EPA/600/D-89/102
CHRONIC EXPOSURE TO OZONE CAUSES RESTRICTIVE LUNG DISEASE
Elaine C. Grose1, Daniel L. Costa1,
Gary E. Hatch1, Kred J. Miller1,
Judy A. Graham2, James D. Crapo3,
Ling-Yi Chang3, Michael A. Stevens4,
Richard H. Jaskot4, Jeffrey S. Tepper4
JEnvironmental Toxicology Division, Health Effects Research Laboratory, U.S.
Environmental Protection Agency, Research Triangle Pa/k, North Carolina 27711.
Environmental Criteria and Assessment Office, Research Triangle Park, North
Carolina 27711.
3Duke University, Department of Medicine, Durham, North rarolina 27710.
4NSI - Technical Services Corp., PO Box 12313, Research Triangle Park,
North Carolina 27709.
DISCLAIMER: The research described in this article has been supported by the
U.S. Environmental Protection Agency, and has been subjected to Agency review.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
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INTRODUCTION
Due to the adverse health effects of ozone (O3) EPA has set a National
Ambient Air Quality Standard (NAAQS) for this pollutant (0.12 ppm O3, hourly
average). During re-evaluation of the criteria for the O3 NAAQS, it became
clear that there were several major uncertainties in the health data base,
Most important of these was the potential for O3 to produce chronic health
effects. Examination of the chronic effects of O3 in the rat is the subject
of this paper.
General conclusions drawn from the entire data base of published studies
were that O3 causes chronic Tunc disease ?nd other effects on lung morphology,
biochemistry, and host defense systems after long-term exposures. Quantitative
interpretation of these few chronic studies for EPA regulatory purposes was
inhibited severely because most of the studies were conducted several years
ago using methods less sensitive than are presently available or because they
were designed to address issues not directly related to EPA needs. Therefore,
O3 cfironic studies relevant to rsgulatory issues were necessary.
Data from the National Aerometric Data Bank (NADB) of the Office of Air
Quality Planning and Standards (OAQPS), EPA, were used to assess diurnal
patterns for O3. The intent of these a.ialyses was to focus on days when
pollution levels were elevated and discern the nature of the diurnal pattern.
For the chronic toxicological studies, the typical pattern was mimicked, but
the absolute concentrations of the O3 in such episodes was not necessarily
maintained. The exposure pattern chosen represented frequently occurring,
worst cases for urban summer environments.
Although a multitude of chronic studies were possible, this study was
designed to address several specific objectives to determine the effects of
chronic exposure to O3 in animals that could be quantitatively extrapolated to
man. These objectives were as follows: (1) to determine the progression of
chronic lung disease in animals exposed to O3 as measured physiologically,
morphometrically, and biochemically; (2) to determine the correlations between
these 3 experimental disciplines; and (3) to determine the reversibi1ity or
progression of lung disease during exposure and clean air post-exposure
periods.
EXPERIMENTAL METHODS
Exposure Faci1itv
The exposure chambers used were of two types: (a) identical, walk-in
environmental rooms (Forma Scientific, Inc.) are described in detail by
Davies et al J and (b) Hazelton 2000 exposure chambers (2m^), two for each
study group (O3, and control). A1] chamber environments were monitored and
controlled by mechanical system- providing temperatures^ of 74 ± 3°C ,and
relative humidities of 60±1Q%, while preventing possible system failures that
would affect study data. The chambers were operated at a ventilation rate of
9-10 air changes per hour. The delivery of 63 to the chambers was regulated
by mass flow controllers, which could be controlled either automatically or
manually. Ozone was generated by passing cylinder-supplied oxygen through a
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silent arc O3 generator (Ozone Research and Equipment Corporation Mode 03V-0)
Redundant components were incorporated to back-up all critical chamber
systems.
Computer control of the exposures was incorporated into the system in
order to achieve: 1) continuous and unassisted control of the exposure
levels, 2) time sequencing of the pollutant concentrations to simulate
environmental patterns, 3) repeatable exposure over the entire study period,
4) detection of abnormal events, and 5) automatic storage of data.
Ozone in the chamber was monitored us.ing continuous chemi 1 uminescent
analyzers (Bendix 8101C for NOx and Bendix Model 8002 for O3) according to EPA
reference methods (RFOA-0176-007 for O3). The analyzers were calibrated
biweekly to a UV-based standard following EPA procedures (EPA, 975, 1979).
