Circulating factors that modify lung cell DNA synthesis following exposure to inhaled oxidants. I. Effect of serum and lavage on lung fibroblasts following exposure of adult rats to 1 ppm ozone

Adult rats were exposed to 1 ppm (1.96 mg/m3) ozone or air for 2 wk. Animals were sacrificed at 3, 5, 7, or 14 d after the onset of exposure, and samples of plasma and lung lavage were obtained. Heat-inactivated plasma, from animals exposed to ozone for 7 or 14 d, significantly increased DNA synthesis by lung fibroblasts compared with plasma from air-exposed animals. Fractionation of plasma and lavage samples indicated that the factor responsible had an isoelectric point of 6.45-6.75 and a molecular weight of 32 +/- 2 kDa. This factor has a dose-dependent effect on lung fibroblast DNA synthesis in culture, but no significant effect on cultured pneumocyte DNA synthesis. The factor is detectable within 72 h of exposure, and may hold some promise as a marker of early oxidant lung injury.     

Surface morphology and morphometry of rat alveolar macrophages after ozone exposure

As the ultrastructural data on the effects of ozone on pulmonary alveolar macrophages (PAM) are lacking, transmission (TEM) and scanning (SEM) electron microscopy were performed on rat PAM present in alveolar lavages following exposure to ozone. Rats were continuously exposed for 7 d to ozone concentrations ranging from 0.25 to 1.50 mg/m3 for 7 d followed by a 5-d recovery period. Additionally, morphometry on lung sections was performed to quantitate PAM. In a second experiment rats were continuously exposed to 1.50 mg O3/m3 for 1, 3, 5, or 7 d. To study the influence of concurrent ozone exposure and lung infection, due to Listeria monocytogenes, rats were exposed for 7 d to 1.50 mg O3/m3 after a Listeria infection. The surface area of lavaged control PAM was uniformly covered with ruffles as shown by SEM and TEM. Exposure to 0.5 mg ozone/m3 for 7 d resulted in cells partly covered with microvilli and blebs in addition to normal ruffles. The number of large size PAM increased with an increase in ozone concentration. After 1 d of exposure, normal-appearing as well as many small macrophages with ruffles and scattered lymphocytes were seen. Lavage samples taken after 5 or 7 d of exposure showed an identical cell composition to that taken after 3 d of exposure. After Listeria infection alone, lavage samples consisted of mainly lymphocytes and some macrophages. Small quantitative changes, such as an increase in the number of polymorphonuclear neutrophils and large-size PAM, occurred in lavages after ozone exposure and infection with L. monocytogenes. Morphometric examination of lung sections revealed a concentration-related increase in the number of PAM, even in animals exposed to 0.25 mg ozone/m3 for 7 d. Centriacinar regions were more severely affected than other regions of lung tissue. By 5 d after termination of exposure to ozone, the number of lysozyme-positive alveolar cells was still significantly increased in centriacinar areas of the lung. The results indicate that ozone exposure causes major changes in the number, size, and surface morphology of PAM in rat lung. Furthermore, the results presented here suggest that changes in alveolar macrophage function are reflected by morphological changes.     

Influence of hyperthyroidism on rat lung cytokine production and nuclear factor-kappaB activation following ozone exposure

