 1
PARTICLE-SIZE DISTRIBUTION OF SULFURIC ACID MIST
Nominal
Condition of Royco Channel
Generation Dilution Number
84 to 90 psig on 10~3 5
pneumatic atomizer;
no heat
3
2
1
lO"4 5
4
3
2
1
84 to 90 psig on 10'4 5
pneumatic atomizer;
inlet to chamber (435
to 445°F) 3
85 psig on diluting air
1
10"s 5
4
3
2
1
Average
Count,
Corrected*
509.5
60,003
63,145
52,927
36,986
18
6,675
8,535
7,647
5,621
3.67
11,088
35.71S3
45,312
36,li>6
0
397
1,631
2,659
2,370
Particle
Size
(u)
3
1.4
0.6
0.4
0.3
3
1.4
0.6
0.4
0.3
3
1.4
0.6
0.4
0.3
3
1.4
0.6
0.4
0.3
Number of
Particles
Greater than
Particle
Size Shown
509.5
60,513
123,658
176,585
213,571
18
6,693
15,228
22,875
28,496
3.67
11,092
46,875
92,187
128,343
0
397
2,028
4,687
7,057
Number of
Particles
Greater than
Particle Size
(Cumulative f5)
0.239
28.34
57.90
82.68
100
0.0631
23.49
53 .44
80.27
100
0.000779
8.64
36.52
71.83
100
0
5.63
28.74
66.42
100
Background count without the atomizer in operation was determined and subtracted.
-------
the aerosolized sulfuric acid particles have diameters of 2.5 or 1.78 u.,
respectively, when generated with and without the inlet airstream-heater.
The above measurements wer,e made by nebulizing a 35$ (w/w) solution of
sulfuric acid. The heated aerosol generator maintained the internal
chamber temperature below 75 F during exposure of animals.
Determination of Gross Toxicity of Sulfuric Acid Mist
From the physicochemical constants for the vapor pressure of sul-
furic acid, we computed the concentration of acid in the aerosol droplets
as a function of ambient humidity. Figure 5 shows that, at 50$ ambient
humidity and at a temperature of 20°C, the equilibrium concentration of
the sulfuric acid droplet is about 43$ (w/w). If the emitted aqueous
aerosol has less than a 43$ sulfuric acid content (w/w) , it will lose
water content and, therefore, particle size. If the concentration of
the sulfuric acid is greater than 43$ (at 20°C and 50$ humidity), the
aerosol will pick up moisture from the environment, and particle size
will increase until an equilibrium concentration of 43$ (w/w) is attained.
An extension of the latter circumstance is likely to pertain once the
sulfuric acid aerosol enters the physiological environment of the mamma-
lian body through respiration. The humidity gradient from the ambient
air (about 50$ humidity) outside the nasal or oral cavity to the saturated
ihtrarespiratory environment (100$ humidity) effects a rapid dilution.
of acid concentration with attendant increase in particle size..
In presenting theoretical growth rates of particle size in Quarterly
Report No* 3, we did not consider the effects of heat transfer and slip
flow. Additional theoretical computations suggest that the time factor
"t" (Equations 4 and 5 in Quarterly Report No. 3) was underestimated by
a factor of 6.4 when effects of heat transfer were considered. This was
calculated from
D C* AH2
t = 1 + V *° V
kMRT2
whore t = time of particle size growth (sec)
D = vapor diffusion coefficient, 0.26 cm2/sec
&I ,= molar heat of vaporization (4.2 x 10" erg/g mole)
0^ = moisture content of ambient air at saturation (4.4' X 10"s g/cm3)
k = thermal conductivity of air (2,600 erg) (sec) (cm) (K° )
-------
70"
60
5
E
5 50
O
UJ
I- 40
<
O 30
IT
I
20
o»
\
20°C \
40°C \ _
I I I I.I I \ \
0 10 20 30 40 SO 60 70 80 90 100
RELATIVE HUMIDITY - H %
FIGURE 5 EQUILIBRIUM CONCENTRATION FOR SULFURIC ACID AEROSOL AS
A FUNCTION OF HUMIDITY
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M = molecular weight of water (18 g/mole)
R = gas constant, 8.31 x 107 ,erg/(°K) (g mole)
T = absolute temperature of ambient air (°K)
We conducted four experiments to study the toxicity of sulfuric
acid mist to guinea pigs. Two concentrations of stock sulfuric acid
were nebulized, each with and without heating the diluting air. Table 2
summarize^ the results of these experiments, in each of which six guinea
pigs were used.
