TOXICOLOOY
AND APPLIED PHARMACOLOGY
51,247-258
(1979)
Ozone-Induced Alterations in Collagen Metabolism of Rat Lungs JEROLDA. LAST, DEBORAHB. GREENBERG, AND WILLIAM California
L. CASTLEMAN
Primate Research Center and Department of Internal University of California, Davis, California 95616
Medicine,
Received April 27, 1979; accepted July 11, 1979 Ozone-Induced Alterations in Collagen Metabolism of Rat Lungs. LAST, J. A., GREENB., AND CASTLEMAN, W. L. (1979). Toxicol. Appl. Pharmacol. 51, 247-258. Rats were exposed to amounts of ozone ranging from 0.5 to 2.0 ppm for intervals of 1, 2, or 3 weeks. Collagen synthesis rates in their lungs were quantitated by biochemical analyses performed with lung minces. Correlative histological observations were made in different lung lobes from the same rats. At all levels of ozone tested, collagen synthesis rates of the lungs were significantly elevated and histologically discernible fibrosis of the alveolar duct walls was observed. Within the range of ozone concentrations studied, the elevation of collagen synthesis rate in exposed rats was a linear function of the level of ozone to which the animals were exposed. We conclude that exposure of rats to near-ambient levels of ozone causes biochemically and histologically discernible fibrotic changes in their lungs, suggesting that such effects may occur at levels of ozone at or near the current ambient air quality standard for this pollutant. BERG, D.
Anywhere that automobile exhaust and sunlight converge there is a potential for ozone pollution of the ambient air; when the particular area is a valley or basin with poor air circulation, the problem tends to be exacerbated. The ambient air quality standard set by the Environmental Protection Agency for total oxidant (essentially entirely ozone or NO, during peak hours of late morning and early afternoon) is 0.08 ppm maximum. This value is routinely exceeded in the Southern California air basin and most large cities of the Southwest (Phoenix, Houston, and El Paso, for example) throughout the summer months. Some locations in the Southern California air basin exceeded this standard more than 200 days of the year in 1974 (Committee on Medical and Biological Effects of Environmental Pollutants, 1977). This standard was also exceeded for 29 days of the year between 1964 and 1973 in several large cities in more northern climates
(Altshuller, 1975). Values of 0.2 ppm have been exceeded rarely in these northern cities, but were exceeded approximately 100 days in 1974 in the South Coast (California) air basin, where values higher than 0.5-0.6 ppm have been recorded. The problem is not unique to the United States; reported values of mean ozone concentration exceeded 0.2 ppm in London and in southern England in 1976 (Apling et al., 1977). It should be recognized that except during severe inversion episodes, where the polluted air is stagnant, peak ozone values occur for only a few hours of the day, usually between a little before noon and 6 PM, due to the requirement for heavy vehicular traffic coupled with sunlight. The present occupational exposure limit is 0.1 ppm for an 8-hr day, with a maximal allowable exposure to 0.3 ppm over a lo-min interval. Exposure of experimental animals to high concentrations of ozone (1 ppm and above) 247
0041~8X/79/140247-12502.00/O Copyright 6 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
248
LAST, GREENBERG, AND CASTLEMAN
has been reported to result in pulmonary fibrosis as defined by morphological criteria (Stokinger et al., 1957). The salient findings in this study were observations of fibrosis of the bronchiolar walls of four guinea pigs killed after 268 days of exposure to about 1.1 ppm of ozone (alkaline KI analysis; the true value was probably about 1.4 ppm) for 6 hr per day, 5 days per week (433 days total on study). Similar changes were observed in rats and hamsters exposed concurrently. Exposure to ozone at near-ambient levels has been suggested in the etiology of pulmonary fibrosis in dogs and rats (Freeman et al., 1973, 1974). In this case, exposure of rats to 0.5-0.6 ppm of ozone for 6 days, for 8 days, or for 3 weeks resulted in an apparent increased deposition of fibroblasts and connective tissue in bronchioles and proximal alveolar ducts. Similar connective tissue was also observed at the junction between the terminal bronchioles and the proximal alveolar ducts after exposure to about 0.9 ppm of ozone for 8 days. The terminal bronchiolar and proximal alveolar duct region is a logical area in which to expect a fibroplastic response to ozone since several studies have demonstrated ozone-induced epithelial cell injury and proliferation, as well as a moderate inflammatory response, in this location (Schwartz et al., 1976; Stephens et al., 1974). Mathematical modeling experiments have suggested that this area of the lung receives the maximum dose when ozone is inhaled (Miller et al., 1978). In the present study biochemical techniques are used to measure ozone-induced changes in the synthetic rate of lung collagen in rats at several levels of ozone, ranging from high to near ambient. The observed levels of collagen synthesis as evaluated by these techniques are correlated with histological indices of pulmonary fibrosis observed in the same rats. The biochemical methodology, which has been described in detail elsewhere (Bradley et al., 1974; Greenberg et al., 1978a), gives quantitative data reflecting biochemical changes in a fundamental metabolic para-
meter within the lungs of the rats at the time they are killed. The rate of pulmonary collagen biosynthesis is a particularly appropriate toxicological parameter to measure since it is known that aberrations in the synthesis and/or degradation of lung collagen are associated with pulmonary fibrosis, an endstage disturbance of pulmonary structure and function (Fulmer and Crystal, 1976). METHODS Ozone generation and monitoring. Techniques used for the generation of ozone from pure oxygen have been described (Schwartz ef al., 1976). Rats were exposed in chambers of the type described by Hinners et al. (1968). They were housed two per stainlesssteel mesh cage and given water and rat chow (Purina) ad libitum. Flow rates were adjusted to give 30 changes of chamber atmosphere per hour. Ozone concentrations throughout the exposures were continuously monitored with a Dasibi meter (Last et al., 1977). Detailed data on typical exposures are presented in Table 1. Replicate exposures were performed under essentially identical regimens. Rats were exposed to levels of ozone between 0.5 and 2.0 ppm for 7 days and up to 14 and 21 days at and below concentrations of 1.5 ppm (Table 1). Animals. Rats were Sprague-Dawley males, chronic respiratory disease-free, from Hilltop (Scottsdale, Pa.), as used in previous experiments from this laboratory (Last et al., 1977). Ages at the start of exposure ranged from about 70 to 150 days. A total of 47 control and 47 exposed rats were studied at the various times, dose levels, and repetitions of this experiment performed. Control animals were housed in identical exposure chambers, as above, but were allowed to breathe filtered air. Rats were killed within an hour of the termination of exposure. At the termination of each exposure period, animals were removed from the chambers and given an overdose of sodium pentobarbital; their chests were then opened. The right apical lobes were tied off with suture thread, and the remaining four lobes were perfused via the pulmonary artery with 0.15 M saline. The left lungs were then tied off with suture thread, removed, and placed in Dulbecco’s modified Eagle’s medium. They were transferred to fresh medium for incubation with isotope in vitro. The right apical lobes were removed for determination of the lung wet weights (Greenberg et aI., 1978a). The biochemical studies performed on the left lung are described in detail elsewhere (Greenberg et al., 1978a). Briefly, the lungs were minced into pieces of
RAT LUNG
COLLAGEN
TABLE
249
METABOLISM
1
EXPOSUREOF RATS TO OZONE Actual ozone concentrations
Nominal ozone concentration (mm)
Days of exposure
Mean mm
SD
Number of samples
2.0
I
2.06
0.07
1359
1.5
1.1
0.8
0.5
7
1.44
0.08
934
14
1.46
0.06
2343
21
1.46
0.06
3161
I
1.08
0.04
1145
14
1.09
0.04
2251
21
1.09
0.04
3233
I
0.78
0.02
1369
14
0.79
0.02
2353
21
0.78
0.03
3339
7
0.50
0.01
844
14
0.50
0.02
1251
21
0.50
0.03
1701