A wet/dry bulb hygrometer provided continuous monitoring of the chamber
temperature and relative humidity. Chart recorders were used to continuously
record the temperature and humidity valupi.
Prior to the study, the spatial distribution of the pollutant in the
exposure chamber (with and without animals) was tested to determine gas
homogeneity. These test data were used to select the location of the analyzer
probes to obtain a representative sample indicative of the chamber
concentration. The analyzer probe was placed in the cage to sample O3 from
the breathing zone of the rats. Spatial testing was performed to cover the
range of gas concentrations to be used during the study. The requirements
were that the range of concentrations within a chamber shall not exceed + 8%
of the target concentration.
Exposure Regimen
The O3 exposure consisted of a background level of 0.06 ppm for a period
of 13 hours, an exposure spike from 0.06 ppm to 0.25 ppm and back to 0.06 ppm
over 9 hours, and a downtime of 2 hours for servicing the facility (Figure 1).
Integration of the spike portion of the curve shows that the exposure was
equivalent to a square wave average of 0.19 ppm. The background exposure level
was maintained on weekends. These concentrations, as well as the pattern of
exposure, do occur in the ambient air. In order to determine the progression
and/or reversibility of lung disease, various exposure lengths and. recovery
periods were chosen (Table 1).
Table 1 Exposure regimen for O3 Chronic Study
EXPOSURE PERIOD
1 wk (5 days)
RECOVERY PERIOD
nor>a
none
EXAMINATION TIMES
immediate
3 wk
3 mo
12 mo
18 mo
1.5 mo
6.0 mo
4.0 mo
immediate and 6 mo
immediate and 4 mo
immediate
immediate and 1.5 mo
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RESULTS
Structural Studies
A wide range of acute responses occurred in the proximal alveolar region
(PAR) of rat lungs. After 1 wk of exposure the volume of alveolar tissue was
increased 32%. This increase consisted of a 16% increase in Type I epithelium,
60% increase in Type II epithelium, 52% increase in interstitial cell volume
and 33% increase in interstitial matrix. The volume of alveolar macrophages
was nearly three fold that of control animals. Qualitative evaluation of the
alveolar septum indicated that the increase in interstitial matrix resulted
mainly from edema. The number, size, and surface area of the alveolar cells
were not significantly changed except for a 32% increase of the number of Type
I epithelial cells in the 03-exposed rats.
After 3 wk of exposure the morphometric parameters from PARs were not
significantly different from controls. Tissue volume was no longer increased,
..however,';the number of;, ilype.J I ,c,e7,7fSwas stillielevated (31%).
Epithelial and interstitial injuries were again evident after three months
of exposure to O3. The volume of the proximal alveolar tissue increased 17%.
These volume changes included a 34% increase in Type I cell epithelium, a 44%
increase in Type II cell epithelium, a 14% increase in interstitial cells and
an 18% increase of interstitial cell matrix. The numbers of Type I and Type II
epithelial cells were elevated 40% and 18%, respectively.
Persistence of epithelial injury and progression of interstitial fibrosis
were observed in the PARs of the 03-exposed rats at 18 mo. A 23% increase in
alveolar tissue volume was measured that consisted of elevated volumes of Type
I epithelium (17%), Type II epithelium (9C%), cellular interstitium (20%), and
interstitial matrix (35%). Large bundles of collagen were evident in the
interstitium. The number of Type I epithelial cells was 47% higher in exposed
rats. These cells covered an alveolar surface that was 38% smaller than in
control animals. The number of Type II epithelial cells increased 65%. The
characteristics of these Type II cells remained unchanged.
Lung reactions to O3 in the proximal alveolar region occurred in a
biphasic manner. An acute response involving epithelial hyperplasia and
hypertrophy, interstitial edema and alveolar macrophage influx was observed 1
wk after O3 exposure. These reactions largely resolved after 3 wk of exposure.
Epithelial and interstitial reactions were observed again after 3 mo of
exposure and these changes either persisted or intensified during the 18 mo
exposure period. The increase in interstitial matrix and the 30% increase of
Type I epithelial cells seen at 3 mo exposure was not fully resolved after 1.5
mo in clean air. However, the majority of epithelial and interstitial
injuries observed after 18 mo exposure to O3, disappeared, if not totally
resolved, after 4 mo recovery in clean air.