Table 2
TOXICITY OF SULFURIC ACID MIST TO GUINEA PIGS
Experiment
Number
2
3
Concentration
of Nebulized
H2S04
(w/w)
50
5.0
35
35
Inlet
Airstream-
Heater
(450°F)
No
Yes
No
Yes
Chamber
Concentration
of H2S04
(mg/m3)
84
60
58
52
Death of
Guinea Pigs
24 hr
24 hr
1 week
1 week
The apparent difference in the concentration of aerosolized sulfuric
acid in the chamber with and without the use of the inlet airstream-
hcater is puzzling. Since neither the rate of acid nebulized into the
chamber nor the airflow rate through the chamber differed, the total
acid concpntration should have remained the same both with and without
the use of the inlet airstream-heater. We are considering possible
explanations for the difference in acid concentration; among them are
the following:
• Because of the reduced median particle size (from 0.73 to 0.46 u.)
and median mass (from 2.5 to 1.78 |j,)--see Figures.3 and 4 — as a
function of use of the inlet airstream-heater, the sample collec-
tion efficiency may have been reduced for the chemical determina-
tion of acid content from the sampled volume of air containing
the relatively smaller aerosol particles generated by the inlet
airstream-heater.
10
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• The reduced particle size generated by the inlet airstream-heater
probably represents greater reactivity of the acid particles
with the hardware components (corrosion and other factors), which
would be an actual loss of acid content in the chamber.
The relative toxicity of the mist generated by the inlet airstream-
heater appears to be greater, which may well be related to the reduced
mean particle size. However, on theoretical grounds, as suggested from
the information in Figure 5, the concentration of the individual sulfuric
acid droplet and its size in the chamber may be different from that reach-
ing the respiratory tissue because of dilution and particle size growth
during transit from the environment of 50$ chamber humidity to the close-
to-saturation humidity of the physiological environment. This change in
the concentration of the acid content of the individual acid droplet and
the attendant particle size growth would not influence the total dose of
acid that enters the nasooropharynx of the individual guinea pig to have
a systemic effect.
Charles Lapple and Clyde Witham aided in preparing and evaluating
the nature of the H2SO4 aerosol.
Examination of Monkeys Exposed to 2 or 9 ppm N02 for Nine Years or to
0.9 ppm O3 for Thirteen Months
Quarterly Report No. 3 presents results of initial studies of this
first group of monkeys. We had found that the mature male monkey Cy,
manifesting clinical respiratory symptoms and signs, developed elevated
erythrocyte and hematocrit counts during the exposure period (Table 6
in QR $3). Cy's CO diffusion rate was found to be reduced relatively
early during the nine years of exposure (Table 12 in QR $3). Functional
studies conducted before sacrifice of Cy revealed that his transpulmonary
resistance to airflow was increased, and the ratio of duration between
"ispiration and expiration favored prolongation of the latter compared
a• 1:1' ratios in control monkeys (Table 12 in QR #3).
A second male monkey, Gr, that had been treated identically but had
loss evidence of disease clinically was sacrificed subsequently. Although
Gr exhibited increased erythrocyte levels during life and reduced rates
ol CO diffusion, his lungs postmortem were neither heavier nor more volumi-
nous than control lungs, but they did have large depressed areas of ata-
lectasis. These areas were easily reinflated through the trachea and
became homogeneous with the rest of the pleural surfaces. Microscopic
examination of the lungs revealed disease quite similar to that seen in
Cy, except for a lack of disruption of alveolar tissue in the peripheral
portions of the parenchyma.
