Serine and Cysteine Proteases in Sulfur Mustard-Exposed Hairless Mouse Skin: Enzymatic Activity and Inhibition Profiles

Inflammatory reactions associated with sulfur mustard (bis(2-chloroethyl)sulfide, SM)-induced skin pathology, including the release of potent proteolytic enzymes capable of destroying cutaneous connective tissue proteins, are not well defined in vivo. In this study, protease activities were measured as potential indicators of SM-induced skin injury in a hairless mouse sulfur mustard vapor exposure model. We also investigated the effects of synthetic protease inhibitors on SM-induced proteolytic activity. Skin homogenates prepared from hairless mouse dorsal skin exposed to SM vapor (1.4 g/m(3)) were assayed for serine and cysteine protease activities using synthetic chromogenic and fluorogenic substrates. Skin samples were obtained 24 h after SM challenge from animals exposed for either 6 or 12 min, while controls were obtained from unexposed skin. With 6 min SM-exposed skin, significantly higher elastase, tryptase, cathepsin B, cathepsin L, and calpain II enzymatic activities were detected compared to the unexposed control. With 12 min SM-exposed skin, significantly higher activities of elastase, tryptase, and calpain II were detected compared to the unexposed control. No significant differences were found between 6 and 12 min SM-induced protease activities, except 6-min cathepsin L, which was higher than the 12 min. Effective inhibitors include the general serine protease inhibitor 3,4-dichloroisocoumarin and specific peptide phosphonate serine protease inhibitors, in addition to peptide ketoamide transition-state cysteine protease inhibitors. These data demonstrate that proteases are involved in the process of SM-induced tissue injury, and suggest that the identification of specific inhibitors should be useful for studying cytotoxic mechanisms and reducing tissue injury caused by SM exposure.