Results from previous studies indicate that hyperthyroidism increases the risk of ozone-induced lung toxicity. To better understand the processes that might contribute to the increased pulmonary inflammatory response to ozone in hyperthyroidism, we evaluated bronchoalveolar lavage fluid levels of selected cytokines in control and hyperthyroid rats after exposure to air or ozone. In addition, we assessed whether there is a relative increase in nuclear factor-kappa B (NF-kappaB) binding activity in cells harvested by bronchoalveolar lavage from hyperthyroid rats following the inhalation of ozone. A hyperthyroid condition was induced by the administration of thyroxine (0.5 mg/kg body weight) for 7 days. Control rats received vehicle injections. The animals were then exposed by inhalation to air or ozone (2 ppm for 3 h) and studied 18 h following the exposure. Bronchoalveolar lavage levels of MIP-2 and MCP-1 were increased in both control and hyperthyroid rats by ozone exposure. However, the increases in hyperthyroid rats were much greater, MIP-2 1.5-fold and MCP-1 11-fold, when compared to levels in controls following ozone. These changes appeared to be relatively specific; bronchoalveolar lavage fluid levels of interleukin (IL)-6, IL-4, and IL-10 were generally low or nondetectable across all of the studied groups at the 18-h postexposure time point. We also found that NF-kappaB binding activity was increased at both 4 and 18 h following ozone exposure in bronchoalveolar lavage cell extracts from hyperthyroid rats relative to the activity in control samples. Collectively, these results suggest that mechanisms contributing to the enhanced pulmonary inflammatory response to ozone in a hyperthyroid state include an increase in NF-kappaB activation and an upregulation of chemokine production.     

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.     

Hyperthyroidism increases the risk of ozone-induced lung toxicity in rats

The risk of lung injury from ozone exposure has been well documented. It is also known that various factors may significantly influence the susceptibility of animals to the toxic effects of ozone. In the present study, we investigated the possibility that hyperthyroidism might be associated with increases in ozone-induced pulmonary toxicity. To create a hyperthyroid condition, mature male Sprague--Dawley rats were given injections of thyroxine (dose range: 0.1 to 1 mg/kg body wt daily for 7 days). Control rats received vehicle injections. The animals were then exposed to air or ozone (dose range: 0.5 to 3 ppm for 3 h). At 18 h postexposure, bronchoalveolar lavage fluid and cells were harvested. In hyperthyroid animals, ozone exposure was associated with three- to sixfold increases in bronchoalveolar lavage fluid lactate dehydrogenase activities and albumin levels as well as the number of polymorphonuclear leukocytes harvested by bronchoalveolar lavage above levels observed in ozone-exposed control rats. Additional results from the present study suggest that these thyroid hormone-linked effects cannot be fully explained by differences in whole-body metabolic rate or changes in the inhaled dose of ozone. These findings indicate that the risk of ozone-induced lung toxicity is substantially increased in a hyperthyroid state and suggest that the susceptibility of the lung to damage from ozone exposure may be significantly influenced by individual thyroid hormone status.     

Changes in lipids of lung lavage in monkeys after chronic exposure to ambient levels of ozone

A group of 8 sub-adult bonnet monkeys (Macaca radiata) was exposed to 0.3 ppm ozone (O3) and another group of 7 monkeys to 0.15 ppm O3 for 8 h/day for 90 days. A third group of 4 monkeys was exposed to 0.15 ppm O3 for 8 h/day for 21 days. The control group consisted of 7 monkeys which breathed filtered air for 90 days. Levels of linoleic and arachidonic acids in the total lipids from lung lavage increased about 2-fold in those exposed to O3 as compared to the levels in the controls. Furthermore, the relative level of cholesterol ester (CE) decreased and phosphatidylcholine (PC) increased markedly with chronic exposure of animals to O3. Enhanced polyunsaturated fatty acid (PUFA) composition in lung lavage and changes in the levels of CE and PC may be related to animals' adaptation to O3-exposure.     

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.     

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.     