Two mature monkeys, Ls and Gy, that also had been exposed to 2 ppm
NO2 for nine years were examined. Neither of these animals had developed
clinical evidence of disease except for elevations in erythrocyte counts
curly during the exposure period. Their lung-to-body weights were not
11
-------
different from the average for control animals. In Ls, the lung capacity,
when equilibrated at 1 atmosphere of pressure, was 10$ greater than the
mean value for control animals. Neither the end-expiratory lung capacity
nor the capacity at atmospheric pressure was increased in Gy. In neither
case could we conclude that the pulmonary capacity of the lungs intra-
thoracically was not reduced by leakage either through the trachea or
pleurae resulting from postmortem manipulation.
Pro- and postmortem physiological studies were conducted principally
by Laszlo T. Juhos and Rowena Mussenden-Harvey.
Cy
-------
in type. Polymorphonuclear leukocytes also were seen occasionally in
the interstitium and among the free cells. In contrast, the more periph-
eral alveolar walls were strikingly attenuated. Whereas epithelium in
the proximal alveoli was readily visible with prominent cells, equivalent
but attenuated alveolar epithelium from the distended areas of the paren-
chyma was identifiable only with considerable uncertainty. Bronchiolar
epithelium was hypertrophic, often hyperplastic, and sporadically meta-
plastic. Capillaries in proximal alveoli were less prominent than normal,
and their endothelial cells could not be distinguished with confidence
from fibroblasts or muscle cells.
Areas characterized by extremely large air spaces were enclosed
by very thin walls that were frequently discontinous from loss of parietal
tissue, as shown in Figure 6. Such areas were characteristic of develop- •
ing emphysema and suggestive of microscopic bullae. Interstitially,
collagen was deposited liberally wherever the alveolar, ductal, or bron-
chiolar epithelium was hyperplastic. This was seen most often in ducts
and in affected alveoli where smooth muscle also was abundant. In con-
trast, collagen was rather sparse in areas of attenuated alveolar walls.
Elastic fibers were uniformly distributed, although in attenuated areas
they often appeared frayed and fractured.
The other lung of the pair was "respired" mechanically while
functional studies were being conducted. Microscopy revealed striking
disruption of the parenchyma in areas of distended and attenuated alveoli.
Alveolar tissue was fragmented, suggesting that the tissue was more
fragile under such stress than normal parenchyma would be.
Microscopic examination of other organs revealed some interest-
ing changes. Randomly situated parenchymal cells, in an otherwise intact
livor, were filled with aggregations of particulate pigment, the nature
oi which is not now known. The pulp of the spleen was "loosely" arranged,
and the germinal centers were indistinct. Basement membranes of renal
fvlomeruli wore sometimes prominent but not remarkably so. The weight
ni' the intact heart was relatively light for the body, possibly reflec-
ting sedentary behavior imposed by a respiratory limitation to physical
activity. The ventricles did not appear disproportionately thick, but
they we ro not weighed separately.
Ls V--NJHU Years of Exposure to 2 ppm NO2
Gross Pathology
The lungs, exposed while in the chest, were pink and full nnd
liiid a ridged suvl'aco conforming to the pattern of the rib cage. The
im>iv ini'latud portions were very light pink and contrasted with a few
smaller, scattered but circumscribed, depressed red blotches of varying
si/.os. The lungs on the surface were otherwise light and fluffy and
appeared somewhat larger than normal. They weighed 50 g and occupied
213.4 ml of space while under intrathoracic pressure. At ambient pressure,
1J
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FIGURE 6 EMPHYSEMA FOLLOWING NINE YEARS OF EXPOSURE TO ABOUT 9 ppm
NO2 IN MONKEY Cy
H&E stain of 4-»i paraffin section
20x magnification
14
-------
the volume fell to 141.4 ml. The volume-to-weight relationship of the
lungs was slightly but significantly increased over normal.
The remaining viscera were not unusual in gross appearance.
