Detoxification of Electrophilic Chemical Threat Agents by Nucleophilic Scavengers

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DESCRIPTION (provided by applicant): One class of chemical threat agents for which medical countermeasures must be developed, the sulfur and nitrogen mustards (SNMs), exert their toxic effects through chemical reactions that involve an electrophilic intermediate. In this respect they are similar to many widely studied chemical carcinogens, and therefore strategies that have been used to detoxify these carcinogens should be applicable to the mustard agents. The primary strategies to be developed in this Project will block the ability of SNMs to form DNA adducts in exposed cells. We will use a combined in vitro/in vivo approach to determine the efficacy of two groups of potential protective agents: nucleophilic scavenging agents and agents that enhance endogenous conjugation reactions, particularly the addition of glutathione to the electrophile by the glutathione-S transferase (GST) enzymes. Two classes of nucleophilic scavenger will be tested: the dithiopurines (DTP) and ellagic acid. Both of these have been shown to completely detoxify the carcinogen BPDE. DTP has also been shown to detoxify melphalan, a nitrogen mustard, and in preliminary studies we have demonstrated facile reaction of DTP with a sulfur mustard, 2-chloroethyl ethyl sulfide. We will use cultured human cells derived from skin and lung to first test these protective compounds against SNMs, with cytotoxicity and mutagenesis as the primary endpoints. The most efficacious compounds will then be tested in two engineered human tissue models, one representing skin, the other representing airway epithelia, with toxic histopathology as the primary endpoint. Finally, the effective compounds will be tested in vivo in mice, using either topical treatment with subsequent analysis of skin, or intratracheal instillation with subsequent analysis of lung tissue. Toxicity endpoints in the mouse tissues will include histopathology (pyknotic/apoptotic nuclei, edema, vesication, infiltration, fibrosis) and induction of mutations in reporter genes carried in the transgenic mutation-reporter mice. These experiments will test the best scavenger, the best GSH/GST enhancer, and a combination of the two compounds. We will also test the sensitivity of human lymphocytes from 300 individuals to SNMs, and correlate the sensitivity with genotypes measured at several GST genetic loci that are polymorphic in the human population. Other biomarkers of exposure, including generalized protein adducts and a specific protein adduct will be analyzed where appropriate. Relevance: We expect to validate a strategy to efficiently detoxify electrophilic chemical threat agents, such as the SNMs and dimethyl sulfate. This will allow us to begin to formulate novel antidotes that could be used to protect personnel responding to an incident involving SNMs, and to stabilize exposed individuals against further damage. In addition, we will obtain data that indicates whether or not individuals with variants of a particular class of genes, the GSTs, are more susceptible to the effects of SNM toxicants.