A comparison of terminal airway remodeling in chronic daily versus episodic ozone exposure

This study compares centriacinar changes by ultrastructural morphology and morphometry following daily versus episodic ozone exposure in rats. Three groups of rats were exposed to air, 0.95 ppm ozone 8 hr daily for 90 days, and 0.95 ppm ozone 8 hr daily in seven successive 5-day episodes separated by 9-day recovery periods for a total of 89 days. Sections from the left lung and dissected acini from the right middle lobe were studied by light and electron microscopy. The centriacinar lesion following episodic exposure was similar but diminished in severity compared to that of rats exposed daily. Damage following episodic exposure appeared to be more than predicted by an exposure regimen which delivered 35% of the total ozone dose during daily exposure. The total volume of affected parenchyma was similar following both exposures. Respiratory bronchiole formation increased following both exposures but this was only statistically significant following daily exposure. The most severe epithelial damage was at the tips of alveolar septa in alveolar ducts distal to the respiratory bronchiole. Interstitial thickness in the injured respiratory bronchiole and proximal alveolar duct increased significantly and similarly following both exposures. Epithelium along the respiratory bronchiole of daily exposed rats was more differentiated. In the episodic group, respiratory bronchiole and alveolar duct epithelium consisted of a range of intermediate, less differentiated bronchiolar or alveolar epithelial cells. The episodic exposure resulted in a diminished lesion, but there appears to be some cumulative effect of repeated exposures with respiratory bronchiolar and alveolar duct epithelium in a more dynamic state of injury and repair.     

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.     

Acute ozone-induced lung injury in neutrophil-depleted rats.

To test the hypothesis that neutrophils contribute to acute, ozone-induced epithelial damage in the lung, rats were depleted of their circulating neutrophils by intraperitoneal injection of a rabbit anti-rat neutrophil serum (ANS) 12 hr prior to an 8-hr exposure to 1.0 ppm ozone. Additional rats were given an injection of normal rabbit serum (NRS) prior to ozone exposure. Exposures were followed by postexposure periods in filtered air for 0, 4, or 16 hr. Control rats were given either ANS or NRS and then exposed only to filtered air. Analysis of bronchoalveolar lavage fluid (BALF) from NRS-treated rats revealed a significant increase in total neutrophils above that of controls at the 4- and 16-hr postexposure times, with a peak increase at 4 hr postexposure. In contrast, there was almost total ablation of the BALF neutrophil response in the ANS-treated rats at all times. Ozone caused an increase in BALF protein, fibronectin, and interleukin-6 above those in controls in both the NRS- and ANS-treated rats, but the only significant difference between the two groups was a level of fibronectin in the neutrophil-depleted animals higher than that in the neutrophil-sufficient animals at the 0-hr postexposure time. Electron microscopic morphometry on lungs fixed by intravascular perfusion demonstrated no significant differences in the volume per surface area epithelial basal lamina (Vs) of necrotic and degenerating epithelial cells in central acini between the neutrophil-depleted and neutrophil-sufficient animals. From these results, we concluded that neutrophils do not play a detectable role in contributing to the early epithelial damage in the lung caused by an acute exposure to ozone.     

Epithelial injury and interstitial fibrosis in the proximal alveolar regions of rats chronically exposed to a simulated pattern of urban ambient ozone.

Electron microscopic morphometry was used to study the development of lung injury during and after chronic (78 weeks) exposure to a pattern of ozone (O3) designed to simulate high urban ambient concentrations that occur in some environments. The daily exposure regimen consisted of a 13-hr background of 0.06 ppm, an exposure peak that rose from 0.06 to 0.25 ppm, and returned to the background level over a 9-hr period, and 2-hr downtime for maintenance. Rats were exposed for 1, 3, 13, and 78 weeks. Additional groups of rats exposed for 13 or 78 weeks were allowed to recover in filtered clean air for 6 or 17 weeks, respectively. Rats exposed to filtered air for the same lengths of time were used as controls. Samples from proximal alveolar regions and terminal bronchioles were obtained by microdissection. Analysis of the proximal alveolar region revealed a biphasic response. Acute tissue reactions after 1 week of exposure included epithelial inflammation, interstitial edema, interstitial cell hypertrophy, and influx of macrophages. These responses subsided after 3 weeks of exposure. Progressive epithelial and interstitial tissue responses developed with prolonged exposure and included epithelial hyperplasia, fibroblast proliferation, and interstitial matrix accumulation. The epithelial responses involved both type I and type II epithelial cells. Alveolar type I cells increased in number, became thicker, and covered a smaller average surface area. These changes persisted throughout the entire exposure and did not change during the recovery period, indicating the sensitivity of these cells to injury. The main response of type II epithelial cells was cell proliferation. The accumulation of interstitial matrix after chronic exposure consisted of deposition of both increased amounts of basement membrane and collagen fibers. Interstitial matrix accumulation underwent partial recovery during follow-up periods in air; however, the thickening of the basement membrane did not resolve. Analysis of terminal bronchioles showed that short-term exposure to O3 caused a loss of ciliated cells and differentiation of preciliated and Clara cells. The bronchiolar cell population stabilized on continued exposure; however, chronic exposure resulted in structural changes, suggesting injury to both ciliated and Clara cells. We conclude that chronic exposure to low levels of O3 causes epithelial inflammation and interstitial fibrosis in the proximal alveolar region and bronchiolar epithelial cell injury.     