Microscopic Pathology
A modest degree of distension of the alveoli was evident in
.some portions of the lungs. The long respiratory bronchioles of Ls were
distinguished by thickening with subepithelial connective tissue, smooth
muscle, and tall, hypertrophic epithelium. The epithelium was variously
columnar or cuboidal or was composed of enlarged, rounded pseudosquamous
or hyperplastic cells. The prominence of the peripheral bronchiolar
epithelium contrasted with the relatively flat, squamous type normally
seen in the monkey. Figure 7, prepared by Dr. Robert J. Stephens, demon-
strates hyperplasia. Collagen was more plentiful than usual both in the
respiratory bronchioles and in the walls of the alveolar ducts. The
alveoli, however, were relatively unaffected. Histologically, elastic
fibers intermingled with collagen and appeared to be "teased apart" at
affected sites such as those underlying hypertrophic epithelium.
Other organs had no remarkable changes.
Rh ^--Thirteen Months of Exposure to 0.9 ppm 03
Gross Pathology
In addition to the group of monkeys exposed to N02, one animal
(Rh) was observed for 13 months during continuous exposure to 0.9 ppm 03 .
We sacrificed Rh to compare pathological effects with those in the NO2
group. Although Rh did not manifest clinical evidence of pulmonary
disease (functional pulmonary tests were not made in vivo), postmortem
her lungs were found to be 34% heavier per unit of body weight than
normal control lungs. Rh's lungs appeared to be inflated at the intra-
thoracic end-expiratory position and proved to be 8.4^1 greater in volume
than normal lungs. They continued to retain 30f: more air than lungs
of control animals under atmospheric conditions.
i In the fixed end-expiratory state, the lungs appeared over-
inflated in the opened thoracic cavity. They were uniformly light pink
except for the angular lobar margins anteriorly, and, at the costophrenic
angle, where the pleurae overlaid white, translucent alveolar tissue
extending as a marginal border 1- to 2-mm in breadth. The lungs were dry
and fluffy. Removal of the tracheal plug initiated release of air under
atmospheric pressure, but the lungs maintained an inflated appearance.
The light-pink surfaces were marked homogeneously with small, spider-
like vessels radiating in several directions from their centers for about
0.5 mm, suggesting an anatomically defined distribution. These vascular
units appeared a few millimeters apart over the .pleural surfaces. The
15
-------
X.
(a) NORMAL ALVEOLAR DUCTAL EPITHELIUM COVERING
MUSCULAR RING
(b) HYPERPLASIA OF ALVEOLAR OUCTAL EPITHELIUM
FIGURE 7 RESPONSE TO EXPOSURE TO 2 ppm NO2 FOR NINE YEARS IN MONKEY Ls
Toluidine Blue stain of ^-^ plastic section
40x magnification
16
-------
posterior surfaces of the right lower and left upper lobes revealed
rotractud (ataloctatic), beefy-red areas occupying much of the pleural
surface. The lungs had at least twice the expected normal volumes at
intrathoracic and atmospheric pressures—237 and 165 ml, respectively.
Microscopic Pathology
The principal changes occurred in the respiratory bronchioles
and alveolar ducts in which the subepithelial interstitial tissue was
considerably thicker and less compact than normal. This area appeared
to be the site of fibroblastic activity. The epithelium at this level
was hypertrophic, having rounded, cuboidal cells in place of the usual
flatter or squamous type. The thickened ductal septa extending into
the proximal alveolar walls also contained fibroblast-like cells. A
characteristic feature of these lungs was the abundance of pigment-loaded
macrophages that aggregated in the affected proximal alveoli and in the
interstitial tissue. In addition to these, other mononuclear cells were
evident within the broad septa. The more proximal, uninterrupted bron-
chioles were well ciliated, whereas the respiratory portions were not.
Deposition of collagen appeared excessive in the thick walls where
"active" fibroblasts could be seen. The remaining viscera did not
appear to have been affected.
Rh had developed evidence of polycythemia during her lifetime,
similar to that seen in the monkeys exposed to N02. The only basis for
comparison of Rh with the monkeys exposed to N02 was that pulmonary dis-
ease was induced by exposure to atmospheric "oxidants."
Pulmonary Cell Renewal Rates
In determing the replication rates of pulmonary cells in monkeys,
Dr. Michael J. Evans found the rates for normal unexposed monkeys to
be similar to those for normal, 11- and 19-month-old rats, as shown in
Table 3.
