CHEMICAL TOXICOLOGY OF ISOCYANATES AND FORMAMIDES

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The central goal of the proposed research is to study chemical aspects of the toxicology of methylisocyanate (MIC), a highly reactive and toxic agent employed industrially in the preparation of carbamate pesticides, and the compound whose accidental release in the city of Bhopal, India, brought about the worst industrial tragedy recorded in history. The project also aims to define the role of MIC and related electrophilic carbamoylating species as mediators of the hepatic damage induced by N-methylformamide (NMF), an experimental antitumor agent, and N,N-dimethylformamide (DMF), a widely-used industrial solvent. The participation of toxic isocyanates in the biological fate and mechanism of action of three antineoplastic drugs, viz. 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU), caracemide and NMF, also will be investigated, with an emphasis on the role of their glutathione (GSH) conjugates as active carbamoylating agents. The specific aims of the project are as follows: (a) To show that, following inhalation exposure of rats to MIC vapor, the glutathione conjugate S-(N-methylcarbamoyl) glutathione (SMG) is present in bile and the corresponding mercapturic acid, S-(N-methylcarbamoyl) -N-acetylcysteine. (AMCC), is excreted in urine. (b) To demonstrate that SMG carbamoylates serum proteins and to establish the nature of the resulting covalent adducts. (c) To establish a mechanistic basis for the rational development of antidotes to MIC poisoning, and to explore the utility of N-acetylcysteine for such use. (d) To elucidate the pathway(s) of metabolic activation of NMF and DMF. (e) To challenge the accepted view that metabolic hydroxylation of DMF at the alpha-carbon atom represents a route of detoxification of this compound. (f) To investigate whether caracemide liberates MIC in vivo, and to establish the origin of the released isocyanate. (g) To determine the in vivo antitumor activity, and the in vitro mutagenicity, of SMG, AMCC and the corresponding cysteine conjugate, S-(N-methylcarbamoyl) cysteine (SMC). (h) To assess the ability of GSH conjugates of reactive isocyanates to serve as active site-directed inhibitors of glutathione reductase. A comprehensive series of in vitro and in vivo experiments is proposed which will employ rats and mice as animal models. Structure elucidation of polar metabolites of the compounds of interest, and of their covalent adducts to proteins, will be based on the use of state-of-the-art analytical techniques, including electrospray ionization and high performance tandem mass spectrometry. Collectively, the proposed studies will provide fundamental information on the chemical toxicology of isocyanates and formamides, and will contribute to our understanding of the role of GSH-dependent processes in the transport reactive, toxic chemicals in vivo.