Suicide inactivation of cytochrome P-450 by methoxsalen. Evidence for the covalent binding of a reactive intermediate to the protein moiety.
Incubation of rat liver microsomes with [3H]methoxsalen and NADPH resulted in the covalent binding of a methoxsalen intermediate to proteins comigrating with cytochromes P-450 UT-A, PB-B/D, ISF-G and PCN-E. Binding was increased by pretreatments with phenobarbital, beta-naphthoflavone (beta NF) and dexamethasone. Such pretreatments also increased the loss of CO-binding capacity either after administration of methoxsalen, or after incubation of hepatic microsomes with methoxsalen and NADPH. Immunoprecipitation of the methoxsalen metabolite-protein adducts in phenobarbital-induced microsomes was moderate with anti-UT-A antibodies, but marked with anti-PB-B/D and anti-PCN-E antibodies. Immunoprecipitation was observed also with anti-ISF-G (anti-beta NF-B) antibodies in beta NF-induced microsomes. Methoxsalen (0.25 mM) inhibited markedly the benzphetamine demethylase activity of phenobarbital-induced microsomes and the erythromycin demethylase activity of dexamethasone-induced microsomes. Whereas methoxsalen itself did not produce any binding spectrum, in contrast either in vivo administration of methoxsalen or incubation in vitro with methoxsalen and NADPH resulted in a low-to-high spin conversion of cytochrome P-450 as suggested by the appearance of a spectrum analogous to a type I binding spectrum. This low-to-high spin conversion was apparently due to a methoxsalen intermediate (probably, covalently bound to the protein and preventing partial sixth ligation of the iron). We conclude that suicide inactivation of cytochrome P-450 by methoxsalen is related to the covalent binding of a methoxsalen intermediate to the protein moiety of several cytochrome P-450 isoenzymes (including UT-A, PB-B/D, PCN-E as well as ISF-G and/or beta NF-B).
Labbe
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Unité de Recherches de Physiopathologie Hépatique (INSERM U24), Hôpital Beaujon, Clichy, France.
Descatoire
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Beaune
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Letteron
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Larrey
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Pessayre
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D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002251:Carbon Tetrachloride / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008730:Methoxsalen / Q000378:metabolism / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D013057:Spectrum Analysis; D014466:Ultraviolet Rays
D065607:Cytochrome P-450 Enzyme Inhibitors; D006418:Heme; D009249:NADP; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D002251:Carbon Tetrachloride; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; D008730:Methoxsalen