Oxidation of the ketoxime acetoxime to nitric oxide by oxygen radical-generating systems

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 >C=N-OH group of amidoximes. The objective of this work was to characterize the oxidation of a ketoxime (ace toxime) and to assess the ability of NOS to catalyze the generation of nitr ic oxide/nitrogen monoxide (. NO) from acetoxime. Acetoxime was oxidized to NO2- (and NO3-) by microsomes enriched with several P450 isoforms, includi ng CYP2E1, CYP1A1, and CYP2B1. Nitric oxide was identified as an intermedia te in the overall reaction. Superoxide dismutase and catalase significantly inhibited the reaction. Exogenous iron increased the microsomal generation of NO2- from acetoxime, while metal chelators (desferrioxamine, EDTA, DTPA ) inhibited it. A Fenton-like system (Fe2+ plus H2O2, pH 7.4) consumed acet oxime with production of NO2- and NO3-, 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-Acet ylacetoxime and O-tert-butylacetoxime were not oxidized by a Fenton system or by liver microsomes to any significant extent. Formation of the 5,5'-dim ethyl-1-pyrroline-N-oxide/. OH adduct by a Fenton system was significantly inhibited by acetoxime, while O-acetylacetoxime and O-tert-butylacetoxime w ere inactive. These results suggest that the . OH-dependent oxidation of ac etoxime initially proceeds via abstraction of a hydrogen atom from its hydr oxyl group, as opposed to the oxidation of its >C=N- function. HepG2 cells with low levels of expression of P450 did not significantly produce NO2- fr om acetoxime, while HepG2 cells expressing CYP2E1 did, and this generation was blocked by a CYP2E1 inhibitor. Acetoxime was inactive either as a subst rate or as an inhibitor of iNOS activity. These results indicate that react ive 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 acetoxi me may be an effective producer of . NO primarily in the liver by a pathway independent of NOS. (C) 2001 Academic Press.