approximately 2 mm3, and the tissue was divided among three replicate vials and incubated with [3H]proline. Labeled hydroxyprohne (Juva and Prockop, 1966), total proline (Troll and Lindsley, 1955), DNA, and protein were determined for each sample. Morphologic studies. The right cardiac, intermediate, and diaphragmatic lung lobes were fixed via the trachea at 30 cm pressure with modified Karnovsky’s fixative (Karnovsky, 1965; Nowell and Tyler, 1971). At least two blocks of lung from right diaphragmatic and cardiac lobes were processed for routine paraffin sectioning. Sections 5 to 7 ,um thick were stained with hematoxylin and eosin (H and E) and by routine methods with Gomori’s reticulin and Van Gieson’s coliagen stains (Luna, 1968). Slides from each animal were randomly numbered and examined without knowledge of the exposure regimen. Lesions present in proximal alveolar ducts were evaluated and scored for three variables: (1) degree of thickening of alveolar duct walls by polypoid or band-like connective tissue, and numbers of fibers in this thickened tissue which stained with (2) Gomori’s reticulin stain and (3) Van Gieson’s stain for collagen. Each factor was assigned a score ranging from 0 (normal) to 3 (most abnormal). The extent of involvement of proximal alveolar ducts within any single rat was further evaluated with a
score ranging from 0 to 3, with 0 corresponding to lesions being present in none of the alveolar ducts and with 3 corresponding to lesions being present in almost all of the alveolar ducts. A total lesion score was assigned to each rat by simple addition of the four scores with the maximum total score possible being 12. Data and calculations. Tissue minces were incubated I, 2, and 3 hr before sampling for PHIhydroxyproline synthesis. The best straight line was fitted to these data (plus a 0-hr value of Opmol of hydroxyproline per gram of protein) by linear regression analysis (least-squares program) (Swinscow, 1977). The calculated collagen synthesis rate used for comparisons between groups of rats was the average slope of the lines derived from each group. The coefficient of variance observed within a given data set averaged, for example, 10% for the combined ‘I-day exposed groups at all levels of ozone tested. All individual data points obtained were used except in those cases where the r2 value upon regression analysis of the best straight line was less than 0.90. Where this occurred, the aberrant data point of a set was discarded and the slope was recalculated using two of the three experimentally determined values of rH]hydroxyproline plus the 0-hr value. Data for lesion scores are arithmetic means of the observations for all of the rats within a given group.
250
LAST,
GREENBERG,
AND
CASTLEMAN
RESULTS Our initial experiments were performed at high concentrations of ozone (about 1.9-2.0 ppm), well into the severely edemagenie dose range, so as to elicit maximal effects. At these high levels of ozone, more than half the exposed rats died in acute respiratory distress during the first week of exposure, thus limiting the duration of an experiment at these concentrations. Such high doses were useful for correlative histological and biochemical preliminary experiments while we were validating the techniques to be used in these studies. As a practical matter, however, 1.5 ppm of ozone was the maximal dose to which rats could be exposed without death of the animals (with possible inadvertent selection of resistant survivors for further study becoming an additional experimental variable). Previous studies (Greenberg et al., 1978a) had documented that lung minces from normal rats and from rats injected with high doses of paraquat incorporated [3H]proline into collagen [3H]hydroxyproline at a linear rate for at least 3 hr of incubation. Thus, data obtained at 0, 1, 2, and 3 hr of incubation could be used to determine rates of incorporation by calculating the slope of a straight line fitted by least squares to the data. We had to validate such an assay technique for lung minces from rats exposed to ozone as well. Data from a typical experiment are shown in Fig. 1, which demonstrates that the time course of incorporation of [3H]proline into collagen hydroxyproline by minces from lungs of rats exposed to 0.8 ppm of ozone for 3 weeks is indeed linear for at least 3 hr of incubation. Thus, rates of synthesis of collagen in vitro by lung minces prepared from rats exposed to ozone could be determined. To study the response of rat lungs to in viva exposures of the animals to various concentrations of ozone, we performed experiments at various levels of this oxidant pollutant ranging from 0.5 to 1.5 ppm. All
100 Lf!? 0
2
3
LLours of incubation
FIG. 1. Time course of incorporation of PHIproline into collagen [3H]hydroxyproline by lung minces
from rats that had breathed only filtered air (0) or had breathed 0.8 ppm of ozone for 21 days (0). Data points are means+ SD of values from three different rats at each time; the lines are fitted to the data by least-squares analysis. The correlation coefficient (r* values) for each line is 0.99.
of the exposed groups of rats studied, at all levels of ozone and at all time points, showed increased lung wet weights as compared with their matched control groups; the magnitude of the increased wet weight was apparently dose dependent. As shown in Fig. 2, exposure of rats to ozone at all concentrations tested, for all time intervals evaluated, resulted in substantial elevations in the in vitro rate of incorporation of [3H]proline into collagen hydroxyproline by lung minces. These elevations are all statistically significant (PC 0.01, one-tailed Student’s t test). When the biochemical results are examined in greater detail, several further generalizations emerge. At all the exposure intervals studied, that is, 1, 2, and 3 weeks, the response of the rats to ozone as evaluated by collagen synthesis rates of their lung minces was related to the level of ozone to which they were exposed. As will be documented below, this response was a linear function of the ozone level. At 0.5 ppm of ozone, the lowest concentration tested in this study, there was a significant biochemical response in these rats
after 1, 2, and 3 weeks of exposure. The magnitude of the response at all ozone levels
RAT
Ozone concentrotion,parts
IOr
LUNG
per million
C
Ozone
concentration,
palh
per million
COLLAGEN
METABOLISM
251
(expressed as percentage of control value) seemed to be greater after 2 weeks of exposure than after 1 week; values were very similar after 2 and 3 weeks of exposure. The highest levels of stimulation of collagen synthesis rate observed are about threefold higher than the control values (after exposure to 2 ppm for 7 days or to 1.5 ppm for 14 and 21 days). In parallel with the measured increase in collagen synthesis rate by the lung slices was an increase in the lesion score in alveolar ducts (Fig. 2). While the lesion score and the collagen synthesis rates are not directly comparable parameters, at least in quantitative terms, the correspondence between these values is striking at all times plotted (Fig. 2). Microscopic lesions were present in all animals exposed to ozone. Such lesions were always present in proximal portions of included alveolar ducts, and sometimes distalmost portions of terminal bronchioles and closely associated interalveolar septa. The lesions induced in rats exposed to 0.82.0 ppm ozone were characterized by moderately diffuse to focal, polypoid thickening of alveolar duct walls and associated interalveolar septa with corresponding narrowing of the ducts and alveoli (Figs. 3 and 4). Thickened’walls contained elongated, spindleshaped cells which were interpreted to be fibroblasts. Between the fibroblasts was fibrillar eosinophilic material of variable density, as well as nonstaining areas of edema fluid. The fibrillar material stained positively with -Gomori’s reticulin method (Fig. 5) and with Van Gieson’s stain for FIG. 2. Biochemical and histological responses of rat lungs to exposure of rats in vivo to various concentrations of ozone for (A) 7, (B) 14, or (C) 21 days. Biochemical data are expressed as the percentage of control value (nmol of [3H]hydroxyproline synthesized per hour per gram of protein) of collagen biosynthesis rate by lung minces (see Methods). Histologic responses are expressed as net lesion score (see Methods). Open bars, histological lesion scores; hatched bars, collagen synthesis rates.