Labeling indices were also measured from PARs of control and exposed rats.
Alveolar cell labeling was increased approximately 2 fold in exposed rats as
compared to controls. Prolonged exposure (3 and 18 mo) did not appear to
increase labeling density. The rate of Type II cell differentiation appeared
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to have increased after O3 exposure, how?vc.-, no change in the rate of Type II
}:ell proliferation was observed. These results correspond to findings in the
morphometric studies. In addition, rates of fibroblast turn over appear to
have increased in the exposed group without a net increase in the size of the
cell population. These results suggest that interstitial cell turn over may
occur earlier than was previously suspected.
The morphometric and cytodynamic studies of rat lungs exposed chronically
to O3 indicated that the alveolar tissue responded to the initial insult of O3
with inflammation and edema. This acute reaction subsided and was not
detectable after 3 wk of O3 exposure. Progressive tissue changes involving
both epithelial and interstitial elements developed on prolonged exDOSure and
persisted in the presence of O3. Deposition of interstitial matrix was
observed after 3 mo of exposure and intensified slightly as the exposure
continued to 18 mo. There was an elevated cell turnover in the PAR throughout
the chronic exposure. However, when rats were placed in clean air, the
majority of both epithelial and interstitial changes were reversed. The extent
of recovery may be dependent 00 the.length of recovery time in clean air.
Pulmonary Functional Studies
Pulmonary function tests were designed to elucidate functional changes
indicative of structural abnormalities. Quiet, resting tidal breathing was
analyzed, and included a measure of tidal volume, frequency, airway flow and
breath timing parameters. No changes were observed in any of these parameters
throughout the study. Static lung volumes, such as vital capacity, residual
Volume, total lung capacity and functional residual capacity, which
Historically are used as indicators of lung damage in the clinic were also
measured. Statistically significant reductions in total lung capacity and
residual volume were detected in the 03-exposed rats after 3 months of
exposure. These depressions in lung volume persisted thereafter through the
18-month examination point. These changes suggest the presence of a
restrictive structural lesion, such as would be expected in a stiff' lung.
However, when the animals were removed from ozone and placed in clean air for
1.5 mo, the animals recovered and the lung volumes returned to normal.
The distribution of air within the ventilated lung was assessed using the
nitrogen washout test. At 3 and 12 months, this test exhibited ventilatory
evidence of a stiffened lung. However, this measure returned to control
values after 18 months probably as the result of compensatory shifts in
regional ventilation.
The distensibil ity, or compliance of the lung was evaluated using the
slow-inflation pressure-volume curve. Alveolar diffusing capacity, an index of
gas exchange was evaluated by measuring the uptake of carbon monoxide. Small
airway integrity was evaluated by analysis of the maximal expiratory flow-
volume (MEFV) curve. Wet and dry lung weights were recorded to detect changes
in tissue growth or edema. None of these parameters were different from
control at any of the examination times.
We speculate that the structural alterations indicated by this set of
unctional data lie as restrictions in the extremes of lung volume. This would
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explain why no changes were observed in the lung compliance measure, as this
is determined at intermediate lung volumes, where elastic properties
theoretically dominate. Furthermore, it appears from these data that some
degree of functional degradation persists as long as the animal is continually
being exposed to ozone. This functional lesion is reversible, however, as
affected pulmonary function returns to normal upon removal of the animal from
03-
Lung Biochemistry Studies
The general concept of the toxic effects of ozone is that it exerts its
effect by oxidation of unsaturated fatty acids in membrane phospholipids
and/or oxidation of the thiol groups and sensitive amino acid residues of
proteins. In designing a study which would be able to detect biochemical
changes in the respiratory tract resulting from exposure to low concentrations
of ozone, we attempted to sample those cells and flu ids., wb.ich would be in
closest contact with the oxidant challenge. These include: alveolar
macrophages, which make up the largest portion of free lavagable cells in the
lung; lung surfactant, whicn is present in the gas exchange regions; 'ana
pulmonary epithelial cells1, present in the whole lung homogenate. This study
sought to find evidences that the cells or fluids in the lung are oxidized or
otherwise altered by the chronic ozone exposure. This was done by analyzing
antioxidant compounds and enzymes that have been shown to be sensitive
indicators of pulmonary damage.