Rat lung recovery from 3 days of continuous exposure to 0.75 ppm ozone

The present study investigated the inflammatory responses and enzyme levels in lungs isolated from male Wistar rats after 3 d of continuous exposure to 0.75 ppm ozone and following 4 d of recovery in air. These times are associated with maximal proliferation of the alveolar type II epithelium and their subsequent transformation to new type I cells. Immediately following ozone exposure, bronchoalveolar lavage demonstrated neutrophil accumulation that was no longer present 4 d later. The number of lavaged macrophages was also found to be increased immediately following ozone exposure, and remained elevated at 4 d postexposure. Whole-lung determinations of key enzymes involved in energy generation (succinate oxidase) and maintenance of lung NADPH and reduced glutathione were corrected for changes in cell number, by use of lung DNA measurements. Immediately following ozone exposure succinate oxidase (SOX), glucose-6-phosphate (G6PD), and 6-phosphogluconate (6PGD) dehydrogenase activities per milligram DNA were significantly enhanced by 76%, 48%, and 21%, respectively. These data suggested that ozone-exposed lungs had cells with increased mitochondria and NADPH-generating capability consistent with the increased metabolic needs of a proliferating epithelium. At 4 d postexposure, only G6PD activity per milligram DNA remained higher by 22% than air-exposed controls. Although both glutathione reductase (GSSG-R) and peroxidase (GSH-Px) activities per lung were elevated in lungs immediately following exposure and 4 d later, when corrected for DNA only GSH-Px activity was significantly increased by 29% in lungs after the postexposure period. Lungs 4 d postexposure therefore had cells relatively enriched in G6PD and GSH-Px that might account for the increased ozone tolerance that has previously been associated with the formation of new type I epithelium.     

Adhesion molecules of blood polymorphonuclear leukocytes and alveolar macrophages in rats: modulation by exposure to ozone.

1. Exposure to elevated levels of ozone results in an infiltration of polymorphonuclear leukocytes (PMNs) into the lungs. The purpose of this study was to investigate whether the ozone-induced inflammatory process is preceded by a change in the expression of adhesion molecules (integrins and selectins) in peripheral blood PMNs and alveolar macrophages in rats. 2. Female Sprague Dawley rats were exposed to air or ozone (1 p.p.m., 2 h). Bronchoalveolar lavage (BAL) was carried out and blood was collected via intracardiac puncture at 0 or 18 h after the exposure. There were no PMN in the BAL fluid at time 0 after the 2 h exposure to ozone. The expression of cell adhesion molecules from the integrin family (represented by CD18) on alveolar macrophages (AM) was lowered. The expression of cell adhesion molecules from the selectin family (represented by CD62L) on blood PMN was not affected by exposure to ozone, while the expression of integrins (CD11b) on blood PMN was lowered. 3. This effect was confirmed by experiments in which plasma of ozone-exposed animals was incubated with PMN from peripheral blood obtained from non-exposed animals. In these experiments, the expression of CD11b on PMNs of non-exposed animals was lower after incubation with plasma from ozone-exposed animals. 4. Our experiments suggest the presence of factor(s) in blood, which cause a decrease in the expression of CD11b on PMNs.     

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.     

Response of T-cell-deficient mice to ozone exposure

The number, appearance, and functional reactivity of T-lymphocytes of mediastinal lymph nodes are altered during experimental ozone inhalation. The purpose of the present work is to determine how the lymph nodes and lungs of a mutant strain of animal, which lacks this type of cell, differ in their response to ozone exposure when compared with animals that possess a normal complement of lymphocytes. We exposed athymic nude (nu/nu) mice or heterozygous (nu/+) euthymic mice to 0.7 ppm ozone for 20 h/d for 7 or 14 d while maintaining control groups in clean air. At 7 d the lymph-node hyperplastic response normally seen in euthymic, ozone-exposed animals was greatly reduced in exposed athymic animals. By both 7 and 14 d, greater damage had occurred in the lungs of ozone-exposed, athymic animals than in similarly exposed euthymic animals. Lung wet weight divided by body weight, which was used as a general indicator of lung damage, increased by substantially more in athymic animals than in conventional animals. In a parallel manner, quantitative microscopic analysis, a more sensitive indicator, revealed a marked increase in the lung lesion volumes. Qualitative histologic analysis showed that the change in the response in the athymic animal was most prominent in the peripheral region of the lung extending from the alveolar duct to the alveoli, and was characterized by a greater acute inflammatory cell reaction. Possible mechanisms by which the T-cell could produce the observed effect include secretion of factors that enhance inherent resistance of the lung's target cells, or alterations in the way the inflammatory response to ozone-mediated damage occurs. The results support the idea that the mediastinal lymph node lymphocyte response is adaptive in nature and aids in protecting the lung from ozone-mediated effects.     

