The respiratory tract is an important target for SM-induced pathology, and understanding the biochemical responses to exposure could lead to improved therapeutic measures to combat the long-term adverse responses. There is relatively little quantitative data on human inhalation exposures. Some early human exposures performed in 1918 and others performed in 1941–1942 and summarized by Watson (2006)
suggest that exposure to SM at concentration × time doses of 1–100 mg × min/m3
caused increasingly severe ocular effects and skin burns, with some evidence of mucosal exfoliation in the nasopharynx. Early exposures of rabbits summarized in the same review indicated that respiratory lesions occurred at exposures of approximately 600 mg × min/m3
. However, methods for assessing the exposure concentrations in this era were very crude. For obvious ethical reasons, no recent human experimental exposures have been performed, and assessments of exposures in battlefield or accidental situations are very limited.
In addition, data for lung injury in animal inhalation exposures until recently have been lacking, in part due to the reactivity of the agent with the upper respiratory tract and resulting uncertainties regarding the doses to the distal lung. The upper respiratory tract in the usual rodent models is much more complex and convoluted than that of humans, and thus bypassing the nasopharynx by intratracheal intubation has been demonstrated to be an effective method of exposing the lung of the rodents to SM vapor (Anderson et al., 1996
; Anderson et al., 1997
; Weber et al., 2009
). Under these conditions, Anderson et al. (1997)
showed extensive evidence of injury as early as 4–6 h following exposure to vapors generated from 0.35 mg of SM in 100 μl of ethanol over the course of a 50-min exposure (actual vapor concentration was not reported; Anderson et al., 1996
; Anderson et al., 1997
). More recently, intratracheal inhalation of 150 mg/m3
SM vapor exposure for 10 min (1500 mg/min/m 3
) causes extensive pathology from 1 day post-exposure (Weber et al., 2009
The results indicate that SM at the concentrations used was noncytotoxic. In contrast, an apparent increase in cell number or WST reduction, possibly suggesting accelerated proliferation or increased metabolic activity was observed at the intermediate concentrations, although this did not reach significance. However, these results indicate that the concentrations tested were generally not overtly toxic.
Inflammatory responses occur as a result of SM exposure in humans and in animal models. It was therefore anticipated that pro-inflammatory cytokine responses would increase. Of the 30 cytokines analyzed in the multiplex, most were below the limits of detection. A statistically significant and dose-dependent increase in IL-8, a key neutrophil chemoattractant chemokine, was observed. IL-6 was also increased in a dose-dependent way albeit not to statistically significant levels. In addition, there were trends to increases in both MCP-1 and G-CSF, which have been demonstrated to be increased in bronchoalveolar lavage fluid from patients with pulmonary fibrosis thought to have been produced due to exposure to SM (Emad & Emad, 2007a
; Emad & Emad, 2007b
). Although individually, the lack of statistical significance makes these results of less interest than the increases in IL-8, taken together they are consistent with mild pro-inflammatory responses. The VEGF response was particularly interesting: this angiogenesis-stimulating cytokine was increased at the lowest concentration of the vapor, but returned towards baseline at the two higher concentrations.
Proteolytic activity has been implicated in the pronounced vesicant response and loss of epithelial cells from the basal lamina in skin and lung tissue. Interestingly, the primary differentiated cultures also showed significant increases in MMP-13, and nonsignificant increases in MMP-1, -7, and -9 at the highest concentrations of the vapor. As suggested for the cytokine results, the consistency among these responses provides some confidence of a trend, that perhaps at higher exposure concentrations would become significant.
SM is known to react with reduced glutathione (Langford et al., 1996
). Depletion of this important cellular antioxidant could lead to activation of redox-dependent transcription factors. HO-1 is a well-established marker for oxidative stress (Li et al., 2002
). However, at the vapor concentrations used here, these cultures did not show significant changes in HO-1 levels.
In summary, the present study indicates activation of inflammatory and proteolytic responses that could contribute to the pathophysiology of this potential chemical weapon, under conditions more closely mimicking the physiological exposures to inhaled SM than any prior studies. Knowledge of these processes could provide insight that could lead to improved strategies for therapeutic intervention. Specifically, these results suggest that interventions in pro-inflammatory cytokine signals or MMP activity could be useful targets for therapeutics.