Long-term consequences of exposure to ozone: I. Lung collagen content

Lung collagen content of rats and monkeys (Macaca fascicularis) exposed to ozone for 1 to 13 weeks and for 1 year, respectively, was quantified by measurement of 4-hydroxyproline in hydrolysates of whole lungs. In addition, ratios of type I to type III collagen in the lungs of the same monkeys were also evaluated by cyanogen bromide peptide mapping techniques. We observed elevated levels of collagen in lungs of both species of animals exposed to ozone. We conclude that elevations in collagen synthesis rates in lungs of rats and monkeys acutely exposed to high levels of ozone are reflected by corresponding increases in lung collagen content over subchronic and chronic time frames. Preliminary results on young rats also suggest that removal of rats from atmospheres containing ozone does not cause reversal of such increases in lung collagen content. To the contrary, recovery periods of up to 6 weeks seem to exacerbate the observed increases in lung collagen content.     

Ozone-induced alterations in collagen metabolism of monkey lungs: Use of biopsy-obtained lung tissue

Open-chest biopsies of lung were performed on three cynomolgus monkeys (Macaca fascicularis) 4–6 weeks before and immediately after exposure to 1.5 ppm of ozone for 1 week. Two control monkeys were subjected to comparable procedures, but were exposed to filtered air for 1 week immediately prior to the second biopsy. Collagen synthesis rate of each lung biopsy specimen was determined. Enhanced rates of collagen synthesis were observed in the lung biopsy specimens obtained from the monkeys exposed to ozone; elevated rates of collagen synthesis were not observed in lung biopsy specimens from the two monkeys undergoing identical surgical procedures but not exposed to ozone. We conclude that exposures of monkeys to high levels of ozone caused increased rates of collagen synthesis in their lungs, suggesting that the ozone-induced fibrotic changes observed previously in rat lungs also occur in lungs anatomically similar to those of humans. This experimental design, in which each animal serves as its own control, is a practical way to perform such exposure experiments using small numbers of valuable animals.     

Residual effects of polybrominated biphenyls following perinatal exposure in rats

Rats were exposed to 0.5 ppm of ozone delivered for 23.5 hr per day for up to 180 days. One group of rats was allowed to breathe filtered air for about 2 months after the conclusion of the exposure. Lung collagen and total-protein synthesis rates were quantitated by biochemical analyses performed with lung minces. Other lung lobes from the same rats were used to quantitate collagen and total protein content. Increased levels of lung protein and of lung collagen (hydroxyproline) content were observed at all times sampled during exposure to ozone (3, 30, 50, 88, 180 days); the observed lung hydroxyproline content at 180 days persisted during the 2 months of postexposure recovery. Collagen synthesis rates measured in lung minces were elevated in the exposed rats at all times sampled, consistent with the observed increased lung collagen content seen throughout the study. The observed biochemical changes were consistent with concurrent morphological observations of the occurrence of mild pulmonary fibrosis.     

Lung collagen and elastin after ozone exposure in vitamin B-6-deficient rats

The effects of vitamin B-6 deficiency and ozone exposure on selected features of connective tissue metabolism in lung were investigated in groups of weanling male rats fed one of three diets: B-6-supplemented, fed ad lib; B-6-deficient, fed ad lib; or B-6-supplemented, restricted to the food intake of deficient rats for 5 weeks. Also, perinatal rat pups were studied that were nursed from dams fed one of the 3 diets from parturition to day 15 of lactation. During the final week of each experiment, half of the rats in each of the groups were exposed to 0.64 ppm of ozone (23.5 h per day). The collagen and elastin content, collagen synthesis rate, total protein synthesis rate, and lysyloxidase activity of lungs were measured. Perinatal pups rendered vitamin B-6-deficient were particularly sensitive to ozone exposure (65% died as compared to fewer than 5% of the ad lib or food-restricted controls). When L-proline incorporation into collagen and total protein was investigated using lung minces, food restriction and B-6-deficiency resulted in about one-half the incorporation normally observed. Total lung lysyl oxidase activity was also decreased in B-6-deficient and food-restricted rats compared to B-6-supplemented rats fed ad lib. Exposure to ozone resulted in increased lysyl oxidase activity and collagen synthesis in lungs from B-6-supplemented rats, but such responses were not observed in B-6-deficient or food-restricted (FR) rats exposed to ozone.     