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.     

Alveolar macrophage-stimulated neutrophil and monocyte migration: effects of in vitro ozone exposure

The ability of rabbit alveolar macrophages (AM) to release factors which stimulate the migration of peripheral blood neutrophils and monocytes was examined, and the influence of in vitro ozone exposure on this secretory activity was investigated. To evaluate the ability of AM to release leukocyte chemotactic activity, AM obtained by bronchoalveolar lavage were established in monolayer or suspension culture, with and without added zymosan, for 2 and 6 hr. The resulting macrophage-conditioned medium was tested for chemotactic activity using modified Boyden-type chambers and rabbit peripheral blood neutrophils or monocytes as the responding cells. The results demonstrate that substrate attachment (monolayer culture) and/or zymosan phagocytosis can stimulate AM to release chemoattractants for monocytes and neutrophils. Additionally, the results suggest that AM are constitutively producing low levels of monocyte chemotactic factors. The effects of in vitro ozone exposure on the secretion of chemotactic activity was investigated by exposing monolayer cultures of AM to air, 0.1, 0.3, or 1.2 ppm ozone for 2 hr. Macrophage-conditioned medium was harvested immediately, 2 and 6 hr postexposure, and tested for chemotactic activity. Exposure to 0.3 and 1.2 ppm ozone significantly increased the AM secretion of factors which stimulated neutrophil migration; additionally, the results strongly suggest that ozone can augment the ability of AM to stimulate monocyte migration. These results imply a role for the AM in the recruitment of inflammatory cells after ozone inhalation.     

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.     

Correlation between cumulative labeling indices measured in the terminal bronchioles and in the centriacinar region in the lungs of rats exposed to oxidant air pollutants

Male Sprague-Dawley rats were exposed on 7 consecutive days for 8 h a night to 0.6 ppm ozone, 10.8 ppm NO₂ or a mixture of the two gases. Cumulative labeling indices were measured in the epithelium of the terminal bronchioles and the adjacent centriacinar region. An excellent correlation in labeling indices between the two sites was found. It is concluded that the labeling index in the terminal bronchioles represents a sensitive measurement for ozone-induced lung injury.     

Cell Proliferation in the Respiratory Tract of Hamsters Continuously Exposed During 4 Weeks to 0.8 PPM Ozone

Abstract

Male Syrian golden hamsters were exposed to 0.8 ppm ozone, 24 h/day for 4 wk. Cumulative labeling indices were measured during each week of exposure in the lung, the intrapulmonary airways, the trachea, and the nasal passages. In the alveolar zone, ozone did not affect labeling indices beyond the centriacinar region. In the terminal bronchioles and large intrapulmonary airways, labeling indices were significantly higher than in controls throughout exposure to ozone. The largest increase was seen during wk I. In the trachea and the nasal passages, a significant increase in cell labeling was only seen during wk I; thereafter the values returned to control levels. It was concluded that exposure to 0.8 ppm ozone produces signs of proliferative activity during a 4-wk continuous exposure in the epithelium of the intrapulmonary airways, but not in other sites.     

Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein

Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O3). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.     

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.     

