Research Project 1. Vesicant-induced Skin Injury Key personnel: Jeffrey D. Laskin, Ph.D., Distinguished Professor, Rutgers University School of Public Health Donald Gerecke, Ph.D., Associate Professor, Rutgers University School of Pharmacy Laurie Joseph, Ph.D., Associate Professor, Rutgers University School of Pharmacy Project Summary/Abstract Mustard vesicants, including sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) and nitrogen mustard (bis(2-chloroethyl)methylamine, HN2) are cytotoxic blistering agents synthesized for chemical warfare. Because they contain highly reactive electrophilic chloroethyl side chains, they readily react with cellular macromolecules like DNA forming monofunctional and bifunctional adducts. By targeting DNA, mustards can compromise genomic integrity by causing single and double strand breaks. Double strand breaks are particularly toxic because they disrupt the physical continuity of the DNA duplex. To protect against genotoxicity following exposure to mustards, cells initiate a DNA damage response (DDR). This involves activation of signaling cascades including ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3-related) and DNA-PKcs (DNA-dependent protein kinase, catalytic unit). Signaling induced by the DDR leads to the recruitment and activation of repair related proteins such as phospho H2AX and phospho p53 to sites of DNA lesions. As a transcriptional activator, p53 can also induce DNA repair proteins, it can also interact with many signaling pathways including those important in cell cycle control and apoptosis. Excessive DNA modifications by mustards can overwhelm DNA repair leading to cytotoxicity and tissue damage. Herein we will investigate DDR signaling pathways induced by mustard vesicants in human keratinocytes and in mouse skin. Two FDA approved drugs, Enoxacin and Amifostine, reported to enhance DNA repair, will be analyzed for their ability to enhance DDR signaling in keratinocytes. Investigations into mechanisms by which Enoxacin and Amifostine enhance DNA repair and potentially protect against cytotoxicity may lead to the development of a novel class of mustard countermeasures.