INHIBITION OF RAT AND HUMAN CYTOCHROME P4502E1 CATALYTIC ACTIVITY ANDREACTIVE OXYGEN RADICAL FORMATION BY NITRIC-OXIDE

Nitric oxide (NO) reacts with heme-containing enzymes, including certa in isoforms of cytochrome P450. Cytochrome P4502E1 (CYP2E1) is induced by ethanol and plays an important role in the toxicity of ethanol and other hepatotoxins. CYP2E1 is also very effective in generating react ive oxygen intermediates such as superoxide radical and H2O2, oxidizin g ethanol to the 1-hydroxyethyl radical, and has a high NADPH oxidase activity. The effect of NO on CYP2E1 catalytic activity and generation of reactive oxygen intermediates was evaluated. Incubating liver micr osomes isolated from rats treated with pyrazole to induce high levels of CYP2E1, with gaseous NO or NO released from a variety of NO donors such as SNAP, DEA/NO, spermine/NO, and GSNO, resulted in a loss of CYP 2E1 catalytic activity with specific substrates such as p-nitrophenol or dimethylnitrosamine. Trapping of NO with hemoglobin resulted in pro tection of CYP2E1 activity against the inactivation by NO. There was n o effect by analogues of the donors which do not release NO nor was th ere any effect by NO on NADPH-cytochrome P450 reductase activity, Inac tivation of CYP2E1 by NO was not prevented by superoxide dismutase or catalase, suggesting that superoxide, H2O2, or peroxynitrite were not responsible for the actions of NO. The inactivated CYP2E1 was not degr aded nor did it lose its epitope sites as shown by Western blot analys is. Associated with loss of CYP2E1 catalytic activity was a decrease i n the formation of superoxide radical and H2O2, in microsomal lipid pe roxidation catalyzed by low, but not high concentration of iron, and i n consumption of NADPH. Oxidation of ethanol to the 1-hydroxyethyl rad ical was also inhibited by NO. ESR experiments indicated the formation of stable heme-NO complexes with CYP2E1. NO appears to compete with O -2 and CO for binding to CYP2E1 as incubation with gaseous NO, or NO d onors inhibited formation of the characteristic CO binding spectrum of P450, Microsomes isolated from a stably transfected HepG2 cell line e xpressing only CYP2E1 were also inactivated by NO, validating interact ion of NO with this isoform of P450. These results indicate that NO in hibits CYP2E1 catalytic activity and generation of reactive radical in termediates. NO may prevent toxicity of agents which require bioactiva tion by P450 isoforms such as CYP2E1 and in generation of reactive int ermediates by CYP2E1. (C) 1997 Academic Press.