In an effort to develop medical countermeasures for exposure of military personnel and civilians, we have been defining the molecular events leading to SM vesication. We have blocked SM-induced toxicity using a genetic approach, and are now adopting a chemical inhibitor-based strategy to block these same pathways. We have made significant headway in elucidating several important pathways by which SM induces cell death in cultured keratinocytes and dermal fibroblasts, as well as in intact mouse and grafted human skin (Rosenthal et al., J Invest Dermatol. 117, 1566-73, 2002). We found that SM induces markers of terminal differentiation as well as apoptosis in NHEK and observed activation of a death receptor pathway for apoptosis, in which Fas receptor and Fas ligand (FasL) play a role, as well as a calmodulin (CaM)/Bcl-2-mediated mitochondrial apoptotic pathway (Rosenthal et al, J Invest Dermatol. 111, 64-71, 1998). Following SM treatment, keratinocytes significantly upregulate levels of both Fas receptor and FasL, which is soon followed by the rapid activation of the upstream caspase-8, mediated by recruitment of the adaptor protein FADD. This is followed by the activation of the executioner caspase-3, -6, and -7. Retroviral constructs expressing a dominant-negative FADD (FADD-DN), as well as antisense to CaM were constructed to block each of the apoptotic pathways. Keratinocytes with reduced levels of CaM or FADD signaling were more resistant to SM-induced PARP cleavage and processing of caspases-3, -6, -7, and -8 into their active forms. Significantly, we have found that altering these pathways in human skin grafted onto nude mice reduces vesication and tissue injury in response to SM (Rosenthal et al., J. Biol. Chem., 278, 8531-8540, 2003). With this understanding of the biochemical pathways for SM vesication, and with the use of our human skin graft system, we are now attempting to alleviate SM-induced toxicity by directly testing specific pharmaceutical inhibitors of these pathways. Towards this end, we have found that pretreatment of keratinocytes with a peptide inhibitor of caspase-3 (Ac-DEVD-CHO) suppressed SM-induced downstream markers of apoptosis. To further analyze the importance of inhibiting the death receptor pathway to block SM toxicity, we utilized Fas- or tumor necrosis factor receptor (TNFR)-neutralizing antibodies to inhibit the recruitment of FADD to the death receptor complex and block the Fas/tumor necrosis factor receptor pathway following SM exposure. Keratinocytes pretreated with Fas- but not TNFR-blocking antibody, demonstrated markedly decreased caspase-3 activity and reduced apoptotic annexin V binding when treated with SM. These results indicate that the death receptor pathway plays a pivotal role in SM-induced apoptosis and is therefore a target for therapeutic intervention to reduce SM injury.