Following the time course of the study from 1 wk thru 18 mo of exposure
the most noticeable change observed was in the aging of both air and ozone
exposed rats. Age alone caused significant treatment-delated increases or
decreases in many of the endpoints studied. Following 1 wk, 3 wk, and 3 mo of
ozone exposure no significant changes were observed as compared to controls.
Since no changes were observed, the protective mechanisms of the lung
(antioxidants, antioxidant enzymes, glutathione, vitamin C and vitamin E) are
assumed to have been able to more than compensate for the oxidative stress
placed on these animals. However, after -12 months of ozone exposure selenium
dependent GSH-peroxidase, GSH-reductase, and NADPH cytochrome C reductase
showed significant increases in activity. In addition, GSH-transferase, GSH-
peroxidase (non-selenium), superoxide dismutase and glucose-6-phosphate
dehydrogenase also showed increases in activity although not statistically
significant. After 18 no of exposure all of these parameters were back to
control levels except for superoxide dismutase which was still slightly
elevated. All of these changes seem to indicate a transient increase in the
susceptibility of the pulmonary cells to the oxidative stress of ozone between
3 mo of exposure and 12 mo. These changes may be attributable to the aging of
the animal and a lessening of it's ability to produce and/or replace the
necessary amounts of antioxidants in the lung tissues to protect against ozone
attack. The reason for the apparent return to normal levels of these enzymes
at 18 mo is not fully understood.
The permeability of the gas exchange region was assessed by measurement of
protein in the lung lavage fluid. There was a general decrease in protein
concentration in lung lavage fluid with increasing age of the rats. Rats
older than 12 mo had half the protein concentrations of 3 mo old rats. The O3
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exposure increased the protein concentration in the lung lavage fluid by 40%
initially in 1 and 3 wk exposed rats and by 20% after 3, 12, and 18 mo of
exposure. This increase in lavagable protein dissipated when the animals were
allowed to reside in clean air. Under conditions of acute O3 exposure, an
increase in lavaguble protein often is thought to indicate the leakage of
plasma front tho hlQcci into thc> air spares. due to damage to the blood-air
barrier of the deep lung.
Phospholipids, which form a surfactant lining of the gas exchange region
of the lung, fluctuated widely in concentration with the age of the rats,
reaching an apparent peak at 3 mo. Ozcne exposure caused deviations from this
fluctuation which represented at most about a 45% change from control. O3
exposed animals had higher phospholipid levels at 1 wk, lower levels at 3 wk
and 3 mo, and higher levels at 12 mo. At 18 mo, phospholipid concentration is
were equal to controls.
Phospholipids in the surfactant of the lung contain carbon-carbon double
bonds .which .are highly reactive w.i.th O3. We examined the content of the major
saturated as well as unsaturated fatty acids in the lung lavage fluid' in order
to determine whether we could detect oxidatiorrcaused—by O3 exposure. It was
evident from the data that after 3 mo of exposure, O3 lowered the content of
all of the fatty acids measured to between 60 and 8G% of control. This change
was not observed at 12 and 18 mo.
Antioxidants, ascorbic acid (vitamin C), uric acid and tocopherol (vitamin
E), form a lining in the respiratory tract through which oxidants must pass
before they can reach the cissues. Tocopherol normally protects carbon-carbon
double bonds in the lung lipids from oxidation. We measured the tocopherol in
a manner which detected only the non-oxidized vitamin. Total lavagable
tocopherol in the eel"! fraction of lung lavage fluid was increased by 34%. The
ratio of tocopherol to phospholipid in the whole lung homogenate was increased
by 55% after 18 mo of exposure (no similar measurement was made at 12 mo). The
fact that tocopherol did not decrease in the lavaged cells and whole lung
suggests that the mechanisms for regeneration of this vitamin in the tissues
are sufficient to cope with the oxidant challenge. The results also indicate
that the lung may even over-compensate by increasing the tocopherol content
above the normal level.
Chronic ozone exposure caused a lowering of lung surfactant tocopherol to
40% of control at 12 mo (p = 0.01) and to 69% of control (p = 0.15) at 18 mo.
