David R. Hemenway

Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat.

To determine whether asbestos inhalation induces the formation of reactive nitrogen species, three groups of rats were exposed intermittently over 2 wk to either filtered room air (sham-exposed) or to chrysotile or crocidolite asbestos fibers. The rats were killed at 1 or 6 wk after exposure. At 1 wk, significantly greater numbers of alveolar and pleural macrophages from asbestos-exposed rats than from sham-exposed rats demonstrated inducible nitric oxide synthase protein immunoreactivity. Alveolar macrophages from asbestos-exposed rats also generated significantly greater nitrite formation than did macrophages from sham-exposed rats. Strong immunoreactivity for nitrotyrosine, a marker of peroxynitrite formation, was evident in lungs from chrysotile- and crocidolite-exposed rats at 1 and 6 wk. Staining was most evident at alveolar duct bifurcations and within bronchiolar epithelium, alveolar macrophages, and the visceral and parietal pleural mesothelium. Lungs from sham-exposed rats demonstrated minimal immunoreactivity for nitrotyrosine. Significantly greater quantities of nitrotyrosine were detected by ELISA in lung extracts from asbestos-exposed rats than from sham-exposed rats. These findings suggest that asbestos inhalation can induce inducible nitric oxide synthase activation and peroxynitrite formation in vivo, and provide evidence of a possible alternative mechanism of asbestos-induced injury to that thought to be induced by Fenton reactions.

Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models.

To evaluate the contribution of reactive nitrogen species to inflammation by asbestos, Fischer 344 rats were exposed to crocidolite or chrysotile asbestos by inhalation to determine whether increases occurred in nitric oxide (NO.) metabolites from alveolar macrophages (AMs). AMs from animals inhaling asbestos showed significant elevations (p < .05) in nitrite/nitrate levels which were ameliorated by NG-monomethyl-L-arginine (NMMA), an inhibitor of inducible nitric oxide synthase (iNOS) activity. Temporal patterns of NO. generation from AMs correlated with neutrophil influx in bronchoalveolar lavage samples after asbestos inhalation or bleomycin instillation, another model of pulmonary fibrosis. To determine the molecular mechanisms and specificity of iNOS promoter activation by asbestos, RAW 264.7 cells, a murine macrophage-like line, and AMs isolated from control rats were exposed to crocidolite asbestos in vitro. These cells showed increases in steady-state levels of iNOS mRNA in response to asbestos and more dramatic increases in both iNOS mRNA and immunoreactive protein after addition of lipopolysaccharide (LPS). After transfection of an iNOS promoter/luciferase reporter construct, RAW 264.7 cells exposed to LPS, crocidolite asbestos and its nonfibrous analog, riebeckite, revealed increases in luciferase activity whereas cristobalite silica had no effects. Studies suggest that NO. generation may be important in cell injury and inflammation by asbestos.

Nitrogen Dioxide Promotes Allergic Sensitization to Inhaled Antigen

Allergen sensitization and allergic airway disease are likely to come about through the inhalation of Ag with immunostimulatory molecules. However, environmental pollutants, including nitrogen dioxide (NO2), may promote adaptive immune responses to innocuous Ags that are not by themselves immunostimulatory. We tested in C57BL/6 mice whether exposure to NO2, followed by inhalation of the innocuous protein Ag, OVA, would result in allergen sensitization and the subsequent development of allergic airway disease. Following challenge with aerosolized OVA alone, mice previously exposed via inhalation to NO2 and OVA developed eosinophilic inflammation and mucus cell metaplasia in the lungs, as well as OVA-specific IgE and IgG1, and Th2-type cytokine responses. One hour of exposure to 10 parts per million NO2 increased bronchoalveolar lavage fluid levels of total protein, lactate dehydrogenase activity, and heat shock protein 70; promoted the activation of NF-κB by airway epithelial cells; and stimulated the subsequent allergic response to Ag challenge. Furthermore, features of allergic airway disease were not induced in allergen-challenged TLR2−/− and MyD88−/− mice exposed to NO2 and aerosolized OVA during sensitization. These findings offer a mechanism whereby allergen sensitization and asthma may result under conditions of high ambient or endogenous NO2 levels.

Comparative Clearance of Quartz and Cristobalite from the Lung

The two silicon dioxide polymorphs, quartz and cristobalite, are known to have different toxicities. The clearance kinetics and biological response of two sources of quartz and one source of pure cristobalite were compared. Models were also developed to show the accumulation of cristobalite in the lungs of Fischer 344 rats as the result of short-term exposures at three different concentrations. The amount of cristobalite cleared from the lung was considerably less than that of the two quartz materials, with little or no clearance after the initial 30 days post exposure. As an indicator of the cellular biological response to the aerosols, total and differential cell counts were measured on bronchoalveolar lavage specimens. Cristobalite showed an early and sustained response with an elevated macrophage, neutrophil, and lymphocyte count through 180 days post exposure. The two quartz materials were not identical in their biological behavior even though they had identical crystal structure and similar trace element analysis. One quartz sample (MIN5) showed an increase in cell response (macrophage, neutrophils, and lymphocytes) approximately 30% that of cristobalite, whereas the other quartz material was not significantly different from control values. In addition, lung hydroxyproline content was greater in the cristobalite-exposed animals.

