Abstract The threats to chemical warfare-associated agents, including arsenicals and nitrogen mustards are increasing exceedingly, and no direct antidote is currently available to mitigate the deleterious cutaneous and systemic responses to prevent mortality. Though the associated cytotoxic effects of most of these agents are mediated due to their ability to act as alkylating agents, a significant knowledge gap exits in the understanding of detailed molecular mechanisms of how these vesicants cause cutaneous and systemic toxic effects, and thus, the development of antidotes. The current proposal is built upon the scientific premise that exposure to various pro- oxidative stressors, including alkylating chemotherapeutic agents, and thermal burn injuries (TBI) when coupled with ethanol (ETOH) produce the potent bioactive lipid mediator, Platelet-activating factor (PAF) by both enzymatically and non-enzymatically via reactive oxygen species (ROS). Studies, including ours, have shown that these PAF agonists induce local inflammation, as well as multi-system organ dysfunction (MOD). Importantly, recent studies have indicated that small membrane-bound vesicles known as microvesicle particles (MVP), generated via acid sphingomyelinase (aSMase) enzyme, are released from cells in response to various stressors. These MVP can act as potent signaling agents due to their ability to carry nuclear and cytoplasmic components. More importantly, the current proposal is built upon our discovery that chemotherapeutic agents, and TBI+ETOH via their ability to damage keratinocytes, generate PAF agonists which travel via MVP to induce the local (cutaneous) and systemic responses. Using antioxidants and PAFR-expressing/null cell lines and pharmacologic/genetic inhibition of aSMase enzyme, our studies have implicated the involvement of the PAFR signaling in aSMase activation resulting in PAF-laden MVP release. Based upon these compelling evidences, we hypothesize that chemical warfare-associated agents via their ability to generate ROS, produce PAF agonists and MVP from human and murine keratinocytes in a PAFR-aSMase-dependent manner, which mediate the cutaneous cytotoxic as well as systemic MOD effects. Two aims are proposed to test our hypothesis. Aim 1 will use validated in vitro and ex vivo models and pharmacologic agents to determine the roles of the PAFR, downstream signaling pathways, and aSMase enzyme in chemical warfare-associated agents-induce PAF agonists and MVP generation. Agents to be tested are nitrogen/sulfur mustards and an arsenical. Aim 2 will use PAFR/aSMase-expressing and deficient mouse models, as well as pharmacological inhibitors of PAFR and aSMase to determine the role of PAF-laden MVP generation in the local and systemic responses by topical nitrogen mustargen exposure. Successful completion of this project will (i) fill important mechanistic gaps in and validate novel tools to allow the modulation of PAF-laden MVP generation to mitigate chemica...