A decrease in tocopherol in the lining layer of the lung would indicate an
impaired defense against further oxidant challenge. The fact that the extra-
cellular tocopherol was decreased suggests that mechanisms for regeneration
extracel 1 ularly may be weaker than those in cells, and thus not able to
compensate for the oxidant exposure.
After 18 mo of O3 exposure, ascorbic acid in the extracellular fluids
lining the inner lung was increased to 233% of control (p = 0.0001). There
was no change in ascorbic acid in the whole lung homogenate at 12 mo (no lung
measurement was made at other times). The ascorbic acid/protein ratio in the
cells lavaged from the lung was increased to 185% of control (P = 0.001) at 12
mo and to 251% of control (p = .02) at 18 mo. Glutathione and uric acid were
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measured in the lung lavage fluid supernatant and cells at 18 mo, but no
changes were observed in these antioxidants due to O3 exposure.
Although the present study indicated that most of the evidence of O3-
induced lung alteration did not persist during a post exposure period in clean
air, the true reversal of the structurally remodeled connective tissues is not
clear. Since the thickened interstitium of the alveolae did not completely
regress to control levels during this period, the question has been raised
whether collagen or elastin fibers/matrix may show irreversible
disorganization or hypertrophy if examined specifically. Hence, with the
tissue sections previously studied still intact, these connective tissues will
be quantitated morphometrically at the deposition target of O3, the
bronchoalveolar junction, in an effort to address this question.
CONCLUSIONS
Through this multifaceted study we have been able to show structurally and
F,u net tonally that chronic exposure to O3 causes restrictive lung disease that
progresses witn continued exposure. T'hie initiation and development of a focal
interstitial fibrosis and a persistence of alveolar epithelial injury was
observed over the 18 mo exposure period. The progression of lung injury
occurred in a biphasic manner. Initially an acute inflammatory response was
observed after 1 wk of exposure, which subsided and was undetectable after 3
wk of exposure. However, epithelial and interstitial reactions were again
observed after 3 mo of exposure to O3 and the lesions persisted or increased
in intensity up to 18 mo of exposure. Functional studies showed persistence
of decreased total lung capacity and residual volumes at 3, 12 and 18 mo,
suggestive of the presence of a restrictive structural lesion as would be
expected with a 'stiff' lung. These functional results correlated well with
the structural findings of interstitial fibrosis, a restrictive lung disease.
Collagen biochemistry studies were not conclusive at any time point thus could
not be correlated to the structural and functional results.
The overall consensus of observations was both a persistence and a
progression of injury with continuous O3 exposure. However, when the animals
were allowed to reside in clean air post exposure, all major lesions were
resolved. This was supported by O3 challenge experiments which showed that
chronic exposure to O3 altered the responsiveness of the airways to O3. This
alteration was observed as an attenuation of the normal response to O3
challenge. As with other parameters studied, the tolerance phenonema was not
observed after a recovery period in clean air.
Several significant changes were observed in the biochemical defense
system of the lung. There appeared to be a persistence of increased epithelial
permeability up to 18 mo of exposure which, like the structural and functional
changes, resolved after a clean air recovery period. Another significant
finding was the lack of persistence or progression of the antioxidant
biochemical lesions. Changes that were observable at 3 or 12 mo of exposure
were not observed after 18 mo of exposure, apparently resolving or
compensating between 12 and 18 mo.
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The primary conclusion of this study is that continuous low level exposure
to O3 causes restrictive lung disease as characterized by development of a
focal interstitial fibrosis. Removal of the animals from the O3 environment to
an environment of clean, filtered air appeared to reverse the disease state to
one of normal structural and functional being. However, one must remember that
people do not breath clear,, filtered air, thus O3 exceedance conditions that
exist in numerous cities in the U.S. would appear to promote a causal
relationship for the potentia1 development of pulmonary interstitial fibrosis.
REFERENCES
1. Davies, D.W., Walsh, L.C. Ill, hiteshew, M.E., Menache, M.G., Miller, F.J.
and Grose, E.C. "Evaluating the toxicity of urban patterns of oxidant
gases I. An automated chronic gaseous animal inhalation exposure
facility." J. Toxicol. Environ. Health 21: 89 (1987).
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