Oxidation of the Ketoxime Acetoxime to Nitric Oxide by Oxygen Radical-Generating Systems

Abstract

Ketoximes undergo a cytochrome P450-catalyzed oxidation to nitric oxide and ketones in liver microsomes. In addition, nitric oxide synthase (NOS) can catalyze the oxidative denitration of the >CN-OH group of amidoximes. The objective of this work was to characterize the oxidation of a ketoxime (acetoxime) and to assess the ability of NOS to catalyze the generation of nitric oxide/nitrogen monoxide ( · NO) from acetoxime. Acetoxime was oxidized to NO−2 (and NO−3) by microsomes enriched with several P450 isoforms, including CYP2E1, CYP1A1, and CYP2B1. Nitric oxide was identified as an intermediate in the overall reaction. Superoxide dismutase and catalase significantly inhibited the reaction. Exogenous iron increased the microsomal generation of NO−2 from acetoxime, while metal chelators (desferrioxamine, EDTA, DTPA) inhibited it. A Fenton-like system (Fe2+ plus H2O2, pH 7.4) consumed acetoxime with production of NO−2 and NO−3, whereas oxidation by superoxide or by H2O2 was inefficient. The results presented suggest a role for hydroxyl radical-like oxidants in the oxidation of acetoxime to nitric oxide. O-Acetylacetoxime and O-tert-butylacetoxime were not oxidized by a Fenton system or by liver microsomes to any significant extent. Formation of the 5,5′-dimethyl-1-pyrroline-N-oxide/ · OH adduct by a Fenton system was significantly inhibited by acetoxime, while O-acetylacetoxime and O-tert-butylacetoxime were inactive. These results suggest that the · OH-dependent oxidation of acetoxime initially proceeds via abstraction of a hydrogen atom from its hydroxyl group, as opposed to the oxidation of its >CN- function. HepG2 cells with low levels of expression of P450 did not significantly produce NO−2 from acetoxime, while HepG2 cells expressing CYP2E1 did, and this generation was blocked by a CYP2E1 inhibitor. Acetoxime was inactive either as a substrate or as an inhibitor of iNOS activity. These results indicate that reactive oxygen species play a key role in the oxidation of acetoxime to · NO by liver microsomes by a mechanism involving H abstraction from the OH moiety by hydroxyl radical-like oxidants and suggest the possibility that acetoxime may be an effective producer of · NO primarily in the liver by a pathway independent of NOS.