Ovalbumin-Induced Airway Inflammation and Fibrosis in Mice Also Exposed to Ozone

A murine model of allergen-induced airway inflammation was used to examine the effects of exposure to ozone on airway inflammation and remodeling. Sensitized BALB/c mice were exposed to ovalbumin aerosol for 4 wk before and after 2 wk of exposure to either 0.2 ppm or 0.5 ppm ozone. Other groups of mice were exposed to ovalbumin aerosol for 6 wk with continuous concurrent exposure to ozone during wk 1–6, or during intermittent concurrent exposure to ozone. Lung inflammation was measured by quantitative differential evaluation of lung lavage cells and by histological evaluation of stained lung sections. Alterations in lung structure (airway fibrosis) were evaluated by quantitative biochemical analysis of microdissected airways. The same total number of cells was observed in lavage fluid from animals exposed for 4 wk to ovalbumin alone or to ovalbumin for 4 wk immediately before or after exposure to 2 wk of 0.2 or 0.5 ppm ozone. Mice exposed to ovalbumin for 6 wk with concurrent exposure to either 0.2 ppm or 0.5 ppm ozone during wk 3–6 had a significant decrease in the total number of cells recovered by lavage. Values as low as 7% of the cell number found in mice exposed to ovalbumin aerosol alone were observed in the mice exposed to ovalbumin plus 0.2 ppm ozone during wk 3–6. There were significant differences in the cell differential counts in the lavage fluid from mice exposed to ovalbumin alone as compared with values from mice exposed to ovalbumin and ozone under all of the protocols studied. When ozone was given for 2 wk prior to ovalbumin exposure (Experiment 1), there were a high percentage of macrophages and low percentages of lymphocytes and eosinophils in the lung lavage. When ozone was given for 2 wk after ovalbumin exposure (Experiment 2), there were a moderate percentage of macrophages, a low percentage of eosinophils, and a high percentage of lymphocytes in the lung lavage. When ozone and ovalbumin were given simultaneously (Experiments 3 and 4), there were a high percentage of macrophages in the lavage with 0.2 ppm ozone and a high percentage of eosinophils. Ozone appears to antagonize the specific inflammatory effects of ovalbumin exposure, especially when given before or during exposure to ovalbumin. Airway remodeling was examined by two different quantitative methods. None of the groups exposed concurrently to ovalbumin and ozone had a significant increase in airway collagen content as compared to the matched groups of mice exposed to ovalbumin alone. The findings were consistent with an additive response of mice to simultaneous exposure to ovalbumin and ozone. Ozone exposure alone for 6 wk did not affect the number of goblet cells in the airways, while mice exposed to ovalbumin aerosol alone for 6 wk had about 25% goblet cells in their conducting airways. Concurrent exposure to ovalbumin and 0.2 ppm ozone caused significant increases in goblet cells (to 43% of total cells) in the conducting airways of the exposed mice. We conclude that when mice with allergen-induced airway inflammation induced by ovalbumin are also exposed to ozone, the lung inflammatory response may be modified, but that this altered response is dependent on the sequence of exposure and the concentration of ozone to which they are exposed. At the concentrations of ozone tested, we did not see changes in airway fibrosis. However, goblet-cell hyperplasia appeared to be increased in mice exposed concurrently to ovalbumin and 0.2 ppm ozone.     

