INACTIVATION OF ETHANOL-INDUCIBLE CYTOCHROME-P450 AND OTHER MICROSOMAL P450 ISOZYMES BY TRANS-4-HYDROXY-2-NONENAL, A MAJOR PRODUCT OF MEMBRANE LIPID-PEROXIDATION

Of the microsomal P450 cytochromes, the ethanol-inducibie isoform, P45 0 2E1, is believed to he predominant in leading to oxidative damage, i ncluding the generation of radical species that contribute to lipid pe roxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensiti vity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After in cubation of a purified cytochrome with HNE, the other components of th e reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylc holine, and NADPH) were added, and the rate of oxygenation of 1-phenyl ethanol to yield acetophenone was assayed. Inactivation occurs in a ti me-dependent and HNE concentration-dependent manner, with P450s 2E1 an d 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4 . At an HNE concentration of 0.24 mu M, which was close to the micromo lar concentration of the enzyme, four of the isoforms were significant ly inhibited, but not P450 2B4. In other experiments, the reductase wa s shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phen obarbital, respectively, are as readily inhibited as the purified form s. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects agains t HNE, and that the inactivation is reversed upon dialysis. Higher lev els of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed in hibition of the various P450s is of sufficient magnitude to cause sign ificant changes in the metabolism of foreign compounds such as drugs a nd chemical carcinogens by the P450 oxygenase system at HNE concentrat ions that occur in biological membranes, In view of the known activiti es of P450 2E1 in generating lipid hydroperoxides and in their beta-sc ission, its inhibition by this product of membrane peroxidation may pr ovide a negative regulatory function.