The turnover ratos for terminal bronchiolar epithelial cells and
Cor Typo 2 alveolar epithelial cells at the time of sacrifice of the
more affected monkeys exposed to either 9 ppm NO2 or 0.9 ppm O3 were
very significantly elevated. In the monkey exposed to 2 ppm NO2 and
in the one exposed to 9 ppm (whose disease was less advanced), evidence
of increased labeling of DNA with tritiated thymidine was uncertain.
The "other" category of cells in Table 3 comprises all other cell types
in the bronchiolar and alveolar areas such as endothelial, migratory,
and fibroblastic cells.
Hematological Changes in Monkeys
The principal hematological effect caused by exposure of monkeys
to N02 and to O3 was an average 'elevation of the red cell counts. In
17
-------
Table 3
PULMONARY CELL RENEWAL RATES
Cell Type (Labeled Cells/1000 Colls)'
Rats (11 mo)
Rats (19 mo)
Monkey
Monkey
Monkey
Monkey
Monkey
Monkey
Monkey
Monkey
An,
Br,
In,
Ls,
cy,
Rh,
Or,
Gy,
Te.rminal
Bronchiolar
, controls' -
, controls'!"
control 0.1
control 0.1
control 0.1
2
9
0
9
2
ppm NO2* 0.6
ppm N02* 4.0
.9 ppm 03** 2.3
ppm NO 2*
ppm N02*
Type 2
Pnoumonocyte
0.2 ± 0.2
0.5
0
0
<0
0
5
8
0
0
± 0.4
.5
.5
.1
.1
.0
.0
.5
.5
Alveolar
Macrophage
0.1 ± 0.1
0.2
0
0
0
1
<0
0
0
0
± 0.2
.1
.1
.1
.0
.1
.3
.0
.0
Other
8.0
3.8
5
6
2
6
19
5
3
2
± 3.3
± 3.0
.0
.5
.0
.0
.0
.6
.5
.5
3000 cells counted.
~Six rats in each group.
^Exposed for about 9 years.
H*
Exposed for about 13 months.
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those animals exposed to 9 ppm NO2» the rise was more than 1 million/nun3
over controls during the first year and about 3 million greater during
Die second year of continuous exposure. During the following four years,
the red cell concentration tended to fall toward normal, and normal levels
wore attained by the ninth year of exposure. Monkeys exposed to 2 ppm
NO2 exhibited a similar pattern, but it was characterized by a slower
rate of. increase, a peak of somewhat less than 2 million over normal
during the second year, and a slow return to normal by the sixth year;
normal levels were maintained into the ninth year.
The hematocrit counts rose with the elevation of red cell counts
in animals exposed to 9 ppm NO2 but to a lesser extent proportionally.
These counts did not change in monkeys exposed to 2 ppm NO2. The hemo-
globin levels rose, as did the red cell counts, only during growth and
maturation of the monkeys but not in relation to elevations in cell
numbers based on exposure to N02. Simultaneously, the mean corpuscular
volumes (MCVs) fell during maturation of both normal and exposed animals,
while their red cell counts were rising as part of their natural growth.
Then, as polycythemia developed with exposure to NO2 (but not in controls),
the MCVs were reduced in proportion to the elevation of cell counts and
elevated to normal as the polycythemia waned during the latter years of
exposure to N02. Accordingly, the microcytosis was exaggerated in the
group of monkeys exposed to 9 ppm N02. The mean corpuscular hemoglobin
concentration remained steady, indicating an increase in overall red
cell surface area during phases of polycythemia based on increased numbers
of smaller cellular units.
The leukocyte counts varied widely but manifested a tendency for
the ratio of polymorphonuclear cells to lymphocytes to rise with eleva-
tions of total white cell counts. This was more pronounced in animals
exposed to 9 ppm NO2.
Hematological studies were conducted by Nazzareno J. Furiosi.
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SUMMARY OF LONG-RANGE STUDIES OF MONKEYS
For "approximately nine years, we have been observing monkeys (Macaca
speciosa) being exposed to either 2 or 9 ppm NO2. These animals have
now reached maturity, and some are middle aged. At least one of these
monkeys appeared to have developed sufficient signs of chronic respira-
tory disease to warrant a pathological study. Several other animals
also were' sacrificed for comparative purposes. Half of.the experimental
animals, including some that were born in the exposure chambers while
their parents were being exposed, are being investigated exhaustively.