Synergistic interaction of ozone and respirable aerosols on rat lungs. II. Synergy between ammonium sulfate aerosol and various concentrations of ozone

Pulmonary responses after continuous exposure of rats to concentrations of ozone (O3) ranging from 0.12 to 0.64 ppm were quantified by measuring tissue collagen synthesis rate, tissue protein and DNA content, and various constituents of bronchoalveolar lavage fluid. After 7 days of exposure to 0.64 ppm of O3, lung collagen synthesis rate and tissue content of protein and DNA were elevated. After shorter durations of exposure to 0.64 ppm of O3, significant elevations were observed in the protein content and the activities of lactate dehydrogenase, acid phosphatase, and N-acetyl-beta-D-glucosaminidase from lavage fluid. After exposure of rats to 0.20 ppm of O3 for 7 days, changes could be detected in both lung collagen synthesis rate and tissue protein content. Total lavagable protein content, a sensitive indicator for O3-induced effects upon the lung, was significantly elevated in lungs of rats exposed to 0.12 or 0.20 ppm of O3. To examine whether a synergistic interaction occurred between 0.20 or 0.64 ppm of O3 and acid aerosols, rats were continuously exposed to O3 with and without concurrent exposure to 5 mg/m3 of ammonium sulfate. A synergistic interaction between 0.20 ppm of O3 and ammonium sulfate aerosol was observed by measurement of total lavagable protein and of lung collagen synthesis rate. These results demonstrate that ammonium sulfate aerosol interacts synergistically with O3 at concentrations of O3 that approach ambient levels.     

The effects of chronic ozone exposure on pulmonary collagen content and collagen synthesis in rats

Male rats were exposed to 0.125, 0.25 or 0.5 ppm of ozone or clean air for up to 1 year. During this exposure period there was little evidence for collagen accumulation in the lungs. However, the rate of incorporation of tritiated proline into both lung collagen and total lung protein was accelerated. These data suggest that exposure to ozone under these conditions results in an increase in the turnover of collagen as well as other lung proteins.     

LUNG COLLAGEN TYPES ARE ALTERED IN RATS CHRONICALLY EXPOSED TO OZONE

A new method has been developed for the quantitative determination of collagen types I and III in the lung, using CNBr digestion to solubilize peptides from these collagens, followed by quantification by an enzyme-linked immunosorbent assay (ELISA) using a polyclonal goat antibody against the 1(I)-CB3 peptide. Collagen type ratios were determined for lung tissue from normal rats and from animals that had been chronically exposed to ozone for durations of exposure of either 90 days or 20 mo. Increases in the relative ratio of type I:III collagen were demonstrable in the lungs of rats exposed for 5 days/wk, 6 h/day, to ozone for 20 mo, even at concentrations of ozone as low as 0.12 ppm.     

Synergistic interaction of ozone and respirable aerosols on rat lungs. III. Ozone and sulfuric acid aerosol

