Covalent interaction of 5-nitroimidazoles with DNA and protein in vitro: mechanism of reductive activation

Human hepatic microsomal enzymes catalyzed the NADPH-dependent anaerobic reductive activation of [1-14C]metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and [4,5-14C]ronidazole [(1-methyl-5-nitroimidazole-2-yl)methyl carbamate] to species that became covalently bound to proteins. Due to the low efficiency of the enzyme-catalyzed covalent binding of metronidazole, the stoichiometry of anaerobic reductive activation was studied with dithionite as the reductant. Two moles of dithionite was consumed per mole of [1-14C]metronidazole for maximal covalent binding to either DNA or immobilized sulfhydryl groups, demonstrating that four electrons are required for the reductive activation of metronidazole. These data implicate the involvement of a hydroxylamine in covalent binding. Maximal covalent binding of [4,5-14C]ronidazole to DNA also required four-electron reduction, consistent with previous studies of the covalent binding of this agent to immobilized sulfhydryl groups [Kedderis et al. (1988) Arch. Biochem. Biophys. 262, 40-48]. Studies of the covalent binding of variously radiolabeled ronidazole molecules to DNA suggested that the imidazole ring was intact while greater than 80% of the 2-carbamoyl group and the C4 proton were not present in the DNA adduct. Studies of both the chemical and human hepatic microsomal reduction of [4-3H]metronidazole demonstrated that covalent binding occurred with the stoichiometric loss of this label, implicating binding at the C4 position. These results suggest that the reductive activation of 5-nitroimidazoles generally proceeds via four-electron reduction to form hydroxylamines followed by nucleophilic attack at C4.