252
LAST,
GREENBERG,
collagen. Small numbers of mononuclear inflammatory cells and occasional neutrophils were also present in the thickened walls. The most consistent thickening of bronchiolar and alveolar duct walls by connective tissue was observed at concentrations higher than
AND
CASTLEMAN
0.8 ppm. The degree of alveolar ductal wall thickening generally decreased with increased length of exposure. This decreased thickness was associated with smaller numbers of spindle-shaped cells (fibroblasts) and with increased density of reticulin or collagen
FIG. 3. (A) Lung from a control rat. A terminal bronchiole (TB) opens into alveolar ddcts (AD). H and E stain. x 160. (B) Lung from a rat exposed to 1.5 ppm ozone for 1 week. There are polypoid and band-like thickenings of alveolar duct walls by connective tissue (arrowheads), with partial occlusion’ of ducts. H and E stain. x 160.
RAT
LUNG
COLLAGEN
METABOLISM
FIG. 4. Polyp in alveolar duct of a rat exposed to 0.8 ppm ozone for 1 week. Spindle-shaped fibroblasts (arrow) are in. the polyp, together with small mononuclear inflammatory cells and occasional neutrophils. The alveolar duct contains aggregates of macrophages and a few neutrophils. H and E stain. x 400.
FIG. 5. Serial section of the field shown in Fig. 4, visualized ,with Gomori’s reticulin stain. Many black reticulin fibers (arrows) are in the polyp. x 400.
253
254
LAST,
GREENBERG,
fibers as demonstrated with Gomori and Van Gieson stains. Minimal or no thickening of walls or evidence of fibrosis was present after exposure of rats to 0.5 ppm for 7 days, although lesions in alveolar ducts characterized by intraluminal and interstitial aggregates of macrophages and other mononuclear cells and increased numbers of cuboidal epithelial cells ,were observed. In rats exposed to 0.5 ppm for 14 and 21 days, there was sometimes minimal thickening of alveolar duct walls with mildly increased amounts of reticulin and collagen. Although the animals purchased for this study were designated as chronic respiratory disease-free, mild to moderate pneumonia was observed in 43% of the ozone-exposed and 12% of the control animals. This pneumonia was characterized by aggregations of mononuclear inflammatory cells and neutrophils in peribronchiolar connective tissue, in closely associated interalveolar septa, and in perivascular connective tissue. Bronchiolar epithelial hyperplasia was also observed. Finally, we have performed initial experiments on the reversibility of the effects observed in the collagen synthesis assay after removal of rats from the exposure chambers. In such studies performed on groups of rats exposed to 1.5 ppm of ozone for 14 or 20 days, then allowed to recover breathing filtered air’ for 6 additional days, we found collagen synthesis rates that were intermediate (39 and 14%, respectively, of the total ozone-induced increase) between the values for the control and the corresponding exposed (without recovery) groups. The observed lesion scores correlated with these decreased synthesis rates. Hence, these biochemical and h&tological changes in rate of lung collagen biosynthesis and lung inflammatory response may be, at least in part, reversible upon removal of the ozone. DISCUSSION In this paper, we have documented that exposure of rats to ozone results in focal
AND
CASTLEMAN
pulmonary fibrosis, mainly in proximal alveolar duct walls. Quantitative biochemical and semiquantitative histological methods were used in this study to determine the relationship between dose and ozone-induced fibrosis. These two types of methodologies complement one another, thus allowing very different aspects of the same process to be studied. The rationale for equating hydroxyproline and collagen synthesis rates in lung minces has been thoroughly discussed elsewhere (Bradley et al., 1974; Greenberg et al., 1978a). The biochemical determination of collagen biosynthesis rate by lung minces is a measurement of the rate at which the lung was making collagen at the instant the animal was killed and, thus, is a measurement that might be expected to rapidly change during the ongoing pathological processes. For example, we have shown elsewhere (Greenberg et al., 1978b) that paraquat, a potent fibrogenic agent, causes rapid changes in this parameter. The magnitude of the changes observed with ozone (Fig. 2) are greater than the stimulation in collagen synthesis rate observed 2 days after administration of high doses of paraquat, but less than the stimulation seen 3 days after injection of paraquat (Greenberg et al., 1978b). On the other hand, the histological determination of a lesion score is a measurement of the extent of accumulation of connective tissue components in proximal alveolar duct walls and is, thus, an assay that might be expected to change relatively slowly during the ongoing pathological processes. The biochemical techniques allow a response of the lung to ozone exposure to be quantitated relatively precisely. However, these measurements might not necessarily be a reliable indicator of lung damage resulting in fibrosis. Increased collagen synthesis rates might alternatively be the result of a nonpatho: logical repair mechanism. The histological observations, on the other hand, unequivocally indicate that there is fibrosis or focal accumulation (synthesis) of collagen as a result of damage to the lung. We might
RAT
LUNG
COLLAGEN
anticipate that the biochemical techniques would be more sensitive indicators of changes in collagen metabolism than are the histological, especially after short-term exposures of rats to relatively low concentrations of ozone. In fact, in the rats sampled after exposure to 0.5 ppm of ozone (Fig. 2), the biochemical determination of collagen synthesis rate is a seemingly more sensitive assay for ozone-induced changes in pulmonary collagen metabolism than is the histological lesion score at the alveolar duct level. After only 1 week, increased levels of collagen synthesis are detected. However, not until the second week is there histologically discernible fibrosis. The same temporal relationship of increased collagen synthesis rate preceding a histologically detectable increase in collagen was probably not observed at higher ozone levels in this study since the response is more severe and probably proceeds more rapidly. It should be pointed out that inflammatory lesions in proximal alveolar ducts were easily recognized in the rats exposed to 0.5 ppm of ozone for 7 days, even though unequivocal thickening of the alveolar duct walls and fibrosis was not observed. Focal fibrosis of walls of bronchioles and alveolar ducts has been demonstrated to occur as a result of exposure to ozone at levels of about 1.0 ppm or above in rats, guinea pigs, and dogs (Freeman et a/., 1973, 1974; Stokinger et al., 1957). The pathogenic mechanisms involved in the induction of this focal fibrosis are poorly understood. In the present study an attempt to semiquantitatively assess ozone-induced fibrosis was made using a lesion scoring system based on several factors. Although local edema and inflammatory cell infiltration at least minimally contribute to the score, the main components of the score (increased amounts of collagen and reticulin fibers and increased numbers of spindle-shaped fibroblasts) are generally accepted morphologic criteria of fibrosis. There was a dose-response in the severity of focal fibrosis as expressed in the lesion score (Fig. 2). The lesion score values
METABOLISM
255
paralleled the ozone-induced increases in rate of collagen synthesis measured biochemically. Histologic changes compatible with mild viral pneumonia were present in a large percentage of the control and ozone-exposed animals in this study (Horsfall and Hahn, 1940; Robinson et al., 1968). Serologic studies were not done on the animals in this study. Serologic analysis of other rats from the same supplier during the experimental time period, however, revealed elevated antibody titers to pneumonia virus of mice (hemagglutination inhibition) and to Sendai virus (complement fixation). Endemic infection of the rats used in this study by one of these viruses was likely. Pneumonia of similar severity was observed in control and ozone-exposed rats, although a greater percentage of the exposed rats had pneumonia. Within all exposure groups ozone-induced lesions usually occurred independently of pneumonic changes and were almost always easily distinguishable from the suspected viral-induced lesions. The lesions in alveolar ducts of rats from the same ozone-exposed groups were of closely comparable severity regardless of whether or not the rat had detectable pneumonia. Although it is highly unlikely that a virus infection was inducing the alveolar duct lesions observed in this study, the possibility that viral-induced damage had an additive or synergistic effect on ozone-induced fibrosis of aIveolar duct walls cannot be excluded. The reproducibility of the observed effects in the biochemical assays (see, for example, Fig. 1) also argue against the changes in collagen metabolism seen in this study being caused by anything other than the ozone exposure per se. In addition, Hussain et al. (1976) have previously presented evidence that collagen synthesis rates in the lung in uivo are elevated after exposure of rats to 0.8 ppm of ozone, suggesting that the present results are not observed solely because of the techniques used or the specific population of rats studied.
