In the current study we have shown that Balb/C mice exposed to Cl2 gas for 5 min develop concentration-dependent airway hyperresponsiveness to inhaled aerosolized MCh. At concentrations of Cl2 greater than 100 ppm there is evidence of epithelial damage with flattening of the cells and the shedding of ciliated cells into the bronchoalveolar lavage fluid. However, at a concentration of Cl2 (100 ppm), despite the lack of gross morphological changes in epithelial cells there was still a substantial degree of airway hyperresponsiveness, an effect potentially attributable to increased oxidative stress. The effect of Cl2 on airway function was attenuated by pre-treating the mice one hour before Cl2 exposure with an intraperitoneal injection of DMTU. Treatment with DMTU 1 hour after exposure to Cl2 also ameliorated the adverse effects on airway function. Oxidative injury to lung tissue was detected 24 hours post-Cl2 exposure and indicated by and increase lipid peroxidation in Cl2 exposed mice, an effect attenuated by pre- or post-Cl2 treatment with DMTU. Additionally, DMTU treatment maintained GSH/GSSG levels at those of control mice, whereas Cl2 only treated mice showed significant changes in both GSH and GSSG at various time points.
Airway hyperresponsiveness has been previously demonstrated to follow Cl2
exposure in both rat and mouse models of irritant induced asthma [15
]. Pathological changes including airway remodeling occur following a single exposure to a high concentration of Cl2
in rats [17
]. It seems likely that epithelial damage is a major contributor to the altered responsiveness to inhaled MCh. The epithelium could serve as a barrier that could reduce access of MCh to the smooth muscle or might attenuate the responsiveness to MCh through the release of relaxant substances such as NO or prostaglandins [18
]. The mechanism of AHR following Cl2
may be similar to that of ozone in that both forms of injury are associated with oxidant damage to the tissues. Natural killer cells and interleukin-17 have been shown recently to be essential in the protection against airway damage and hyperresponsiveness following repeated ozone exposures [21
potentially causes toxicity through its highly reactive nature. However, it is also know to cause damage through the generation of hydrochloric acid (HCl). Indeed HCl has been shown to cause airway hyperresponsiveness in mice when administered into the airways, by mechanisms that have been suggested to relate to epithelial barrier function. However, it has been shown that HCl is much less toxic than Cl2
so it is likely that the effects of Cl2
induced oxidants are more likely to account for its adverse effects [22
Irrespective of the mechanism of Cl2
induced airway hyperresponsiveness, DMTU was highly effective in preventing its development when given either as a pre-treatment or as a rescue treatment. Assuming that the therapeutic effects of DMTU are indeed mediated by anti-oxidant properties, the data suggest that the initial direct oxidative stress caused by Cl2
is only part of the oxidative burden and that another source of reactive oxygen is important in the time period between 1 and 24 h following Cl2
exposure. For example, secondary activation of neutrophils, macrophages or epithelium and various chemokines, cytokines and growth factors they secrete could conceivably contribute to airway damage in a mechanism similar those shown for respiratory viral infection [23
Measures of oxidant injury such as nitric oxide production, as reflected in BAL nitrates/nitrites, and protein carbonylation were not detectably different from control animals at 24 hours after Cl2
exposure, consistent with a relatively mild injury compared to previous results [7
]. However, presence of oxidative stress was apparent following assessment of lung tissue levels of 4-HNE, an indication of lipid peroxidation. 4-HNE levels were reduced to baseline by pre- and post-Cl2
treatment with DMTU, suggesting that lipid peroxidation is a prolonged effect of exposure to Cl2
further supporting the conclusion that the amelioration of markers of airway injury is likely mediated by anti-oxidant properties of DMTU.
Glutathione is an important endogenous antioxidant and changes in its intracellular and extracellular concentrations are expected following an oxidant challenge such as Cl2
. Generally oxidant stress is noted to diminish GSH both intracellularly and extracellularly in the lung (reviewed in [24
]) although glutathione increases as an adaptive response to oxidative stress associated for example with cigarette smoking or pulmonary infection [25
]. We found that Cl2
exposure induced rapid and transient changes in glutathione concentrations. Ten minutes following exposure there was a surge in both intra- and extra-cellular GSH levels in BAL, presumably attributable to GSH synthesis and export into the extracellular milieu. Additionally, Cl2
may induce lysis of pulmonary cells, especially epithelial cells which might also contribute to the large amount of extracellular GSH. Epithelial cells are known to contain high levels of GSH [25
] and high doses of Cl2
have been shown to cause epithelial cell shedding and/or lysis. However the changes in GSH observed in the current experiment occurred in the absence of significant changes in epithelial cell counts in BAL fluid or in epithelial cell numbers enumerated in the airway walls themselves. The changes in GSH were transient and had resolved by 1 hour. The rapid rise in GSH was prevented by pre-treatment with DMTU prior to Cl2
exposure, suggesting a measure of relief against the effects of oxidative stress.
In addition to the early spike in GSH concentration in BAL cells and fluid, we also noted a significant increase in GSH in its oxidized form, glutathione disulfide (GSSG), both intra-and exrtacellularly at 10 minutes, presumably indicative of oxidative stress in the lung. These changes were abrogated by DMTU supporting the idea that the mechanism of protection was through neutralization of oxygen metabolites. Furthermore, the protection provided by delayed treatment with DMTU further suggests that delayed oxidative stress is also a significant contributor to the response to injury. By 1 and 24 hours, GSH levels were restored but GSSG levels showed a significant decrease in chlorine exposed groups. It is not clear what the significance of this finding is for airway function. Despite the GSSG levels being depleted at this time point, the ratio of GSH/GSSG was higher in chlorine exposed mice compared with controls and DMTU treated animals. The anti-oxidants ascorbic acid, desferroxamine and N-acetyl-L-cysteine have been show to ameliorate the injury caused by Cl2
in the rat [9
]. In these experiments there was evidence of depletion of GSH by Cl2
, an observation that we have not confirmed. However the exposure in the rat was substantially greater (400 ppm for 30 minutes).
Consideration of oxidative stress as a target in irritant-induced asthma caused by potent oxidants is reasonable. However, oxidative stress-induced damage may also contribute to other forms of asthma. Asthmatic subjects manifest evidence of oxidative stress, as evidenced by a variety of changes including increased superoxide generation from leukocytes, increased total nitrites and nitrates, increased protein carbonyls, increased nitric oxide in exhaled breath condensate, increased lipid peroxidation products and decreased protein sulfhydryls in plasma [26
]. They also show increased superoxide dismutase activity in red blood cells, increased total blood glutathione, and decreased glutathione peroxidase activity in red blood cells and leukocytes. A recent epidemiological study of childhood asthma demonstrated significant decreases in glutathione and amino acid precursors of glutathione as well as various other components of both enzymatic and non-enzymatic endogenous antioxidant defense mechanisms [27
]. Thioredoxin, a reducing protein, may also inhibit experimental allergic asthma and airway remodeling [28
In conclusion, exposure to modest levels of Cl2 (100 ppm) leads to an increase in airway responsiveness in mice. Mice exposed to Cl2 showed increases in total inflammatory cells, in particular neutrophils and lymphocytes. Despite lack of increases in nitrate/nitrite or carbonylated proteins, lipid peroxidation levels (4-HNE) were significantly higher in Cl2 exposed animals. Importantly, there was also evidence of a salutary treatment effect when DMTU was administered as late as 1 hour after the exposure to Cl2 suggesting that oxidative damage is an ongoing process following the initial injury. Treatment with anti-oxidants shortly after acute exposure to highly irritant oxidant substances such as Cl2 may have therapeutic utility.