Repeated episodes of ozone inhalation attenuates airway injury/repair and release of substance P,but not adaptation

To determine the impact of repeated episodes of ozone exposure on physiologic adaptation, epithelial injury/repair, and tracheal substance P levels, adult rats were subjected to episodes of ozone (5 days, 1 ppm, 8 h/day) followed by 9 days of filtered air for four cycles. Rats were sampled on days 1 and 5 of each episode and 9 days after day 5 of episodes 1, 2, and 4. One hour before being euthanized each rat was injected with 5-bromo-2'-deoxyuridine to label proliferating cells. Each 5-day episode showed a characteristic pattern of rapid shallow breathing (days 1 and 2), epithelial injury, and interstitial and intraluminal inflammation. In contrast, the neutrophil component of inflammation, tracheal substance P release, and cell proliferation became attenuated with each consecutive episode of exposure. Concurrent with this cyclic and attenuated response there was progressive hypercellularity and hyperplasia in all airways studied and a progressive remodeling present in the terminal bronchioles. Our findings are consistent with the notion that the cumulative distal airway lesion is at least in part the result of a depressed cell proliferative response to injury in these airways. This depressed cell proliferative response may be in part the result of diminished neutrophil inflammation and/or release of mitogenic neuropeptides in response to ozone-induced injury.     

Short-term exposure to aged and diluted sidestream cigarette smoke enhances ozone-induced lung injury in B6C3F1 mice.

To determine the effects of aged and diluted sidestream cigarette smoke (ADSS) as a surrogate of environmental tobacco smoke (ETS) on ozone-induced lung injury, male B6C3F1 mice were exposed to (1) filtered air (FA), (2) ADSS, (3) ozone, or (4) ADSS followed by ozone (ADSS/ozone). Exposure to ADSS at 30 mg/m3 of total suspended particulates (TSP) for 6 h/day for 3 days, followed by exposure to ozone at 0.5 ppm for 24 h was associated with a significant increase in the number of cells recovered by bronchoalveolar lavage (BAL) compared with exposure to ADSS alone or ozone alone. The proportion of neutrophils and lymphocytes, as well as total protein level in BAL, was also significantly elevated following ADSS/ozone exposure, when compared with all other groups. Within the centriacinar regions of the lungs, the percentage of proliferating cells identified by bromodeoxyuridine (BrdU) labeling was unchanged from control, following exposure to ADSS alone, but was significantly elevated following exposure to ozone (280% of control) and further augmented in a statistically significant manner in mice exposed to ADSS/ozone (402% of control). Following exposure to ozone or ADSS/ozone, the ability of alveolar macrophages (AM) to release interleukin (IL)-6 under lipopolysaccharide (LPS) stimulation was significantly decreased, while exposure to ADSS or ADSS/ozone caused a significantly increased release of tumor necrosis factor alpha from AM under LPS stimulation. We conclude that ADSS exposure enhances the sensitivity of animals to ozone-induced lung injury.     

Alteration in surface structure of Clara cells and pulmonary cytochrome P-450b level in rats exposed to ozone.

Changes in the surface structure of Clara cells in the terminal bronchioles following exposure of rats to 0.4 ppm ozone (O3) for 14 days were evaluated and compared to the content of pulmonary cytochrome P-450, an enzyme active in xenobiotic metabolism. Exposure to O3 caused a striking alteration of Clara cells in the terminal bronchiole. After 6 h exposure apical protrusions of Clara cells enlarged and these Clara cells formed clusters. However after 24 h exposure, the Clara cells decreased in number and flattened. They increased in number and enlarged again during the subsequent period of exposure. By the 14th day of O3 exposure the number of Clara cells had increased significantly. The content of cytochrome P-450b (IIB1), a main isozyme of pulmonary cytochromes P-450 of rats, was determined by an immuno-blotting method using anti-cytochrome P-450b antibody. The cytochrome P-450b in the rats exposed to O3 increased significantly to 1.37- and 1.81-times that of the control on the 7th and 14th days, respectively. Immuno-electron microscopy demonstrated that cytochrome P-450b was localized abundantly in endoplasmic reticulum of Clara cells. Morphological alterations in Clara cells appear to be closely related with changes in the cytochrome P-450b content of the lung.     

Inflammatory cell availability affects ozone-induced lung damage.