256
LAST,
0
0.2
GREENBERG,
0.4
AND
0.8 Ozone
concentration,
CASTLEMAN
1.2 parts
1.6
I
2.0
per million
FIG. 6. Collagen biosynthesis rates of rat lung minces as a function of concentration of ozone to which the rats had been exposed in oivo. Lines are fitted to the data by linear regression analysis (least-squares program). O-0, 7-day exposures (r2 = 0.79); l - - -0, 1Cday exposures (r2 = 0.98); A- ---A, 21-day exposures (r’ = 0.95). Apparent “threshold” values are determined by linear extrapolation to zero effect levels (i.e., values of 100 % of control).
The biochemical data obtained in this study may also be used to calculate the theoretical “threshold levels” of ozone required to elicit stimulation of collagen synthesis rate in rat lungs after exposure for 1, 2, or 3 weeks, as shown in Fig. 6. The assumption made for the purposes of these calculations was that the dose-response curve remains linear for. all concentrations of ozone, including extrapolations’ to lower levels than those actually tested. Such linear extrapolations probably overestimate the actual level at which minimal effects might occur (Barth et al., 1971; Committee on Medical and Biological Effects of Environmental Pollutants, 1977). The minimal effective concentrations of ozone thus calculated range from about 0.1 to 0.2 ppm, concentrations actually commonly encountered during pollution episodes in the Southern California air basin (Committee on Medical and Biological Effects of Environmental Pollutants, 1977), and close to the ambient air quality standard for ozone (0.08 ppm) currently in force (Altshuller, 1975). The 7- and 1Cday exposures, for which we have the
most data, extrapolate to values of 0.13 and 0.10 ppm, respectively, for no-effect levels in this assay. It is important to emphasize that this “threshold level” at least in part reflects the detection limit for a specific assay. Thus, the limiting factor in the calculation of this “threshold” may be in the assumption of linear response to extrapolated values or in the “noise” level of the assay, which included factors such as the sensitivity of detection of differences in lung mince [3H]hydroxyproline levels and the biological variability of individual rats. Conversely, the limiting factor may be in the “signal” itself, i.e., the inherent capacity of the rat lung to respond to insult by low levels of ozone by increasing its collagen synthesis rate independently of other parameters that might be affected. In any event, this calculated threshold level of ozone must be viewed as either a potential underestimation or overestimation, of the levels of ozone that can provoke changes in collagen metabolism by rat lungs upon continuous exposure to these levels of ozone for the durations studied herein. More sensitive histological (ultrastructural
257
RAT LUNG COLLAGEN METABOLISM
morphometry) and biochemical (larger numbers of rats, lower levels of ozone exposure, comparisons of collagen synthesis and degradative rates) methods must be applied to these questions to more precisely define the lowest levels of ozone exposure that provoke pulmonary fibrosis. We believe the methods described here could easily, effectively, and productively be applied toward evaluating other fibrosisinducing pneumotoxins. Threshold levels of known pneumotoxins could be determined in this way, as well as the potential damaging effects (on lung collagen metabolism) of putative pneumotoxins. By using both the histological and biochemical techniques described here, the ongoing damage as well as that already produced can be monitored. While we believe the biochemical measurements to be more convenient and sensitive for quantitative evaluations, the histological studies allow conclusions to be drawn as to whether or not true pulmonary damage, more specifically pulmonary fibrosis, is being measured. Since preparation of this manuscript, the ambient air quality standard for ozone has been raised by the EPA from 0.08 ppm to 0.12 ppm. ACKNOWLEDGMENTS We thank June Wong for excellent technical assistance in performing the biochemical assays. This work was supported in part by National Institutes of Health Grants ES-00628 and ES-01713, and by a grant from the American Lung Association.
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AND
CASTLEMAN
(1976). Pulmonary responses of rats to ambient levels of ozone. Lab. Invest. 34, 565-578. STEPHENS, R. J., SLOAN, M. F., EVANS, M. J., AND FREEMAN, G. (1974). Early responses of lung to low levels of ozone. Amer. J. Pathol. 74, 31-58. STOKINGER, H. E., WAGNER, W. D., AND DOBROGORSKI, 0. J. (1957). Ozone toxicity studies. III. Chronic injury to lungs of animals following exposure at a low level. Arch. Environ. Health 16, 514-522. SWINSCOW, T. D. V. (1977). Statistics at Square One, 2nd ed., Brit. Med. Assoc., London. TROLL, W., AND LINDSLEY, J. (1955). The colorimetric determination of proline in tissue and body fluids. J. Biol. Chem. 215, 655-660.
AND APPLIED PHARMACOLOGY
51,247-258
(1979)
Ozone-Induced Alterations in Collagen Metabolism of Rat Lungs JEROLDA. LAST, DEBORAHB. GREENBERG, AND WILLIAM California
L. CASTLEMAN
Primate Research Center and Department of Internal University of California, Davis, California 95616
Medicine,
Received April 27, 1979; accepted July 11, 1979 Ozone-Induced Alterations in Collagen Metabolism of Rat Lungs. LAST, J. A., GREENB., AND CASTLEMAN, W. L. (1979). Toxicol. Appl. Pharmacol. 51, 247-258. Rats were exposed to amounts of ozone ranging from 0.5 to 2.0 ppm for intervals of 1, 2, or 3 weeks. Collagen synthesis rates in their lungs were quantitated by biochemical analyses performed with lung minces. Correlative histological observations were made in different lung lobes from the same rats. At all levels of ozone tested, collagen synthesis rates of the lungs were significantly elevated and histologically discernible fibrosis of the alveolar duct walls was observed. Within the range of ozone concentrations studied, the elevation of collagen synthesis rate in exposed rats was a linear function of the level of ozone to which the animals were exposed. We conclude that exposure of rats to near-ambient levels of ozone causes biochemically and histologically discernible fibrotic changes in their lungs, suggesting that such effects may occur at levels of ozone at or near the current ambient air quality standard for this pollutant. BERG, D.
