Recent studies suggest that oxidative stress contributes to the dermatotoxicity of many chemical hazards including vesicants such as sulfur mustards.  In skin cells, oxidative stress arises via the formation of reactive oxygen intermediates (ROI) generated by a variety of inflammatory cytokines and lipid mediators.  In previous studies we showed that topical antioxidants and anti-inflammatory agents readily suppress chemical-induced injury in a  mouse skin model.  These agents also block edema and production of cytokines including TNF-a, IL-1 and IL-6, as well as cyclooxygenase-2 and prostaglandin E synthase.  In the present studies we characterized vesicant-induced oxidative stress in cultures of differentiating keratinocytes and determined if agents that induce intracellular antioxidants protect against cytotoxicity.  Primary mouse keratinocytes were cultured in low calcium Keratinocyte Growth Medium.  To initiate differentiation, calcium (0.15 mM) was added directly to the culture medium.  After 24 hr, cells were treated with sulforaphane (5-10 mM), an inducer of antioxidant response genes including thioredoxin reductase, followed by increasing concentrations of half mustard (2-chloroethyl ethyl sulfide, CEES) or nitrogen mustard.  We found that sulforaphane was a potent inducer of thioredoxin reductase mRNA and protein in the cells as measured by real-time PCR and enzymatic activity, respectively.  Despite increases in thioredoxin reductase activity, sulforaphane provided minimal protection against vesicant toxicity.  This was due to the inhibitory actions of CEES on thioredoxin reductase activity, a critical selenium-containing enzyme important in protecting cells against oxidative stress.  Inhibitory activity was found with insulin as the substrate as well as 5, 5’-dithiobis(2-nitrobenzoic acid).  The inhibitory effects were also observed using keratinocyte enzyme, as well as purified rat liver and human recombinant thioredoxin reductase.  The effects of CEES were not dependent on selenium because a mutant human recombinant enzyme without selenium was also inhibited by the vesicant.  Taken together, our results suggest that vesicating agents not only cause cell damage but also inhibit wound repair processes by blocking a key enzyme required for maintaining cellular redox status.  Thioreductase reductase may serve as an important target to block vesicant-induced skin toxicity. *This work was supported by the UMDNJ/Rutgers University CounterAct Research Center of Excellence NIH grant U54AR055073.