Previously we have demonstrated that a synergistic interaction, as evaluated by several biochemical, toxicological, and morphological responses of the lung, results from exposure of rats to ozone (O3) in conjunction with moderate concentrations of acidic, but not neutral, aerosols. To extend these studies, groups of rats were continuously exposed to either O3 or sulfuric acid aerosol alone, or to combinations of these pollutants. Pulmonary responses from these rats were measured by assay, after exposures for 6 hr to 7 days, of total lavageable protein content, total lung tissue protein content after 5, 7, or 9 days of exposure, or apparent collagen synthesis rates from lung tissue after 7 days of exposure. While the lavageable protein content from rats exposed for 3 days to 0.1 or 1.0 mg/m3 of sulfuric acid aerosol alone was not different from control values, significant elevations from control values were observed from groups exposed to 0.12, 0.20, or 0.64 ppm of O3. Synergy was demonstrated by this assay upon exposure of rats to 0.20 ppm of O3 in conjunction with 0.1, 0.5, or 1.0 mg/m3 of sulfuric acid aerosol. Similarly, the tissue protein content from rats exposed to 0.1 or 1.0 mg/m3 of sulfuric acid aerosol alone was indistinguishable from control values. Significant elevations from control values were observed by this assay from groups of rats exposed to 0.64 or 0.20 ppm of O3, and a synergistic interaction was demonstrated between 0.64 ppm of O3 and 1.0 mg/m3 of sulfuric acid aerosol. Furthermore, synergy was observed by quantification of increased total lung protein between 0.20 ppm of O3 + 40 micrograms/m3 and higher concentrations of sulfuric acid aerosol. Values of the lung collagen synthesis rate from rats exposed to 0.1, but not 1.0, 0.5, or 0.04 mg/m3 of sulfuric acid aerosol were significantly higher than values from lungs of control animals. Significant elevations from control values were also observed by this assay from groups of rats exposed to 0.64 or 0.20 ppm of O3. A synergistic interaction was demonstrated by the collagen synthesis rate assay between groups of rats exposed to 0.64 ppm of O3 + 0.20 mg/m3 and higher concentrations of sulfuric acid aerosol or between groups exposed to 0.20 ppm of O3 + 40 micrograms/m3 and higher concentrations of sulfuric acid aerosol. These results demonstrate synergy between O3 and sulfuric acid aerosol upon exposure to concentrations of each pollutant at or near peak hourly ambient levels in polluted urban atmospheres.     

Effects of chronic exposure to ozone on collagen in rat lung

Pulmonary fibrosis is a consequence of severe injury from some toxic agents including high doses of ozone. It is not known, however, whether chronic exposure to low doses of ozone, such as those encountered in polluted ambient atmospheres, could also result in abnormal accumulations of lung collagen. Rats were exposed to ozone for 20 hr per day, 7 days per week for 3, 6, 12, and 18 months at concentrations of 0.12, 0.25, or 0.50 ppm. Controls were exposed under identical conditions to purified air. Upon removal from the chambers, rats were euthanized and lung tissue slices incubated with [14C]proline. The incorporation of 14C into hydroxyproline and the total hydroxyproline content of lung tissue were measured as estimates of lung collagen synthesis and content, respectively. The formation of labeled hydroxyproline tended to decrease significantly with time in controls and at the three ozone doses. There were, however, no significant dose-related changes at any of the time points tested. Total lung hydroxyproline increased with age in all groups, but no dose-related changes were detected at any time point. It was concluded that chronic exposure of rats to ozone at concentrations which approximate ambient urban concentrations did not affect normal age-related changes in either synthesis or accumulation of lung collagen.     

Long-term consequences of exposure to ozone: II. Structural alterations in lung collagen of monkeys

The effects of chronic exposure to ozone on lung collagen crosslinking were investigated in two groups of juvenile cynomolgus monkeys exposed to 0.61 ppm of ozone 8 hrs per day for 1 year. One group was killed immediately after the exposure period; the second exposed group breathed filtered air for 6 months after the ozone exposure before being killed. Previous studies of these monkeys had revealed that lung collagen content was increased in both exposed groups (J. A. Last et al., (1984). Toxicol. Appl. Pharmacol. 72, 111–118). In the present study specific collagen crosslinks were quantified in order to determine whether the excess collagen in the lungs of these animals was structurally normal or abnormal. In the group killed immediately after exposure, the difunctional crosslink dehydrodihydroxylysinonorleucine (DHLNL) was elevated, as was the ratio of DHLNL to dehydrohydroxylysinonorleucine (HLNL). Lung content of the mature nonreducible crosslink hydroxypyridinium was also increased in this group. In the group killed after a 6-month postexposure period, lung content of the difunctional crosslinks DHLNL and HLNL was indistinguishable from control values. However, lung hydroxypyridinium content was significantly increased. The changes in collagen crosslinking observed in the group killed at the termination of exposure are characteristic of those seen in lung tissue in the acute stage of experimental pulmonary fibrosis. The changes seen in the postexposure group suggest that while the lung collagen being synthesized at the time the animals were killed was apparently normal, “abnormal” collagen synthesized during the period of ozone exposure was irreversibly deposited in the lungs. This study suggests that long-term exposure to relatively low levels of ozone may cause irreversible changes in lung collagen structure.     