Anywhere that automobile exhaust and sunlight converge there is a potential for ozone pollution of the ambient air; when the particular area is a valley or basin with poor air circulation, the problem tends to be exacerbated. The ambient air quality standard set by the Environmental Protection Agency for total oxidant (essentially entirely ozone or NO, during peak hours of late morning and early afternoon) is 0.08 ppm maximum. This value is routinely exceeded in the Southern California air basin and most large cities of the Southwest (Phoenix, Houston, and El Paso, for example) throughout the summer months. Some locations in the Southern California air basin exceeded this standard more than 200 days of the year in 1974 (Committee on Medical and Biological Effects of Environmental Pollutants, 1977). This standard was also exceeded for 29 days of the year between 1964 and 1973 in several large cities in more northern climates
(Altshuller, 1975). Values of 0.2 ppm have been exceeded rarely in these northern cities, but were exceeded approximately 100 days in 1974 in the South Coast (California) air basin, where values higher than 0.5-0.6 ppm have been recorded. The problem is not unique to the United States; reported values of mean ozone concentration exceeded 0.2 ppm in London and in southern England in 1976 (Apling et al., 1977). It should be recognized that except during severe inversion episodes, where the polluted air is stagnant, peak ozone values occur for only a few hours of the day, usually between a little before noon and 6 PM, due to the requirement for heavy vehicular traffic coupled with sunlight. The present occupational exposure limit is 0.1 ppm for an 8-hr day, with a maximal allowable exposure to 0.3 ppm over a lo-min interval. Exposure of experimental animals to high concentrations of ozone (1 ppm and above) 247
0041~8X/79/140247-12502.00/O Copyright 6 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
248
LAST, GREENBERG, AND CASTLEMAN
has been reported to result in pulmonary fibrosis as defined by morphological criteria (Stokinger et al., 1957). The salient findings in this study were observations of fibrosis of the bronchiolar walls of four guinea pigs killed after 268 days of exposure to about 1.1 ppm of ozone (alkaline KI analysis; the true value was probably about 1.4 ppm) for 6 hr per day, 5 days per week (433 days total on study). Similar changes were observed in rats and hamsters exposed concurrently. Exposure to ozone at near-ambient levels has been suggested in the etiology of pulmonary fibrosis in dogs and rats (Freeman et al., 1973, 1974). In this case, exposure of rats to 0.5-0.6 ppm of ozone for 6 days, for 8 days, or for 3 weeks resulted in an apparent increased deposition of fibroblasts and connective tissue in bronchioles and proximal alveolar ducts. Similar connective tissue was also observed at the junction between the terminal bronchioles and the proximal alveolar ducts after exposure to about 0.9 ppm of ozone for 8 days. The terminal bronchiolar and proximal alveolar duct region is a logical area in which to expect a fibroplastic response to ozone since several studies have demonstrated ozone-induced epithelial cell injury and proliferation, as well as a moderate inflammatory response, in this location (Schwartz et al., 1976; Stephens et al., 1974). Mathematical modeling experiments have suggested that this area of the lung receives the maximum dose when ozone is inhaled (Miller et al., 1978). In the present study biochemical techniques are used to measure ozone-induced changes in the synthetic rate of lung collagen in rats at several levels of ozone, ranging from high to near ambient. The observed levels of collagen synthesis as evaluated by these techniques are correlated with histological indices of pulmonary fibrosis observed in the same rats. The biochemical methodology, which has been described in detail elsewhere (Bradley et al., 1974; Greenberg et al., 1978a), gives quantitative data reflecting biochemical changes in a fundamental metabolic para-
meter within the lungs of the rats at the time they are killed. The rate of pulmonary collagen biosynthesis is a particularly appropriate toxicological parameter to measure since it is known that aberrations in the synthesis and/or degradation of lung collagen are associated with pulmonary fibrosis, an endstage disturbance of pulmonary structure and function (Fulmer and Crystal, 1976). METHODS Ozone generation and monitoring. Techniques used for the generation of ozone from pure oxygen have been described (Schwartz ef al., 1976). Rats were exposed in chambers of the type described by Hinners et al. (1968). They were housed two per stainlesssteel mesh cage and given water and rat chow (Purina) ad libitum. Flow rates were adjusted to give 30 changes of chamber atmosphere per hour. Ozone concentrations throughout the exposures were continuously monitored with a Dasibi meter (Last et al., 1977). Detailed data on typical exposures are presented in Table 1. Replicate exposures were performed under essentially identical regimens. Rats were exposed to levels of ozone between 0.5 and 2.0 ppm for 7 days and up to 14 and 21 days at and below concentrations of 1.5 ppm (Table 1). Animals. Rats were Sprague-Dawley males, chronic respiratory disease-free, from Hilltop (Scottsdale, Pa.), as used in previous experiments from this laboratory (Last et al., 1977). Ages at the start of exposure ranged from about 70 to 150 days. A total of 47 control and 47 exposed rats were studied at the various times, dose levels, and repetitions of this experiment performed. Control animals were housed in identical exposure chambers, as above, but were allowed to breathe filtered air. Rats were killed within an hour of the termination of exposure. At the termination of each exposure period, animals were removed from the chambers and given an overdose of sodium pentobarbital; their chests were then opened. The right apical lobes were tied off with suture thread, and the remaining four lobes were perfused via the pulmonary artery with 0.15 M saline. The left lungs were then tied off with suture thread, removed, and placed in Dulbecco’s modified Eagle’s medium. They were transferred to fresh medium for incubation with isotope in vitro. The right apical lobes were removed for determination of the lung wet weights (Greenberg et aI., 1978a). The biochemical studies performed on the left lung are described in detail elsewhere (Greenberg et al., 1978a). Briefly, the lungs were minced into pieces of
RAT LUNG
COLLAGEN
TABLE
249
METABOLISM
1
EXPOSUREOF RATS TO OZONE Actual ozone concentrations
Nominal ozone concentration (mm)
Days of exposure
Mean mm
SD
Number of samples
2.0
I
2.06
0.07
1359
1.5
1.1
0.8
0.5
7
1.44
0.08
934
14
1.46
0.06
2343
21
1.46
0.06
3161
I
1.08
0.04
1145
14
1.09
0.04
2251
21
1.09
0.04
3233
I
0.78
0.02
1369
14
0.79
0.02
2353
21
0.78
0.03
3339
7
0.50
0.01
844
14
0.50
0.02
1251
21
0.50
0.03
1701
approximately 2 mm3, and the tissue was divided among three replicate vials and incubated with [3H]proline. Labeled hydroxyprohne (Juva and Prockop, 1966), total proline (Troll and Lindsley, 1955), DNA, and protein were determined for each sample. Morphologic studies. The right cardiac, intermediate, and diaphragmatic lung lobes were fixed via the trachea at 30 cm pressure with modified Karnovsky’s fixative (Karnovsky, 1965; Nowell and Tyler, 1971). At least two blocks of lung from right diaphragmatic and cardiac lobes were processed for routine paraffin sectioning. Sections 5 to 7 ,um thick were stained with hematoxylin and eosin (H and E) and by routine methods with Gomori’s reticulin and Van Gieson’s coliagen stains (Luna, 1968). Slides from each animal were randomly numbered and examined without knowledge of the exposure regimen. Lesions present in proximal alveolar ducts were evaluated and scored for three variables: (1) degree of thickening of alveolar duct walls by polypoid or band-like connective tissue, and numbers of fibers in this thickened tissue which stained with (2) Gomori’s reticulin stain and (3) Van Gieson’s stain for collagen. Each factor was assigned a score ranging from 0 (normal) to 3 (most abnormal). The extent of involvement of proximal alveolar ducts within any single rat was further evaluated with a
score ranging from 0 to 3, with 0 corresponding to lesions being present in none of the alveolar ducts and with 3 corresponding to lesions being present in almost all of the alveolar ducts. A total lesion score was assigned to each rat by simple addition of the four scores with the maximum total score possible being 12. Data and calculations. Tissue minces were incubated I, 2, and 3 hr before sampling for PHIhydroxyproline synthesis. The best straight line was fitted to these data (plus a 0-hr value of Opmol of hydroxyproline per gram of protein) by linear regression analysis (least-squares program) (Swinscow, 1977). The calculated collagen synthesis rate used for comparisons between groups of rats was the average slope of the lines derived from each group. The coefficient of variance observed within a given data set averaged, for example, 10% for the combined ‘I-day exposed groups at all levels of ozone tested. All individual data points obtained were used except in those cases where the r2 value upon regression analysis of the best straight line was less than 0.90. Where this occurred, the aberrant data point of a set was discarded and the slope was recalculated using two of the three experimentally determined values of rH]hydroxyproline plus the 0-hr value. Data for lesion scores are arithmetic means of the observations for all of the rats within a given group.