Identifying whether or not neutrophils have a role to play in the early stages of acute lung epithelial injury brought about by inhalation of reactive substances continues to be a major area of investigation. In this study, the availability of circulating neutrophils was manipulated by treatment with either cyclophosphamide or rabbit antiserum against rat neutrophils, prior to exposures to air, a single high ozone exposure of 1 or 2 ppm for 3 h, or a continuous exposure to 0.8-1.0 ppm for up to 48 h. Although cyclophosphamide treatment resulted in undetectable levels of neutrophils in the blood, the recovery of tissue marginated-interstitial neutrophils of 1 x 10(6) cells by collagenase tissue digestion was not significantly diminished at the onset of air and ozone exposures. Cyclophosphamide treatment alone did not cause any permeability damage to air-exposed rat lungs, but did ameliorate ozone-induced increases in bronchoalveolar lavage (BAL) neutrophil and albumin recoveries after both short-term and 1 d of continuous ozone exposure. In contrast to cyclophosphamide, antiserum treatment resulted in greater than a 90% decrease in neutrophil recoveries from both blood and lung tissue at the onset of air and ozone exposures. Antiserum treatment also abrogated ozone-induced neutrophil accumulations in lung lavageable spaces following both single and continuous ozone exposures, but did not significantly affect ozone-associated lung permeability damage indicated by unaltered BAL fluid albumin recoveries. These data demonstrated that under experimental conditions when neutrophils remain within lung tissue marginated and interstitial pools, reduction in circulating blood neutrophil availability is associated with a concomitant decrease in ozone-induced lung damage.     

Structural and functional localization of airway effects from episodic exposure of infant monkeys to allergen and/or ozone

Both allergen and ozone exposure increase asthma symptoms and airway responsiveness in children. Little is known about how these inhalants may differentially modify airway responsiveness in large proximal as compared to small distal airways. We evaluated whether bronchi and respiratory bronchioles from infant monkeys exposed episodically to allergen and/or ozone differentially develop intrinsic hyperresponsiveness to methacholine and whether eosinophils and/or pulmonary neuroendocrine cells play a role. Infant monkeys were exposed episodically for 5 months to: (1) filtered air, (2) aerosolized house dust mite allergen, (3) ozone 0.5 ppm, or (4) house dust mite allergen + ozone. Studying the function/structure relationship of the same lung slices, we evaluated methacholine airway responsiveness and histology of bronchi and respiratory bronchioles. In bronchi, intrinsic responsiveness was increased by allergen exposure, an effect reduced by bombesin antagonist. In respiratory bronchioles, intrinsic airway responsiveness was increased by allergen + ozone exposure. Eosinophils were increased by allergen and allergen + ozone exposure in bronchi and by allergen exposure in respiratory bronchioles. In both airways, exposure to allergen + ozone resulted in fewer tissue eosinophils than did allergen exposure alone. In bronchi, but not in respiratory bronchioles, the number of eosinophils and neuroendocrine cells correlated with airway responsiveness. We conclude that episodically exposing infant monkeys to house dust mite allergen with or without ozone increased intrinsic airway responsiveness to methacholine in bronchi differently than in respiratory bronchioles. In bronchi, eosinophils and neuroendocrine cells may play a role in the development of airway hyperresponsiveness.     

Effects of low levels of NO2 on terminal bronchiolar cells and its relative toxicity compared to O3

This report describes structural changes occurring in the terminal bronchioles of rats exposed to low levels of NO2 continuously for 6 weeks. In addition, the relative susceptibility of epithelial cells to oxidants and the comparative toxicity of NO2 and O3 are discussed. Terminal bronchioles isolated from rats exposed 5 days/week to 2.0 ppm NO2 (plus two 1-hr daily spikes to 6.0 ppm) were found to have 19% less ciliated cells per unit area of epithelial basement membrane. The remaining ciliated cells had a reduced mean surface area (-29%). The shape of the Clara cell changed with reduced size of the dome protrusions but increased cell contact with the basement membrane. These data indicate that exposure to 2.0 ppm NO2 (+ spikes) for 6 weeks caused injuries to cilia and ciliated cells and possible Clara cell differentiation in the terminal bronchioles of adult rats. Exposures of adult or juvenile rats to 0.5 ppm NO2 (+ two 1-hr daily spikes 5 days/week to 1.5 ppm) did not cause morphologically measurable injuries in the terminal bronchioles. The severity of the concentration-dependent epithelial cell reactions to NO2 and O3 in adult rat terminal bronchioles were compared to those occurring in the proximal alveolar regions (PAR). Epithelial cells in the PAR appeared to be more susceptible to oxidant insult since both 0.5 ppm NO2 and 0.25 ppm O3 were found to cause epithelial injury only in the PAR. Comparison of epithelial reactions to 6-week exposures to either NO2 or O3 indicated that 0.25 ppm O3 caused four times as much increase in the number of type I epithelial cells as did 2 ppm (+spikes) NO2. Therefore, O3 could be 40 times more toxic than NO2 in the PAR on the basis of the inspired concentration and the focal response. On the other hand, there was no loss of ciliated cells following the 0.25 ppm O3 exposure. This suggests that the ratio of O3 to NO2 toxicity in the terminal bronchioles is considerably less than 10. The relative toxicity of the two oxidant gases appears to be site specific.     