Age-related morphometric differences in responses of rat lungs to ozone

The influence of age on morphologic changes in lungs of rats exposed to ozone was studied in female Sprague-Dawley rats, aged 60 and 444 days. Rats of both age groups were exposed continuously for 72 hr to either 0.35 or 0.80 ppm ozone, or to filtered air. Tissues were evaluated using light microscopic morphometry and scanning electron microscopy. The lungs from ozone-exposed 60-day-old rats had larger volume fractions of centriacinar lesions than lungs from exposed 444-day-old rats. Within each age group there was an observed dose response, with rats exposed to 0.80 ppm ozone having larger volume fractions of lesions than those exposed to 0.35 ppm. Only the 444-day-old rats lost body weight during the exposure period. They also had smaller fixed lung volumes than same-aged controls. All 60-day-old rats gained weight during the exposure period, although rats exposed to 0.80 ppm ozone gained less than filtered air controls. Lesions observed in both age groups of female rats were qualitatively similar to those previously described in young adult male rats. We conclude that there are age-related differences in the morphometric responses of rats to ozone exposure. Younger rats had larger proportions of centriacinar lesions and macrophages while older rats had greater body weight and lung volume changes.     

Application of the EPR spin-trapping technique to the detection of radicals produced in vivo during inhalation exposure of rats to ozone.

Ozone is known to induce lipid peroxidation of lung tissue, although no direct evidence of free radical formation has been reported. We have used the electron paramagnetic resonance (EPR) spin-trapping technique to search for free radicals produced in vivo by ozone exposure. The spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was administered ip to male Sprague-Dawley rats. The rats were then exposed for 2 hr to either 0, 0.5, 1.0, 1.5, or 2.0 ppm ozone with 8% CO2 to increase their respiratory rate. A six-line 4-POBN/radical spin adduct signal (aN = 15.02 G and a beta H = 3.27 G) was detected by EPR spectroscopy in lipid extracts from lungs of rats treated with 4-POBN and then exposed to ozone. Only a weak signal was observed in the corresponding solution from rats exposed to 0 ppm ozone (air with CO2 only). The concentration of the radical adduct increased as a function of ozone concentration. After administration of 4-POBN, rats were exposed for either 0.5, 1.0, 2.0, or 4.0 hr to either 0 or 2.0 ppm ozone (with CO2). The radical adduct concentration of the ozone-exposed groups at exposure times of 2.0 and 4.0 hr was significantly different from that of the corresponding air control groups. A correlation was observed between the radical adduct concentration and the lung weight/body weight ratio. These results demonstrate that ozone induces the production of free radicals in rat lungs during inhalation exposure and that radical production may be involved in the induction of pulmonary toxicity by ozone. This is the first direct evidence for ozone-induced free radical production in vivo.     

Ozone exposure, food restriction and protein deficiency: changes in collagen and elastin in rodent lung

Two groups of weanling or young adult rats were fed ad lib casein-based diets containing 4 or 16% protein. Food was restricted in a third group (fed the 16% protein diet) to the amount consumed daily by rats (adult or weanlings) fed the 4% diet. After 3 weeks (weanlings) or 1, 3 or 5 weeks (adults), one-half of the rats in each group were exposed to 0.64 ppm (1.28 mg/m3) of ozone for 7 days (23.5 h each day). Several parameters were then evaluated related to lung connective tissue metabolism including: (1) total lung hydroxyproline, (2) total lung elastin, (3) apparent rates for lung collagen synthesis and elastin accumulation and (4) lung and body weights. In general, the response to protein deficiency and food restriction was more pronounced than to ozone exposure. Protein deficiency and food restriction resulted in decreased lung size and collagen content. However, the ability of lung to respond to ozone (in relative terms) was not altered by changes in diet as assessed by changes in lung weight or the collagen synthetic rate.     