Ovalbumin-induced airway inflammation and fibrosis in mice also exposed to ozone

A murine model of allergen-induced airway inflammation was used to examine the effects of exposure to ozone on airway inflammation and remodeling. Sensitized BALB/c mice were exposed to ovalbumin aerosol for 4 wk before and after 2 wk of exposure to either 0.2 ppm or 0.5 ppm ozone. Other groups of mice were exposed to ovalbumin aerosol for 6 wk with continuous concurrent exposure to ozone during wk 1-6, or during intermittent concurrent exposure to ozone. Lung inflammation was measured by quantitative differential evaluation of lung lavage cells and by histological evaluation of stained lung sections. Alterations in lung structure (airway fibrosis) were evaluated by quantitative biochemical analysis of microdissected airways. The same total number of cells was observed in lavage fluid from animals exposed for 4 wk to ovalbumin alone or to ovalbumin for 4 wk immediately before or after exposure to 2 wk of 0.2 or 0.5 ppm ozone. Mice exposed to ovalbumin for 6 wk with concurrent exposure to either 0.2 ppm or 0.5 ppm ozone during wk 3-6 had a significant decrease in the total number of cells recovered by lavage. Values as low as 7% of the cell number found in mice exposed to ovalbumin aerosol alone were observed in the mice exposed to ovalbumin plus 0.2 ppm ozone during wk 3-6. There were significant differences in the cell differential counts in the lavage fluid from mice exposed to ovalbumin alone as compared with values from mice exposed to ovalbumin and ozone under all of the protocols studied. When ozone was given for 2 wk prior to ovalbumin exposure (Experiment 1), there were a high percentage of macrophages and low percentages of lymphocytes and eosinophils in the lung lavage. When ozone was given for 2 wk after ovalbumin exposure (Experiment 2), there were a moderate percentage of macrophages, a low percentage of eosinophils, and a high percentage of lymphocytes in the lung lavage. When ozone and ovalbumin were given simultaneously (Experiments 3 and 4), there were a high percentage of macrophages in the lavage with 0.2 ppm ozone and a high percentage of eosinophils. Ozone appears to antagonize the specific inflammatory effects of ovalbumin exposure, especially when given before or during exposure to ovalbumin. Airway remodeling was examined by two different quantitative methods. None of the groups exposed concurrently to ovalbumin and ozone had a significant increase in airway collagen content as compared to the matched groups of mice exposed to ovalbumin alone. The findings were consistent with an additive response of mice to simultaneous exposure to ovalbumin and ozone. Ozone exposure alone for 6 wk did not affect the number of goblet cells in the airways, while mice exposed to ovalbumin aerosol alone for 6 wk had about 25% goblet cells in their conducting airways. Concurrent exposure to ovalbumin and 0.2 ppm ozone caused significant increases in goblet cells (to 43% of total cells) in the conducting airways of the exposed mice. We conclude that when mice with allergen-induced airway inflammation induced by ovalbumin are also exposed to ozone, the lung inflammatory response may be modified, but that this altered response is dependent on the sequence of exposure and the concentration of ozone to which they are exposed. At the concentrations of ozone tested, we did not see changes in airway fibrosis. However, goblet-cell hyperplasia appeared to be increased in mice exposed concurrently to ovalbumin and 0.2 ppm ozone.     

Assessments of lung toxicity to Acrawax C following acute inhalation exposure

Acrawax is a trademark for a series of synthetic waxes which are used as flatteners in paint, and lubricants in plastics, and these materials have been routinely regarded as nuisance dusts. Due to a paucity of information regarding the pulmonary toxicity of this material, we investigated the effects of acute inhalation of Acrawax C in rats. CD rats were exposed to aerosols of Acrawax C for 6 hours at 112 mg/m3. Fluids and cells from sham and exposed animals were recovered by bronchoalveolar lavage (BAL) and measured for cellular and biochemical parameters at 0, 24, 48, 172 hrs (8 days), and 1 month postexposure. Pulmonary macrophages (PM) were cultured and studied for in vitro and in vivo phagocytosis, as well as surface morphology. The lungs of additional animals exposed to Acrawax were fixed for assessment by histopathology, and transmission electron microscopy. Our results showed that Acrawax C exposure produced a mild inflammatory response at 24 hours postexposure, but cell differentials were not significantly different from controls at 48 hrs after exposure. BAL levels of lactate dehydrogenase, alkaline phosphatase and protein were slightly different from controls only at 8 days postexposure, and had returned to control values by 1 month of recovery. Acrawax exposure had no adverse effects on either morphology or the phagocytic capacity of pulmonary macrophages recovered from exposed animals. Histopathologic analysis of lung tissue from Acrawax C-exposed rats revealed normal lung architecture. Based on acute studies, our results suggest that the response to inhaled Acrawax C is not substantially different from the response to other nuisance dusts such as carbonyl iron and titanium dioxide.     