INTERSPECIES DIFFERENCES IN TIME COURSE OF PULMONARY TOXICITY FOLLOWING REPEATED EXPOSURE TO OZONE

To compare the extent and time course of pulmonary injury and repair in 3 rodent species, rats, mice and guinea pigs were continuously exposed for 3, 7, 28, and 56 days to 400 and 800 mug O3/m3 (0.2 and 0.4 ppm). Recovery from 28 days of exposure was studied at 3, 7, and 28 days after exposure. Pulmonary injury and repair was studied at various time points by histology, electron microscopy, morphometry, and biochemistry. In all 3 species a concentration-related centriacinar inflammation occurred, with a maximum after 3 days of exposure. The number of alveolar macrophages and the pulmonary cell density in the centriacinar region increased progressively until 56 days of exposure, with the guinea pig the most sensitive species. Only the mouse displayed a concentration and exposure-time dependent hypertrophy of bronchiolar epithelium. After 56 days of exposure to 800 mug O3/m3 in the rat and the guinea pig, giant lamellar bodies in type II cells were present. Exposures for 3 and 7 days at near ambient ozone concentrations (400mug O3/m3) resulted in significantly elevated lung enzyme activities in the mouse, and in significant histological and morphometric changes in all 3 species. In rat and guinea pigs exposures for 56 days resulted in alveolar duct fibrosis. The highest biochemical response and the slowest recovery from ozone exposure were seen in the mouse. Histology, morphometry, and biochemistry revealed a total recovery from a 28-day exposure period in rats after 28 days, while in guinea pigs the ductular septa were still thickened and in mice all enzyme activities were still elevated in comparison with control values. In conclusion, the response of mice to ozone was evaluated as most severe, followed by those of guinea pigs and least in rats.     

Respiratory Toxicity of Diacetyl in C57Bl/6 Mice

Diacetyl, a component of artificial butter flavoring, is a potentialetiological agent of obliterative bronchiolitis (OB); however,the toxic dose and mechanisms of toxicity remain controversial.We evaluated the respiratory toxicity of diacetyl in a murinemodel using several exposure profiles relevant to workplaceconditions at microwave popcorn packaging plants. Male C57Bl/6mice were exposed to inhaled diacetyl across several concentrationsand duration profiles, or by direct oropharyngeal aspiration.Effects of diacetyl on the respiratory tract were evaluatedby histopathology and BALF analyses. Subacute exposure to 200or 400 ppm diacetyl for 5 days caused deaths, necrotizing rhinitis,necrotizing laryngitis and bronchitis. Reducing the exposureto 1 h/day (100, 200, 400 ppm) for 4 weeks resulted in lessnasal and laryngeal toxicity, but led to peribronchial and peribronchiolarlymphocytic inflammation. A similar pattern was observed withintermittent high-dose exposures at 1200 ppm (15 min, twicea day, 4 weeks). Subchronic exposures to 100 ppm (6 h/day, 12weeks) caused moderate nasal injury, and peribronchial lymphocyticinflammation accompanied by epithelial atrophy, denudation,and regeneration. Treatment with 400 mg/kg by oropharyngealaspiration to bypass the nose caused foci of fibrohistiocyticproliferation with little or no inflammation at the junctionof the terminal bronchiole and alveolar duct. Depending on theroute and duration of exposure, diacetyl causes significantepithelial injury, peribronchial lymphocytic inflammation, orfibrohistiocytic lesions in the terminal bronchioles. Collectivelythese results indicate that clinically relevant diacetyl exposuresresult in a pattern of injury that replicates features of humanOB.     

Response of guinea pig respiratory tract to inhalation of submicron zinc oxide particles generated in the presence of sulfur dioxide and water vapor

Guinea pigs were exposed for 3 hr to submicron zinc oxide aerosols generated in the presence of water vapor and 1 ppm sulfur dioxide. After exposure to the aerosol containing 5 mg/m³ of zinc oxide, the animals developed transient pulmonary edema accompanied by increased permeability of the respiratory epithelium to macromolecules and increased DNA synthesis in the terminal bronchioles, indicated by an elevated labeling index on autoradiographs. Peribronchiolar edema with cellular infiltrates was present after exposure to the aerosol containing 25 mg/m³ of zinc oxide and significant increases in the labeling index were noted for epithelial cells of the bronchi, bronchioles, and alveoli. The changes coincided with functional abnormalities detected in complementary physiological studies.