The overall aim of our proposed studies is to reveal the molecular mechanisms of cytotoxicity induced by mustard agents such as sulfur mustard (2, 2'-dichlorodiethyl sulfide). Sulfur mustard is a potent vesicant known to damage lung tissue. Toxicity is the result of alkylation of critical proteins that maintain the integrity of the lung, resulting in inflammation and oxidative stress. However, the underlying mechanisms mediating the actions of sulfur mustard are not fully understood. The thioredoxin (Trx) system, composed of thioredoxin reductase (TrxR) and thioredoxin, is a major thiol-regulating system in cells and plays an important role in many cellular functions including antioxidant defense, redox regulation, and cell growth control. Inhibition of this system can lead to oxidative stress and inhibition of cell proliferation as well as necrosis and apoptosis. In preliminary experiments we found that 2-chloroethyl ethyl sulfide (GEES), a model sulfur mustard vesicant, is a potent inhibitor of thioredoxin reductase in lung epithelial cells. We hypothesize that vesicants target critical sulfhydryl's in thioredoxin reductase and/or thioredoxin resulting in enzyme inhibition and in turn creating oxidative stress in target cells. To test our hypothesis, we initially plan to examine the link between cytotoxicity and the function of the thioredoxin system after vesicant exposure (using the monofunctional vesicant CEES and a bifunctional nitrogen mustard HN-2) in lung epithelial cells. Then, the interaction between vesicants and thioredoxin reductase and/or thioredoxin will be characterized using purified enzymes. PUBLIC HEALTH RELEVANCE: Sulfur mustard, a vesicant first employed during World War I as a chemical weapon, remains a significant civilian and military threat. There are currently no approved treatments for exposure to sulfur mustard and related vesicants. Successful therapy for sulfur mustard poisoning will depend on the development of drugs that target one or more of its sites of action. This research is designed to identify sites of action of sulfur mustard that can be targeted for drug treatment.