Asbestos-Induced Lung Inflammation and Epithelial Cell Proliferation Are Altered in Myeloperoxidase-Null Mice

Asbestos fibers are carcinogens causing oxidative stress and inflammation, but the sources and ramifications of oxidant production by asbestos are poorly understood. Here, we show that inhaled chrysotile asbestos fibers cause increased myeloperoxidase activity in bronchoalveolar lavage fluids (BALF) and myeloperoxidase immunoreactivity in epithelial cells lining distal bronchioles and alveolar ducts, sites of initial lung deposition of asbestos fibers. In comparison with sham mice, asbestos-exposed myeloperoxidase-null (MPO-/-) and normal (MPO+/+) mice exhibited comparable increases in polymorphonuclear leukocytes, predominately neutrophils, in BALF after 9 days of asbestos inhalation. Differential cell counts on BALF revealed decreased proportions of macrophages and increased lymphocytes in all mice exposed to asbestos, but numbers were decreased overall in asbestos-exposed myeloperoxidase-null versus normal mice. Asbestos-associated lung inflammation in myeloperoxidase-null mice was reduced (P < or = 0.05) in comparison with normal asbestos-exposed mice at 9 days. Decreased lung inflammation in asbestos-exposed myeloperoxidase-null mice at 9 days was accompanied by increases (P < or = 0.05) in Ki-67- and cyclin D1-positive immunoreactive cells, markers of cell cycle reentry, in the distal bronchiolar epithelium. Asbestos-induced epithelial cell proliferation in myeloperoxidase-null mice at 30 days was comparable to that found at 9 days. In contrast, inflammation and epithelial cell proliferation in asbestos-exposed normal mice increased over time. These results support the hypothesis that myeloperoxidase status modulates early asbestos-induced oxidative stress, epithelial cell proliferation, and inflammation.

Concomitant exposure to carbon black particulates enhances ozone‐induced lung inflammation and suppression of alveolar macrophage phagocytosis

The goal of this study was to investigate whether coexposures to carbon black and O3 result in a toxicologic interaction in the lungs as quantitated by the inflammatory response and alveolar macrophage (AM) phagocytosis. This aim was accomplished through inhalation coexposures of Swiss mice for 4 h to target concentrations of 10 mg/m3 of carbon black and 1.5 ppm O3, or exposure to either agent alone. As a control for the coexposure experiments, mice were also exposed for 4 h to carbon black, followed immediately thereafter by exposure for 4 h to O3, or vice versa. At 24 h after exposure, the lungs of the animals were lavaged for quantitation of total and differential cell counts and assessment of AM Fc-receptor-mediated phagocytosis. Exposure to carbon black did not result in an inflammatory response, nor had it any effect on AM phagocytosis. Ozone exposure resulted in an inflammatory response in the lungs and suppression of AM phagocytosis. Both biologic parameters were significantly enhanced following combined exposure to the particle and the gas. Carbon black exposure either before or after O3 had no significant effect on AM phagocytosis as compared to O3 exposure alone. These data demonstrate the toxicologic interaction of coexposures to an inert particle and O3 on well-accepted biologic markers pulmonary toxicity. The mechanism for the enhanced biologic effect may be that the carbon black particle acts as a carrier mechanism for O3 to areas in the distal lung not accessible to O3 in the gaseous phase or that O3 alters the physicochemistry of the particulate from a nontoxic to a toxic form.

Design, development and test results of a horizontal flow inhalation toxicology facility

A horizontal flow chamber was developed for the purposes of conducting inhalation toxicology research. The chamber utilizes a unique diffuser system and pre-mixing of contaminants to achieve uniform exposure. The use of fecal catch pans eliminates exposure of animals to fecal debris from overhead animals. The chamber performance shows a rapid equilibrium in startup and shutdown.