The first five monkeys examined in this experiment comprised three
unexposed control animals and one each from groups that had been exposed
to either 2 or 9 ppm N02 for nine years. Included for comparison was
one that had been exposed for more than one year to 0.9 ppm O3. The
findings suggest that
• The lesions produced by N02 and by O3 in monkeys closely resemble
those occurring in rats exposed to higher concentrations of N02
for up to two years and in rats exposed to 0.9 ppm O3 for much
shorter periods.
• The attenuation and disruption of peripheral alveolar structural
tissue in one monkey that was exposed to 9 ppm N02 appear to be
coincident with architectural changes in the smallest airways
and associated proximal alveoli. The changes suggest that air-
flow between the peripheral alveoli and the bronchial airways
was impeded by intervening features obstructive .to airflow. The
changes are both structural (due to hypertrophic epithelium and
extracellular tissue in small airways and in adjacent alveolar
walls) and inflammatory (due to free macrophages, other "inflam-
matory" cells, and organizing secretion and fibrinous exudate).
' • . . • i i ,
• The enlarged air spaces resulting from disruption and loss of
alveolar tissue in the animal that was exposed to 9 ppm NO2 is
characteristic of chronic pulmonary emphysema.
• The morphological changes in the lungs of the monkey exposed to
2.ppm NO2 for nine years are similar but much less advanced.
There is no evidence of destruction of parenchyma.
• The effects of O3 on pumonary tissue also are found mainly in
the smallest airways, but the affected level occupies a slightly
more peripheral segment of the airway system (the respiratory
bronchiole and the alveolar duct) than do the effects of N02.
This difference was seen also in the rat. The macrophage response
20
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to injury of tissue by 03 is more striking in the monkey than
in the rat, although this may be attributable to large differ-
ences in relative dosage and duration of exposure.
•>. • In three of five exposed animals, residual lung volumes were
increased significantly over normal residual volumes.
The overall objective of these long-range experiments is to develop
a perspective of the insidious and protracted development of chronic
obstructive pulmonary disease in man. These investigations are a logical
extension of those in which a parallel disease was induced during the
relatively short lifetime of the rat through exposure to either N02 or
03. The Macaca speciosa has an estimated life span intermediate between
that of man and the rat; thus, we included this species to introduce
temporal and genetic reality into the perspective of studies of chronic
obstructive lung disease. The first compelling clinical evidence of
developing pulmonary disease in the monkeys coincided with the adminis-
trative termination of the 1975 contract period.
Thus, in retrospect, we are describing threshold effects in terms
of the total development of chronic obstructive pulmonary disease. Four
monkeys, two of which were exposed to 9 ppm and two to 2 ppm NO2, are
now being compared with four similar animals that lived under parallel
conditions in clean air. All four exposed animals developed similar
morphological pathogenetic evidence of pulmonary disease, but only one
had reached a state consistent with the early stage of emphysema seen
in humans. These borderline but highly significant observations indicate
the need for further specific definition of the roles of NO2 and 03 in
the induction of the etiologically obscure and intractable chronic obstruc-^
tive pulmonary disease.
Following completion of the investigation of half of the experimental
animals, we will design a meaningful approach for studying the remaining
monkeys. The unqualified raw data provided in earlier quarterly reports
are being examined for validity and statistical significance. Such data
i'nclude hematologic factors such as erythrocyte and leukocyte counts,
differentials, hematocrits, hemoglobin levels, fragility tests, mean
corpuscular volumes and mean corpuscular hemoglobin concentrations, and
blood chemistry values for methemoglobin, ATP, 2,3-DPG, glutathione
peroxidase and reductase, G-6-PDH, and red cell cholinesterase. Circu-
lating blood gas levels are being reviewed, and vitamin E concentrations
found portmortem in tissue, the lungs, the liver, and muscle are being
evaluated also and will be reported.
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