The goals of this program are to elucidate the mechanism of action of 2-nitropropane (2-NP), an important industrial chemical, a known human hepatotoxin and a hepatocarcinogen in rats. In the previous 2 years of this program, several mechanisms were examined, including the possibility that the compound is metabolized to 2-azoxypropane, which might represent the proximate carcinogen, and the possibility that nitrite, derived metabolically from 2-NP, reacts with endogenous amines to form carcinogenic N-nitroso compounds. No evidence for the support of these mechanisms was found. However, during these studies the observation was made that 1-electron oxidation of 2-NP anion to the 2-NP radical results in the production of reactive species, presumably hydroxyl radicals, capable of oxidizing thymidine to thymidine glycol, hydroxymethyldeoxyuridine, thymine and thymine glycol in a model system. The 2-NP anion was also found to be a potent mutagen for S. typhimurium TA102, a special tester strain with A-T base pairs at the locus of mutation, indicating that 2-NP can similarly cause oxidative damage to DNA thymidine in a biological system. The present specific aims are to examine the concept that the in vivo production of 2-NP free radicals and/or derived oxygen radicals induces hepatotoxicity and hepatocarcinogenicity by oxidative damage to subcellular components including DNA. To this end, the formation of the 2-NP dimer, as evidence for the conversion of 2-NP to a free radical form, will be examined in vivo, as will the ability of 2-NP to cause depletion of liver GSH, to cause lipid peroxidation and to induce DNA damage in the form of strand breaks, altered bases and 2-NP radical-DNA base adducts. The ability of agents which modify liver GSH levels on the toxic and genotoxic effects of 2-NP will be examined. Since the proposed free radical mechanism implies a threshold effect, the dose dependence of 2-NP on carcinogenicity in Sprague Dawley rats and B6C3F1 mice will be determined, as will the effects of depletion and supplementation in the important cellular antioxidants vitamin E and selenium. In related studies, the hypothesis that acetoxime is hepatocarcinogenic through metabolic N-oxidation to 2-NP, and hence to damaging free radicals, will also be tested. It has been estimated that more than 185,000 workers have been occupationally exposed to 2-NP. The clarification of the mechanism were by 2-NP causes cellular damage, and of factors influencing this process, can be important for the development of prophylactic and antidotal agents.
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