Acrolein Inhalation Suppresses Lipopolysaccharide-Induced Inflammatory Cytokine Production but Does Not Affect Acute Airways Neutrophilia

Acrolein is a reactive unsaturated aldehyde that is produced during endogenous oxidative processes and is a major bioactive component of environmental pollutants such as cigarette smoke. Because in vitro studies demonstrate that acrolein can inhibit neutrophil apoptosis, we evaluated the effects of in vivo acrolein exposure on acute lung inflammation induced by LPS. Male C57BL/6J mice received 300 μg/kg intratracheal LPS and were exposed to acrolein (5 parts per million, 6 h/day), either before or after LPS challenge. Exposure to acrolein either before or after LPS challenge did not significantly affect the overall extent of LPS-induced lung inflammation, or the duration of the inflammatory response, as observed from recovered lung lavage leukocytes and histology. However, exposure to acrolein after LPS instillation markedly diminished the LPS-induced production of several inflammatory cytokines, specifically TNF-α, IL-12, and the Th1 cytokine IFN-γ, which was associated with reduction in NF-κB activation. Our data demonstrate that acrolein exposure suppresses LPS-induced Th1 cytokine responses without affecting acute neutrophilia. Disruption of cytokine signaling by acrolein may represent a mechanism by which smoking contributes to chronic disease in chronic obstructive pulmonary disease and asthma.

ASBESTOS EXPOSURE INDUCES MCP-1 SECRETION BY PLEURAL MESOTHELIAL CELLS

We showed previously that both crocidolite and chrysotile asbestos inhalation induced a persistent macrophage in ammatory response within the pleural space of the rat. We postulated that the stimulus for pleural macrophage recruitment after asbestos exposure was the induction of monocyte chemoattractant protein-1 (MCP-1) synthesis by pleural mesothelial cells. To test this hypothesis, rat pleural mesothelial cells (RPMC) were cultured with or without chrysotile or crocidolite asbestos fibers (8mug/cm2) in the presence (50ng/mL) or absence of either tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). MCP-1 mRNA expression was assessed by RT-PCR in RPMC cultured for 2 to 24 hours, and MCP-1 protein secretion was measured by ELISA in conditioned medium from 24-hour and 48-hour cultures. Crocidolite and chrysotile fibers induced MCP-1 mRNA expression in RPMC which was maximal after 12 hours in the absence of cytokines, but which peaked after 2 hours when RPMC were challenged with asbestos+TNF-alpha or IL-1beta. Both types of asbestos also significantly increased MCP-1 protein secretion after 24 and 48 hours (P <. 0001), an effect that was potentiated by cytokine stimulation. Rats exposed by inhalation to either chrysotile or crocidolite asbestos fibers also had greater amounts of MCP-1 protein in their pleural lavage fluid than did sham-exposed rats. These findings suggest that MCP-1 secretion by RPMC may have a role in the initiation and/or potentiation of asbestos-induced pleural injury.We showed previously that both crocidolite and chrysotile asbestos inhalation induced a persistent macrophage inflammatory response within the pleural space of the rat. We postulated that the stimulus for pleural macrophage recruitment after asbestos exposure was the induction of monocyte chemoattractant protein-1 (MCP-1) synthesis by pleural mesothelial cells. To test this hypothesis, rat pleural mesothelial cells (RPMC) were cultured with or without chrysotile or crocidolite asbestos fibers (8 micrograms/cm2) in the presence (50 ng/mL) or absence of either tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). MCP-1 mRNA expression was assessed by RT-PCR in RPMC cultured for 2 to 24 hours, and MCP-1 protein secretion was measured by ELISA in conditioned medium from 24-hour and 48-hour cultures. Crocidolite and chrysotile fibers induced MCP-1 mRNA expression in RPMC which was maximal after 12 hours in the absence of cytokines, but which peaked after 2 hours when RPMC were challenged with asbestos + TNF-alpha or IL-1 beta. Both types of asbestos also significantly increased MCP-1 protein secretion after 24 and 48 hours (P < .0001), an effect that was potentiated by cytokine stimulation. Rats exposed by inhalation to either chrysotile or crocidolite asbestos fibers also had greater amounts of MCP-1 protein in their pleural lavage fluid than did sham-exposed rats. These findings suggest that MCP-1 secretion by RPMC may have a role in the initiation and/or potentiation of asbestos-induced pleural injury.

Inhalation of acid coated carbon black particles impairs alveolar macrophage phagocytosis

A flow-past nose-only inhalation system was used for the co-exposure of mice to carbon black aerosols (CBA) and sulfur dioxide (SO2) at varying relative humidities (RH). The conversion of SO2 to sulfate (SO4(-2)) on the CBA, at a fixed aerosol concentration, was dependent on RH and SO2 concentration. The effect of the aerosol-gas mixture on alveolar macrophage (AM) phagocytosis was assessed three days following exposure for 4 h. Exposure to 10 mg/m3 CBA alone at low RH (10%) and high RH (85%), to 10 ppm SO2 alone at both RH, and to the mixture at low RH had no effect on AM phagocytosis. In contrast, AM phagocytosis was significantly suppressed following co-exposure at 85% RH, the only circumstance in which significant chemisorption of the gas by the aerosol and oxidation to SO4(-2) occurred. The results suggest that fine carbon particles can be an effective vector for the delivery of toxic amounts of SO4(-2) to the periphery of the lung.