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Adult Sprague-Dawley (SD) male rats were exposed for a single 3 h period to air, ozone (O₃) or O₃) followed by tobacco smoke (O₃/TS). For pulmonary effects, bronchoalveolar lavage (BAL) cells and fluid were analyzed. Data revealed a significant increase in polymorphonuclear leukocytes (PMN), total protein and albumin concentrations in the O₃ group, reflecting inflammatory and toxic responses. A subsequent exposure to TS attenuated PMN infiltration into the airspaces and their recovery in the BAL. A similar reduction was observed for BAL protein and albumin in the O₃/TS group, but it was not statistically significant. We also observed a significant increase in BAL total antioxidant capacity following O₃ exposure, suggesting development of protective mechanisms for oxidative stress damage from O₃. Exposure to TS attenuated the levels of total antioxidant capacity. Lung tissue protein analysis showed a significant reduction of extracellular superoxide dismutase (EC-SOD) in the O₃ or O₃/TS group and catalase in the O₃/TS group. TS further altered O₃-induced EC-SOD and catalase protein expression, but the reductions were not significant. For effects in the central nervous system (CNS), we measured striatal dopamine levels by HPLC with electrochemical detection. O₃ exposure produced a nonsignificant decrease in the striatal dopamine content. The effect was partially reversed in the O₃/TS group. Overall, the results show that the toxicity of O₃ in the lung is modulated by TS exposure, and the attenuating trend, though nonsignificant in many cases, is contrary to the synergistic toxicity predicted for TS and O₃, suggesting limited cross-tolerance following such exposures.

Inflammation is a common feature in the pathogenesis of cigarette smoke-associated diseases. The recruitment of inflammatory cells into the lung following cigarette smoke exposure presents a risk of tissue damage through the release of toxic mediators, including proteolytic enzymes and reactive oxygen species. This review represents a toxicological approach to investigation of cigarette smoke-induced lung injury, with a focus on laboratory studies and an emphasis on inflammatory mechanisms. The studies discussed in this review analyze the role of inflammation and inflammatory mediators in the development of injury. In cases where information relating to cigarette smoke is limited, examples are taken from other models of lung injury applicable to cigarette smoke. The primary aim of the review is to summarize published work so as to permit (1) an evaluation of chronic lung injury and inflammatory responses in animal models, (2) a discussion of inflammatory mediators in the development of chronic injury, and (3) identification of immunological mechanisms of injury. These studies discuss the currently understood roles of cytokines, cell adhesion molecules, and oxidative stress in inflammatory reactions and lung injury. A role for lipocortin 1 (annexin 1), a naturally occurring defense factor against inflammation, is discussed because of the possibility that impaired synthesis and degradation of lipocortin 1 will influence immune responses in animals exposed to cigarette smoke either by augmenting T helper cell Th1 response or by shifting Th1 to Th2 response. While Th1 augmentation will increase the risk for development of emphysema, Th1 to Th2 shift will favor development of asthma.

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.

The airway epithelial lining serves as an efficient barrier against penetration of exogenous particles and macromolecules. Disruption of this barrier following O3 exposure represents a state of compromised epithelial defenses leading to increased transmucosal permeability. Although the barrier disruption following an acute exposure is transient in nature, the brief period of disruption caused by O3, an oxidant air pollutant, provides an opportunity for facilitated entry of a potentially toxic particulate copollutant(s) across the airway epithelia. The subsequent deposition and retention of the copollutant(s) in the subepithelial compartment for prolonged periods adds the risk of injury due to chronic exposure following an acute episode. Toxicological studies from several laboratories have demonstrated alterations in epithelial permeability, suggestive of barrier disruption, in animals and humans exposed to O3. Inflammatory cells represent another important component of pulmonary defenses, but upon activation these cells can both induce and sustain injury. The recruitment of these cells into the lung following O3 exposure presents a risk of tissue damage through the release of toxic mediators by activated inflammatory cells. Several studies have reported concomitant changes in permeability and recruitment of the inflammatory cells in the lung following O3 exposure. In these studies, an inflammatory response, as detected by an increase in the number of polymorphonuclear leukocytes in the bronchoalveolar lavage (BAL) or in lung parenchyma, was accompanied by either an increased tracer transport across the airway mucosa or an elevation in the levels of total protein and/or albumin in the BAL. The magnitude of response and the time at which the permeability changes and inflammatory response peaked varied with O3 concentration, exposure duration, and the mode of analysis. The responsiveness to O3 also appeared to vary with the animal species, and increased under certain conditions such as physical activity and pregnancy. Some of the effects seen after an acute exposure to O3 were modified upon repeated exposures. The responses following repeated exposures included attenuation, persistence, or elevation of permeability and inflammation. Mechanistic studies implicate chemotactic factors, cellular mediators, and cell-surface-associated molecules in the induction of inflammation and lung injury. In discussing these studies, this review serves to introduce the mucosal barrier functions in the lung, evaluates inflammatory and permeability consequences of O3, addresses mechanisms of inflammatory reactions, and offers alternate viewpoints.