250
LAST,
GREENBERG,
AND
CASTLEMAN
RESULTS Our initial experiments were performed at high concentrations of ozone (about 1.9-2.0 ppm), well into the severely edemagenie dose range, so as to elicit maximal effects. At these high levels of ozone, more than half the exposed rats died in acute respiratory distress during the first week of exposure, thus limiting the duration of an experiment at these concentrations. Such high doses were useful for correlative histological and biochemical preliminary experiments while we were validating the techniques to be used in these studies. As a practical matter, however, 1.5 ppm of ozone was the maximal dose to which rats could be exposed without death of the animals (with possible inadvertent selection of resistant survivors for further study becoming an additional experimental variable). Previous studies (Greenberg et al., 1978a) had documented that lung minces from normal rats and from rats injected with high doses of paraquat incorporated [3H]proline into collagen [3H]hydroxyproline at a linear rate for at least 3 hr of incubation. Thus, data obtained at 0, 1, 2, and 3 hr of incubation could be used to determine rates of incorporation by calculating the slope of a straight line fitted by least squares to the data. We had to validate such an assay technique for lung minces from rats exposed to ozone as well. Data from a typical experiment are shown in Fig. 1, which demonstrates that the time course of incorporation of [3H]proline into collagen hydroxyproline by minces from lungs of rats exposed to 0.8 ppm of ozone for 3 weeks is indeed linear for at least 3 hr of incubation. Thus, rates of synthesis of collagen in vitro by lung minces prepared from rats exposed to ozone could be determined. To study the response of rat lungs to in viva exposures of the animals to various concentrations of ozone, we performed experiments at various levels of this oxidant pollutant ranging from 0.5 to 1.5 ppm. All
100 Lf!? 0
2
3
LLours of incubation
FIG. 1. Time course of incorporation of PHIproline into collagen [3H]hydroxyproline by lung minces
from rats that had breathed only filtered air (0) or had breathed 0.8 ppm of ozone for 21 days (0). Data points are means+ SD of values from three different rats at each time; the lines are fitted to the data by least-squares analysis. The correlation coefficient (r* values) for each line is 0.99.
of the exposed groups of rats studied, at all levels of ozone and at all time points, showed increased lung wet weights as compared with their matched control groups; the magnitude of the increased wet weight was apparently dose dependent. As shown in Fig. 2, exposure of rats to ozone at all concentrations tested, for all time intervals evaluated, resulted in substantial elevations in the in vitro rate of incorporation of [3H]proline into collagen hydroxyproline by lung minces. These elevations are all statistically significant (PC 0.01, one-tailed Student’s t test). When the biochemical results are examined in greater detail, several further generalizations emerge. At all the exposure intervals studied, that is, 1, 2, and 3 weeks, the response of the rats to ozone as evaluated by collagen synthesis rates of their lung minces was related to the level of ozone to which they were exposed. As will be documented below, this response was a linear function of the ozone level. At 0.5 ppm of ozone, the lowest concentration tested in this study, there was a significant biochemical response in these rats
after 1, 2, and 3 weeks of exposure. The magnitude of the response at all ozone levels
RAT
Ozone concentrotion,parts
IOr
LUNG
per million
C
Ozone
concentration,
palh
per million
COLLAGEN
METABOLISM
251
(expressed as percentage of control value) seemed to be greater after 2 weeks of exposure than after 1 week; values were very similar after 2 and 3 weeks of exposure. The highest levels of stimulation of collagen synthesis rate observed are about threefold higher than the control values (after exposure to 2 ppm for 7 days or to 1.5 ppm for 14 and 21 days). In parallel with the measured increase in collagen synthesis rate by the lung slices was an increase in the lesion score in alveolar ducts (Fig. 2). While the lesion score and the collagen synthesis rates are not directly comparable parameters, at least in quantitative terms, the correspondence between these values is striking at all times plotted (Fig. 2). Microscopic lesions were present in all animals exposed to ozone. Such lesions were always present in proximal portions of included alveolar ducts, and sometimes distalmost portions of terminal bronchioles and closely associated interalveolar septa. The lesions induced in rats exposed to 0.82.0 ppm ozone were characterized by moderately diffuse to focal, polypoid thickening of alveolar duct walls and associated interalveolar septa with corresponding narrowing of the ducts and alveoli (Figs. 3 and 4). Thickened’walls contained elongated, spindleshaped cells which were interpreted to be fibroblasts. Between the fibroblasts was fibrillar eosinophilic material of variable density, as well as nonstaining areas of edema fluid. The fibrillar material stained positively with -Gomori’s reticulin method (Fig. 5) and with Van Gieson’s stain for FIG. 2. Biochemical and histological responses of rat lungs to exposure of rats in vivo to various concentrations of ozone for (A) 7, (B) 14, or (C) 21 days. Biochemical data are expressed as the percentage of control value (nmol of [3H]hydroxyproline synthesized per hour per gram of protein) of collagen biosynthesis rate by lung minces (see Methods). Histologic responses are expressed as net lesion score (see Methods). Open bars, histological lesion scores; hatched bars, collagen synthesis rates.