Glutathione pathway enzyme activities and the ozone sensitivity of lung cell populations derived from ozone exposed rats

Rats were exposed to 1.5 +/- 0.1 mg ozone/m3 for 4 days and the activities of glucose-6-P dehydrogenase (G6PDH), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were measured in the cytosolic fraction of lungs from exposed and control rats. Enzyme activities were also measured in isolated alveolar macrophages and type II cells. After ozone exposure enzyme activities, expressed per gram of protein, showed the following changes. G6PDH activity was increased (P less than 0.001) in the whole rat lung and showed the same tendency in isolated alveolar macrophages and type II cells. GR activity did not change significantly neither in whole lungs, nor in isolated cell populations. GSHPx activity was increased (P less than 0.001) in whole lung homogenates, and was also markedly increased in both alveolar macrophages (P less than 0.05) and type II cells (P less than 0.01) isolated from ozone-exposed rats. From these results it was concluded that biochemical changes measured in whole lung homogenates might reflect biochemical changes that occur within specific cell types. Furthermore, it was demonstrated, using an in vitro ozone exposure system, that lung cell populations isolated from ozone-exposed rats, in spite of their marked increase in GSHPx activity, did not show a decreased ozone sensitivity compared to cells from unexposed rats, as determined by trypan blue exclusion or phagocytosis. So an increase in GSHPx activity might not be related to an increased cellular resistance to ozone.     

Alveolar permeability to protein in rats differentially susceptible to ozone

Sprague-Dawley rats susceptible (DS) to NaCl-induced hypertension suffer higher mortality when exposed daily to 2.0 ppm ozone than do hypertension-resistant (DR) rats, independent of salt in the diet or systemic blood pressure. To investigate one possible contribution to this differential sensitivity to ozone, alveolar permeabilities to serum albumin were measured both in ozone-exposed and in control DS and DR rats. Female rats aged 5-7 weeks maintained on a low-salt (0.4% NaCl) diet were injected intravenously with 125I-bovine serum albumin and were then exposed to either 2.0 ppm ozone or air for 5 h. After pentobarbital anesthesia, the rats were exsanguinated and their lungs were lavaged in situ with saline. Lavage fluids and blood samples were measured for radioactivity using a NaI-well gamma counter. The results indicated that while DS and DR control rats have similar pulmonary permeabilities to 125I-albumin, the lungs of the ozone-exposed DS animals were 63% (p less than 0.02) more permeable than those of DR rats exposed to ozone. Sloughing of epithelial tissue, mucous formation and an accumulation of macrophages in the end-airways were more pronounced among ozone-exposed DS animals than in DR-ozone-exposed rats. This increased damage among DS rats correlated well with the increased protein permeability levels. In similar studies, Sprague-Dawley (D) rats were more variable in their response to ozone than either inbred strain. However, the results appeared generally more like those of the DS animals, suggesting that the trait selected by inbreeding may have been resistance rather than sensitivity to ozone-induced lung injury.     

Concentration-response relationships of rat lungs to exposure to oxidant air pollutants: a critical test of Haber's Law for ozone and nitrogen dioxide.

Exposure protocols were designed to ask whether lung damage in rats exposed to either ozone or nitrogen dioxide is proportional to dose rate or to cumulative dose. Thus, the response of rats to a constant product of concentration of oxidant air pollutant and time of exposure (C x T) was evaluated for 3-day exposures over a fourfold range of concentrations of ozone (0.2-0.8 ppm) or of nitrogen dioxide (3.6-14.4 ppm) for exposure durations of 6-24 hr per day. The response of rat lungs was quantified by changes in total protein content of lung lavage supernatants or by changes in content of specific cell types in lung lavage pellets. The results of these experiments clearly demonstrate that acute lung damage is a function of cumulative dose (that is, C x T product) for the three highest dose rates tested. However, when exposure duration is extended to include the entire 24-hr period (the lowest dose rate tested), there is a marked attenuation of pulmonary response. Rats were also exposed to mixtures of ozone and nitrogen dioxide with the C x T product held constant. Our results clearly demonstrate that when rats are exposed to combinations of ozone and nitrogen dioxide, lung damage is a function of peak concentration rather than a function of cumulative dose. This deviation from Haber's Law is attributed to a concentration-dependent, synergistic (greater than additive) response to this specific mixture of oxidant air pollutants.     