Recovery of lung pyridine nucleotides following acute exposure of adult and aged rats to ozone.

Male, pathogen-free Fischer 344 rats aged 6 and 24 mo were exposed to 1.5 or 3.0 ppm for 8 h and recovery rates of diphosphonucleotides (NAD+ and NADH) and triphosphonucleotides (NADP+ and NADPH) were measured and compared to controls. Recovery after 0.5 ppm was not examined because no significant changes occurred in either age group after this lower exposure. At zero time (immediately after exposures) both concentrations are depressed in adults and aged animals except for NADH in aged animals at 3.0 ppm; NADP+ in adults at 1.5 and 3.0 ppm was decreased, but not significantly. For NAD+ and NADH, recovery of whole lung concentrations is complete by 24 h following an 8-h exposure to 1.5 or 3.0 ppm of ozone. Only after 3.0 ppm of ozone was the ratio of the reduced to oxidized form (NADH/NAD+) still elevated after 24 h; however, it also returned to control levels by 96 h. For the triphosphonucleotides, an 8-h exposure to 1.5 ppm of ozone resulted in a sustained depression of whole lung concentrations of NADPH throughout the 96-h recovery period. Also, only after the 1.5 ppm exposure was the reduced to oxidized ratio (NADPH/NADP+) significantly depressed throughout the 96-h recovery period. Unexpectedly, recovery of whole lung levels returned to normal within 24 h after the 8-h exposure to both the 1.5 and the 3.0 ppm concentrations. With the exception of the sustained effect on NADPH levels, these data indicate that di- and triphosphonucleotide concentrations rapidly return to normal in the lung after severe, acute oxidant injury. There were no differences in recovery rates between the adult and the aged groups.     

Acute pulmonary effects of combined exposure to carbon nanotubes and ozone in mice

Ozone (O3) is a well-investigated gaseous air pollutant known to produce acute and chronic toxicity in the respiratory system. Whether prior exposure to nanoparticles influences the toxicity of O3 has not been well investigated. To determine if there are toxicological interactions between particulate and gas exposures, we examined acute pulmonary effects of a 3-h ozone exposure (0.5 ppm) in female C57Bl mice that had been preexposed to a single dose of 20 microg multiwall carbon nanotubes (CNT) by pharyngeal aspiration 12 h earlier. A total of four groups were compared: (1) PBS/air-control, (2) PBS/O3, (3) CNT/air, and (4) CNT/O3. Analyses of the bronchoalveolar lavage fluid (BALF) and lung tissue samples collected at 5 and 24 h post O3 exposure were performed for various markers of cytotoxicity and inflammation using standard enzyme-linked immunosorbent assay (ELISA) and immunoblot procedures. The results showed a pronounced cellular response and increase in various cytotoxicity/inflammatory markers in the lungs of CNT-exposed mice. Ozone by itself produced minimal effects, but in CNT-exposed animals there was a significant increase in total brochoalveolar lavage (BAL) cells and polymorphonuclear leukocytes. Additionally, protein, lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and mucin levels in BALF at 5 and 24 h were higher in CNT-exposed animals than in corresponding air-exposed controls or animals exposed to O3 alone. A comparable increase over the controls was also observed in the CNT/O3 group, but neither an additive nor a synergistic interaction was observed in mice that received sequential exposure to CNT and ozone. In fact, some CNT-induced cytotoxic/inflammatory responses were attenuated in mice following exposure to both CNT and low levels of ozone. These results are contrary to enhanced responses that were anticipated and may represent the development of "cross-tolerance" reported by others for some sequentially administered pollutants.     