252
LAST,
GREENBERG,
collagen. Small numbers of mononuclear inflammatory cells and occasional neutrophils were also present in the thickened walls. The most consistent thickening of bronchiolar and alveolar duct walls by connective tissue was observed at concentrations higher than
AND
CASTLEMAN
0.8 ppm. The degree of alveolar ductal wall thickening generally decreased with increased length of exposure. This decreased thickness was associated with smaller numbers of spindle-shaped cells (fibroblasts) and with increased density of reticulin or collagen
FIG. 3. (A) Lung from a control rat. A terminal bronchiole (TB) opens into alveolar ddcts (AD). H and E stain. x 160. (B) Lung from a rat exposed to 1.5 ppm ozone for 1 week. There are polypoid and band-like thickenings of alveolar duct walls by connective tissue (arrowheads), with partial occlusion’ of ducts. H and E stain. x 160.
RAT
LUNG
COLLAGEN
METABOLISM
FIG. 4. Polyp in alveolar duct of a rat exposed to 0.8 ppm ozone for 1 week. Spindle-shaped fibroblasts (arrow) are in. the polyp, together with small mononuclear inflammatory cells and occasional neutrophils. The alveolar duct contains aggregates of macrophages and a few neutrophils. H and E stain. x 400.
FIG. 5. Serial section of the field shown in Fig. 4, visualized ,with Gomori’s reticulin stain. Many black reticulin fibers (arrows) are in the polyp. x 400.
253
254
LAST,
GREENBERG,
fibers as demonstrated with Gomori and Van Gieson stains. Minimal or no thickening of walls or evidence of fibrosis was present after exposure of rats to 0.5 ppm for 7 days, although lesions in alveolar ducts characterized by intraluminal and interstitial aggregates of macrophages and other mononuclear cells and increased numbers of cuboidal epithelial cells ,were observed. In rats exposed to 0.5 ppm for 14 and 21 days, there was sometimes minimal thickening of alveolar duct walls with mildly increased amounts of reticulin and collagen. Although the animals purchased for this study were designated as chronic respiratory disease-free, mild to moderate pneumonia was observed in 43% of the ozone-exposed and 12% of the control animals. This pneumonia was characterized by aggregations of mononuclear inflammatory cells and neutrophils in peribronchiolar connective tissue, in closely associated interalveolar septa, and in perivascular connective tissue. Bronchiolar epithelial hyperplasia was also observed. Finally, we have performed initial experiments on the reversibility of the effects observed in the collagen synthesis assay after removal of rats from the exposure chambers. In such studies performed on groups of rats exposed to 1.5 ppm of ozone for 14 or 20 days, then allowed to recover breathing filtered air’ for 6 additional days, we found collagen synthesis rates that were intermediate (39 and 14%, respectively, of the total ozone-induced increase) between the values for the control and the corresponding exposed (without recovery) groups. The observed lesion scores correlated with these decreased synthesis rates. Hence, these biochemical and h&tological changes in rate of lung collagen biosynthesis and lung inflammatory response may be, at least in part, reversible upon removal of the ozone. DISCUSSION In this paper, we have documented that exposure of rats to ozone results in focal
AND
CASTLEMAN
pulmonary fibrosis, mainly in proximal alveolar duct walls. Quantitative biochemical and semiquantitative histological methods were used in this study to determine the relationship between dose and ozone-induced fibrosis. These two types of methodologies complement one another, thus allowing very different aspects of the same process to be studied. The rationale for equating hydroxyproline and collagen synthesis rates in lung minces has been thoroughly discussed elsewhere (Bradley et al., 1974; Greenberg et al., 1978a). The biochemical determination of collagen biosynthesis rate by lung minces is a measurement of the rate at which the lung was making collagen at the instant the animal was killed and, thus, is a measurement that might be expected to rapidly change during the ongoing pathological processes. For example, we have shown elsewhere (Greenberg et al., 1978b) that paraquat, a potent fibrogenic agent, causes rapid changes in this parameter. The magnitude of the changes observed with ozone (Fig. 2) are greater than the stimulation in collagen synthesis rate observed 2 days after administration of high doses of paraquat, but less than the stimulation seen 3 days after injection of paraquat (Greenberg et al., 1978b). On the other hand, the histological determination of a lesion score is a measurement of the extent of accumulation of connective tissue components in proximal alveolar duct walls and is, thus, an assay that might be expected to change relatively slowly during the ongoing pathological processes. The biochemical techniques allow a response of the lung to ozone exposure to be quantitated relatively precisely. However, these measurements might not necessarily be a reliable indicator of lung damage resulting in fibrosis. Increased collagen synthesis rates might alternatively be the result of a nonpatho: logical repair mechanism. The histological observations, on the other hand, unequivocally indicate that there is fibrosis or focal accumulation (synthesis) of collagen as a result of damage to the lung. We might
RAT
LUNG
COLLAGEN
anticipate that the biochemical techniques would be more sensitive indicators of changes in collagen metabolism than are the histological, especially after short-term exposures of rats to relatively low concentrations of ozone. In fact, in the rats sampled after exposure to 0.5 ppm of ozone (Fig. 2), the biochemical determination of collagen synthesis rate is a seemingly more sensitive assay for ozone-induced changes in pulmonary collagen metabolism than is the histological lesion score at the alveolar duct level. After only 1 week, increased levels of collagen synthesis are detected. However, not until the second week is there histologically discernible fibrosis. The same temporal relationship of increased collagen synthesis rate preceding a histologically detectable increase in collagen was probably not observed at higher ozone levels in this study since the response is more severe and probably proceeds more rapidly. It should be pointed out that inflammatory lesions in proximal alveolar ducts were easily recognized in the rats exposed to 0.5 ppm of ozone for 7 days, even though unequivocal thickening of the alveolar duct walls and fibrosis was not observed. Focal fibrosis of walls of bronchioles and alveolar ducts has been demonstrated to occur as a result of exposure to ozone at levels of about 1.0 ppm or above in rats, guinea pigs, and dogs (Freeman et a/., 1973, 1974; Stokinger et al., 1957). The pathogenic mechanisms involved in the induction of this focal fibrosis are poorly understood. In the present study an attempt to semiquantitatively assess ozone-induced fibrosis was made using a lesion scoring system based on several factors. Although local edema and inflammatory cell infiltration at least minimally contribute to the score, the main components of the score (increased amounts of collagen and reticulin fibers and increased numbers of spindle-shaped fibroblasts) are generally accepted morphologic criteria of fibrosis. There was a dose-response in the severity of focal fibrosis as expressed in the lesion score (Fig. 2). The lesion score values
METABOLISM
255
paralleled the ozone-induced increases in rate of collagen synthesis measured biochemically. Histologic changes compatible with mild viral pneumonia were present in a large percentage of the control and ozone-exposed animals in this study (Horsfall and Hahn, 1940; Robinson et al., 1968). Serologic studies were not done on the animals in this study. Serologic analysis of other rats from the same supplier during the experimental time period, however, revealed elevated antibody titers to pneumonia virus of mice (hemagglutination inhibition) and to Sendai virus (complement fixation). Endemic infection of the rats used in this study by one of these viruses was likely. Pneumonia of similar severity was observed in control and ozone-exposed rats, although a greater percentage of the exposed rats had pneumonia. Within all exposure groups ozone-induced lesions usually occurred independently of pneumonic changes and were almost always easily distinguishable from the suspected viral-induced lesions. The lesions in alveolar ducts of rats from the same ozone-exposed groups were of closely comparable severity regardless of whether or not the rat had detectable pneumonia. Although it is highly unlikely that a virus infection was inducing the alveolar duct lesions observed in this study, the possibility that viral-induced damage had an additive or synergistic effect on ozone-induced fibrosis of aIveolar duct walls cannot be excluded. The reproducibility of the observed effects in the biochemical assays (see, for example, Fig. 1) also argue against the changes in collagen metabolism seen in this study being caused by anything other than the ozone exposure per se. In addition, Hussain et al. (1976) have previously presented evidence that collagen synthesis rates in the lung in uivo are elevated after exposure of rats to 0.8 ppm of ozone, suggesting that the present results are not observed solely because of the techniques used or the specific population of rats studied.