Method of measuring deposition of unlabeled monodisperse microspheres in rat lungs

A system to simultaneously measure the total deposition of four different sizes of monodisperse microspheres in normal and damaged lungs of rats was developed and tested. The system reproducibly measured the deposition of microspheres in control rats, and the procedure was shown to be sufficiently sensitive to measure ozone-induced changes in deposition rates. Rats exposed to 1.2 ppm ozone 6 h/d for 2 consecutive days showed greater deposition of the 1.09-micron-, 2.02-micron-, and 2.99-micron- but not of the 0.48-micron-diameter microspheres when compared to controls. After 8 consecutive days of exposure to the same concentration of ozone, there were no differences in deposition rates between control and ozone-exposed rats. Respiratory physiology and lung histopathology data provided evidence that subtle changes in the airway architecture and/or aerodynamics were likely to be responsible for the differential deposition rates as a function of the duration of ozone exposure.     

Comparison of acute ozone-induced nasal and pulmonary inflammatory responses in rats

The centriacinar pulmonary lesion induced by ozone has been extensively characterized, but little is known about the effects of this oxidant gas in the upper airways. The present study was designed to compare the effects of acute ozone exposure in the nose and lungs of rats. We examined the cellular inflammatory responses in the nasal cavity and lower respiratory tract by means of nasal and bronchoalveolar lavage and morphometric quantitation of neutrophils within the nasal mucosa and pulmonary terminal bronchioloalveolar duct regions (i.e., centriacinar). Rats were exposed to 0.0, 0.12, 0.8, or 1.5 ppm ozone for 6 hr and were sacrificed immediately or 3, 18, 42, or 66 hr following exposure. Eighteen hours after exposure, increased numbers of neutrophils, as compared to controls, were recovered from nasal lavage fluid (NLF) of rats exposed to 0.12 ppm ozone. There was no change in the number of neutrophils recovered from bronchoalveolar lavage fluid (BALF) at any time after exposure. Rats exposed to 0.8 ppm ozone had more neutrophils in NLF than controls immediately after exposure, but no concomitant increase in BALF neutrophils at that time. However, as the number of neutrophils in BALF increased (maximum at 42 hr), the number of neutrophils recovered from NLF decreased (minimum at 42 hr). Rats exposed to 1.5 ppm ozone had no significant increases in nasal neutrophils in NLF at any time after exposure but had greatly increased numbers of neutrophils in BALF 3, 18, and 42 hr after exposure. The number of neutrophils recovered by nasal and bronchoalveolar lavage accurately reflected the tissue neutrophil response at sites within the nasal cavity and lung that were injured by acute ozone exposure. Our results suggest that at high ozone concentrations (0.8 and 1.5 ppm), the acute nasal inflammatory response is attenuated by a simultaneous, competing, inflammatory response within the centriacinar region of the lung. Analysis of nasal lavage fluid for changes in cellular composition may be a useful indicator of acute exposure to ambient levels of ozone, but at higher ozone levels, the nasal cellular inflammatory response may underestimate the effects of ozone on nasal and pulmonary epithelia.     