Ozone (O3) exposure of rats increases airway epithelial permeability. We hypothesized that this increased permeability may be mediated by the epithelial cell cytoskeleton. To test this hypothesis, we studied the effect of cytoskeletal disruption on the transmucosal transport of tracers from airway lumen to blood and compared the results with the effects of O3 exposure. No increase in transport occurred following disruption of microtubules by vinblastine, but disruption of microfilaments with cytochalasin D resulted in increased transport of radiolabeled tracers [99mTc- and 111In-labeled diethylenetriamine-pentacetate (DTPA) and 125I-labeled bovine serum albumin (BSA)]. In control rats, both horseradish peroxidase (HRP) and BSA, localized by cytochemistry and autoradiography, respectively, were detected on the epithelial cell surfaces and in endocytic vesicles. In rats treated with cytochalasin D or exposed to O3, the tracer molecules also penetrated the intercellular spaces, though the apical tight junctions remained devoid of the tracers. Increased numbers of endocytic vesicles containing HRP and aggregation of 125I-labeled BSA autoradiographic grains in the subepithelial region were also seen after either treatment. We conclude that destabilization of cytoskeletal elements following O3 exposure is a possible mechanism of increased transmucosal transport, which may be a combined effect of accelerated transport through both endocytic and paracellular pathways.

Full-length tracheas from Sprague-Dawley rats were exposed to cytoskeleton-active drugs in short-term organ culture, and the permeability of the tracheal epithelium was measured by instilling radiotracers into the lumen and assay of the radioactivity appearing in the external bathing medium. In vitro treatment with cytochalasin D (cyto D, 2-10 x 10(-6) M) increased the rate of movement of [14C]mannitol across the epithelium. Exposure to vinblastine (VB, 10(-4) M) alone had no significant effect. However, VB in combination with cyto D increased the permeability in a dose-dependent manner. In vivo exposure to ozone (O3, 0.8 or 2.0 ppm, 2 h) had only a slight effect on the rate of movement of the tracer as measured in vitro immediately after exposure. At 24 h postexposure there was no significant difference in permeability between ozone- and air-exposed tracheas. Prior in vivo O3 exposure sensitized the tracheas to the in vitro effects of cyto D; treatment of O3-exposed tracheas with cyto D immediately after O3 exposure produced a greater than additive effect on permeability measured in vitro. VB at concentrations up to 10(-4) M had no enhancing effect on permeability in O3-exposed tracheas. Sham exposure to clean air did not affect permeability compared to untreated (shelf) controls. Electron microscopic studies demonstrated penetration of horseradish peroxidase into intercellular spaces in the tracheas treated in vitro with cyto D or cyto D plus VB. Cyto D is known to affect intracellular microfilaments that have attachments at or near the cell surface, while VB affects microtubules associated with internal cellular structures. Therefore, the synergistic effect on tracheal permeability observed with O3 and cyto D, but not with O3 and VB, suggests that O3 may change cell surface structures associated with the microfilamentous cytoskeleton.