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.     

Acute lung injury following inhalation exposure to nerve agent VX in guinea pigs

A microinstillation technique of inhalation exposure was utilized to assess lung injury following chemical warfare nerve agent VX [methylphosphonothioic acid S-(2-[bis(1-methylethyl)amino]ethyl) O-ethyl ester] exposure in guinea pigs. Animals were anesthetized using Telazol-meditomidine, gently intubated, and VX was aerosolized using a microcatheter placed 2 cm above the bifurcation of the trachea. Different doses (50.4 microg/m3, 70.4 micro g/m(m3), 90.4 microg/m(m3)) of VX were administered at 40 pulses/min for 5 min. Dosing of VX was calculated by the volume of aerosol produced per 200 pulses and diluting the agent accordingly. Although the survival rate of animals exposed to different doses of VX was similar to the controls, nearly a 20% weight reduction was observed in exposed animals. After 24 h of recovery, the animals were euthanized and bronchoalveolar lavage (BAL) was performed with oxygen free saline. BAL was centrifuged and separated into BAL fluid (BALF) and BAL cells (BALC) and analyzed for indication of lung injury. The edema by dry/wet weight ratio of the accessory lobe increased 11% in VX-treated animals. BAL cell number was increased in VX-treated animals compared to controls, independent of dosage. Trypan blue viability assay indicated an increase in BAL cell death in 70.4 microg/m(m3) and 90.4 microg/m(m3) VX-exposed animals. Differential cell counting of BALC indicated a decrease in macrophage/monocytes in VX-exposed animals. The total amount of BAL protein increased gradually with the exposed dose of VX and was highest in animals exposed to 90.4 microg/m(m3), indicating that this dose of VX caused lung injury that persisted at 24 h. In addition, histopathology results also suggest that inhalation exposure to VX induces acute lung injury.     

Lung function and airway inflammation in rats following exposure to combustion products of carbon-graphite/epoxy composite material: comparison to a rodent model of acute lung injury

Pulmonary function and inflammation in the lungs of rodents exposed by inhalation to carbon/graphite/epoxy advanced composite material (ACM) combustion products were compared to that of a rodent model of acute lung injury (ALI) produced by pneumotoxic paraquat dichloride. This investigation was undertaken to determine if short-term exposure to ACM smoke induces ALI; and to determine if smoke-related responses were similar to the pathogenic mechanisms of a model of lung vascular injury. We examined the time-course for mechanical lung function, infiltration of inflammatory cells into the lung, and the expression of three inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Male Fischer-344 rats were either exposed to 26.8-29.8 g/m(3) nominal concentrations of smoke or were given i.p. injections of paraquat dichloride. Measurements were determined at 1, 2, 3, and 7 days post exposure. In the smoke-challenged rats, there were no changes in lung function indicative of ALI throughout the 7-day observation period, despite the acute lethality of the smoke atmosphere. However, the animals showed signs of pulmonary inflammation. The expression of TNF-alpha was significantly increased in the lavage fluid 1 day following exposure, which preceded the maximum leukocyte infiltration. MIP-2 levels were significantly increased in lavage fluid at days 2, 3, and 7. This followed the leukocyte infiltration. IFN-gamma was significantly increased in the lung tissue at day 7, which occurred during the resolution of the inflammatory response. The paraquat, which was also lethal to a small percentage of the animals, caused several physiologic changes characteristic of ALI, including significant decreases in lung compliance, lung volumes/capacities, distribution of ventilation, and gas exchange capacity. The expression of TNF-alpha and MIP-2 increased significantly in the lung tissue as well as in the lavage fluid. Increased MIP-2 levels also preceded the maximum neutrophil infiltration. The differences in the time-course and primary site of TNF-alpha, MIP-2, and IFN-gamma expression; and the differences in the temporal relationship between their expression and infiltration of inflammatory cells may have accounted for the differences in lung function between paraquat treated and ACM smoke exposed animals.