Time study on development and repair of lung injury following ozone exposure in rats

The aim of this study was to investigate the time course of lung injury in rats during acute and subchronic ozone exposure and during postexposure recovery. Rats were continuously exposed to 0.4 ppm ozone ( approximately 0.8 mg O(3)/m(3)) for 1, 3, 7, 28, or 56 days. Recovery from 3 days of exposure was studied at day 7, 14, and 28; recovery from 7 days of exposure was studied at day 14, 28, and 56, recovery from 28 days of exposure was studied at day 35 and 56, and recovery from 56 days of exposure was studied at day 136. The study included a correlated biochemical and morphological analysis of inflammatory responses, structural changes, and collagen content. The acute inflammatory response, as measured by an increase of polymorphonuclear cells and plasma protein in bronchoalveolar lavage (BAL) fluid, reached a maximum at day 1 and resolved largely within 6 days during ongoing exposure. Numbers of macrophages in BAL fluid increased progressively up to day 56, and slowly returned to near control levels when exposure was followed by postexposure recovery. Histological examination and morphometry of the lungs revealed centriacinar inflammatory responses throughout ozone exposure. Centriacinar thickening of septa was observed at day 7. Ductular septa, thickened progressively at days 7, 28, and 56 of exposure, showed increased collagen upon exposure at day 28, which was further enhanced at exposure at day 56. Increased collagen content in lungs, as measured biochemically by hydroxyproline concentration, was observed at exposure day 56. Collagen content was not different from control at day 56 when 7 or 28 days of exposure was followed by postexposure recovery. After continuous ozone exposure, respiratory bronchioles were present in an increasing degree, and remained present after a recovery period. The results of this study clearly show that after continuous exposure to O(3) some acute effects, such as protein and albumin content, and neutrophil influx in BAL fluid, returned to control levels within a few days. However, other parameters, such as the alveolar macrophage response and structural changes such as the presence of terminal bronchioles, thickening of ductular septa by enhanced cellularity, and collagen formation, persisted or progressively increased during continued exposure. Postexposure recovery seems to partly resolve these subchronic responses (macrophages response, septal cellularity), whereas other effects (collagen increase and respiratory bronchioles formation) do not disappear.     

TIME STUDY ON DEVELOPMENT AND REPAIR OF LUNG INJURY FOLLOWING OZONE EXPOSURE IN RATS

The aim of this study was to investigate the time course of lung injury in rats during acute and subchronic ozone exposure and during postexposure recovery. Rats were continuously exposed to 0.4 ppm ozone (~0.8 mg O 3 /m 3) for 1, 3, 7, 28, or 56 days. Recovery from 3 days of exposure was studied at day 7, 14, and 28; recovery from 7 days of exposure was studied at day 14, 28, and 56, recovery from 28 days of exposure was studied at day 35 and 56, and recovery from 56 days of exposure was studied at day 136. The study included a correlated biochemical and morphological analysis of inflammatory responses, structural changes, and collagen content. The acute inflammatory response, as measured by an increase of polymorphonuclear cells and plasma protein in bronchoalveolar lavage (BAL) fluid, reached a maximum at day 1 and resolved largely within 6 days during ongoing exposure. Numbers of macrophages in BAL fluid increased progressively up to day 56, and slowly returned to near control levels when exposure was followed by postexposure recovery. Histological examination and morphometry of the lungs revealed centriacinar inflammatory responses throughout ozone exposure. Centriacinar thickening of septa was observed at day 7. Ductular septa, thickened progressively at days 7, 28, and 56 of exposure, showed increased collagen upon exposure at day 28, which was further enhanced at exposure at day 56. Increased collagen content in lungs, as measured biochemically by hydroxyproline concentration, was observed at exposure day 56. Collagen content was not different from control at day 56 when 7 or 28 days of exposure was followed by postexposure recovery. After continuous ozone exposure, respiratory bronchioles were present in an increasing degree, and remained present after a recovery period. The results of this study clearly show that after continuous exposure to O 3 some acute effects, such as protein and albumin content, and neutrophil influx in BAL fluid, returned to control levels within a few days. However, other parameters, such as the alveolar macrophage response and structural changes such as the presence of terminal bronchioles, thickening of ductular septa by enhanced cellularity, and collagen formation, persisted or progressively increased during continued exposure. Postexposure recovery seems to partly resolve these subchronic responses (macrophages response, septal cellularity), whereas other effects (collagen increase and respiratory bronchioles formation) do not disappear.