Oxidative Denitrification of 2-Nitropropane in Mouse Liver Microsomes (Cumene Hydroperoxide, Cytochrome P-450).

2-Nitropropane (2NP) is a widely used industrial chemical to which a significant number of workers may be exposed. Human liver toxicity has been attributed to 2NP. Inhalation studies in rats have shown the presence of liver tumors. Hepatic metabolism resulting in nitrite release has been shown and , through a cytochrome P-450 (P450) mediated oxidative mechanism, acetone was found as second product. Denitrosation of nitrosamines by a P450-mediated reductive mechanism has also been reported. This pathway could potentially contribute to 2NP metabolism. It was the purpose of this dissertation to elucidate the reaction mechanism involved in hepatic P450-mediated 2NP metabolism. NADPH-dependent nitrite release from 2NP similar to that reported in chemically induced rat liver microsomes was observed in uninduced liver microsomes from five mouse strains. All subsequent investigations were performed in hepatic microsomes from male CD(,1) mice. Involvement of P450 was confirmed by in vivo P450 induction and destruction, in vitro inhibition by the P450 inhibitors SKF-525A, metyrapone and carbon monoxide, and the presence of a Type I binding spectrum. Acetone was identified as the second product of the reaction. The involvement of multiple P450 isozymes was suggested by two pH and substrate optima, by differential response to inhibitors dependent on pH, and by pH differences in specific activity within mouse strains. Further support for an oxidative mechanism was shown by the ability of cumene hydroperoxide (CHP) supplemented hepatic microsomes to support the reaction with nitrite and acetone as products. During the CHP-supported reaction, P450 remains in the oxidized state thus excluding the possibility of a reductive mechanism contributing to 2NP metabolism. This is the first report of CHP-supported denitrification of a xenobiotic. The involvement of multiple P450 isozymes was suggested by two pH and substrate optima and differential response to the inhibitors n-octylamine, butylated hydroxyanisole and SKF-525A. The predominance of an oxidative mechanism for 2NP metabolism in uninduced mouse liver microsomes is strongly suggested by these investigations, though the possibility of some reductive metabolism in the NADPH-dependent reaction cannot be excluded.PhDHealth sciencesUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/160692/1/8520939.pd