256
LAST,
0
0.2
GREENBERG,
0.4
AND
0.8 Ozone
concentration,
CASTLEMAN
1.2 parts
1.6
I
2.0
per million
FIG. 6. Collagen biosynthesis rates of rat lung minces as a function of concentration of ozone to which the rats had been exposed in oivo. Lines are fitted to the data by linear regression analysis (least-squares program). O-0, 7-day exposures (r2 = 0.79); l - - -0, 1Cday exposures (r2 = 0.98); A- ---A, 21-day exposures (r’ = 0.95). Apparent “threshold” values are determined by linear extrapolation to zero effect levels (i.e., values of 100 % of control).
The biochemical data obtained in this study may also be used to calculate the theoretical “threshold levels” of ozone required to elicit stimulation of collagen synthesis rate in rat lungs after exposure for 1, 2, or 3 weeks, as shown in Fig. 6. The assumption made for the purposes of these calculations was that the dose-response curve remains linear for. all concentrations of ozone, including extrapolations’ to lower levels than those actually tested. Such linear extrapolations probably overestimate the actual level at which minimal effects might occur (Barth et al., 1971; Committee on Medical and Biological Effects of Environmental Pollutants, 1977). The minimal effective concentrations of ozone thus calculated range from about 0.1 to 0.2 ppm, concentrations actually commonly encountered during pollution episodes in the Southern California air basin (Committee on Medical and Biological Effects of Environmental Pollutants, 1977), and close to the ambient air quality standard for ozone (0.08 ppm) currently in force (Altshuller, 1975). The 7- and 1Cday exposures, for which we have the
most data, extrapolate to values of 0.13 and 0.10 ppm, respectively, for no-effect levels in this assay. It is important to emphasize that this “threshold level” at least in part reflects the detection limit for a specific assay. Thus, the limiting factor in the calculation of this “threshold” may be in the assumption of linear response to extrapolated values or in the “noise” level of the assay, which included factors such as the sensitivity of detection of differences in lung mince [3H]hydroxyproline levels and the biological variability of individual rats. Conversely, the limiting factor may be in the “signal” itself, i.e., the inherent capacity of the rat lung to respond to insult by low levels of ozone by increasing its collagen synthesis rate independently of other parameters that might be affected. In any event, this calculated threshold level of ozone must be viewed as either a potential underestimation or overestimation, of the levels of ozone that can provoke changes in collagen metabolism by rat lungs upon continuous exposure to these levels of ozone for the durations studied herein. More sensitive histological (ultrastructural
257
RAT LUNG COLLAGEN METABOLISM
morphometry) and biochemical (larger numbers of rats, lower levels of ozone exposure, comparisons of collagen synthesis and degradative rates) methods must be applied to these questions to more precisely define the lowest levels of ozone exposure that provoke pulmonary fibrosis. We believe the methods described here could easily, effectively, and productively be applied toward evaluating other fibrosisinducing pneumotoxins. Threshold levels of known pneumotoxins could be determined in this way, as well as the potential damaging effects (on lung collagen metabolism) of putative pneumotoxins. By using both the histological and biochemical techniques described here, the ongoing damage as well as that already produced can be monitored. While we believe the biochemical measurements to be more convenient and sensitive for quantitative evaluations, the histological studies allow conclusions to be drawn as to whether or not true pulmonary damage, more specifically pulmonary fibrosis, is being measured. Since preparation of this manuscript, the ambient air quality standard for ozone has been raised by the EPA from 0.08 ppm to 0.12 ppm. ACKNOWLEDGMENTS We thank June Wong for excellent technical assistance in performing the biochemical assays. This work was supported in part by National Institutes of Health Grants ES-00628 and ES-01713, and by a grant from the American Lung Association.
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ALTSHULLER, A. P., AND HORTON, R. J. M. (1971). Discussion [of national ambient air quality standards]. J. Air PolIut. Co&r. Assoc. 21, W-548. BRADLEY, K. H., MCCONNELL, S. D., AND CRYSTAL, R. G. (1974). Lung collagen composition and synthesis. J. Biol. Chem. 249, 26742683. Committee on Medical and Biological Effects of Environmental Pollutants (1977). Ozone and Other Photochemical Oxidants, pp. 126194. Nat. Acad. Sci., Washington, D.C. FREEMAN, G., JUHOS, L. T., FIJRIOSI, N. J., MUSSENDEN, R., STEPHENS,R. J., AND EVANS, M. J. (1974). Pathology of pulmonary disease from exposure to interdependent ambient gases (nitrogen dioxide and ozone). Arch. Environ. Health 29, 203-210. FREEMAN, G., STEPHENS, R. J., COFFIN, D. L., AND STARA, J. F. (1973). Changes in dogs’ lungs after long-term exposure to ozone. Arch. Environ. Health 26, 209-216. FULMER, J. D., AND CRYSTAL, R. G. (1976). The biochemical basis of pulmonary function. In The Biochemical Basis of Pulmonary Function (R. G. Crystal, ed.), pp. 419-466. Dekker, New York. GREENBERG, D. B., LYONS, S. A., AND LAST, J. A. (1978a). Paraquat-induced changes in the rate of collagen biosynthesis of rat lung explants. J. Lab. Clin. Med. 92, 1033-1042. GREENBERG, D. B., REISER, K. M., AND LAST, J. A. (1978b). Correlation of biochemical and morphologic manifestations of acute pulmonary fibrosis in rats administered paraquat. Chest 74, 421425. HINNERS, R. G., BURKART, J. K., AND PUNTE, C. L. (1968). Animal inhalation exposure chambers. Arch.
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HORSFALL, F. L., JR., AND HAHN, R. G. (1940). Latent virus in normal mice capable of producing pneumonia in its natural host. J. Exp. Med. 71, 391-408. HUSSAIN, M. Z., CROSS,C. E., MUSTAFA, M. G., AND BHATNAGAR, R. S. (1976). Hydroxyproline contents and prolyl hydroxylase activities in lungs of rats exposed to low levels of ozone. Life Sci. 18, 897-904. JUVA, K., AND PROCKOP, D. J. (1966). Modified procedure for the assay of 3H- or V-labeled hydroxyproline. Anal. Biochem. 15, 77-83. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A. LAST, J. A., JENNINGS,M. D., SCHWARTZ, L. W., AND CROSS, C. E. (1977). Glycoprotein secretion by tracheal explants cultured from rats exposed to ozone. Amer. Rev. Resp. Dis. 116, 695-703. 1 LUNA, L. G. (1968). Manual of Histologic Staining Methods
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