title: Activation of phenacetin O-deethylase activity by alpha-naphthoflavone in human liver microsomes.
abstract: 1. The roles of different human cytochrome P450s (CYP) in phenacetin O-deethylation were investigated using human liver microsomes and recombinant proteins. Phenacetin O-deethylase (POD) activities in human liver microsomes at substrate concentrations of 10 and 500 microM were inhibited by 0.1 and 1 microM alpha-naphthoflavone and activated by 10 and 100 microM alpha-naphthoflavone. The activation of POD activity in human liver microsomes by alphanaphthoflavone was inhibited by 100 microM aniline, anti-CYP2E1 antibody, 1 microM ketoconazole and anti-CYP3A4 antibody. 2. In recombinant CYP from human B-lymphoblast cells, POD activities at a phenacetin concentration of 500 microM were detected for CYP2E1 and CYP3A4, as well as CYP1A2, CYP1A1, CYP2C19, CYP2C9 and CYP2A6. In recombinant CYP from human B-lymphoblast cells or baculovirus-infected insect cells and in reconstituted systems, a requirement of cytochrome b5 (b5) for POD activities catalysed by CYP2E1 and CYP3A4 was observed. The activation of POD activity by alpha-naphthoflavone was observed for CYP3A4, but not for CYP2E1. Co-expression of b5 with CYP3A4 enhanced the activation of POD activity by alpha-naphthoflavone. 3. In the absence of alpha-naphthoflavone, the POD activity in pooled human liver microsomes at 500 microM phenacetin was significantly inhibited (p<0.0001) by 10 microM fluvoxamine, but not by 1 microM ketoconazole. In the presence of alpha-naphthoflavone, the activity was significantly inhibited (p<0.0001) by 1 microM ketoconazole, but not by 10 microM fluvoxamine. 4. Inter-individual differences in the effects of alpha-naphthoflavone on POD activity in human liver microsomes were observed, and the involvement of CYP3A4 as well as CYP1A2 in POD activity in human liver was identified even at a low substrate concentration.
authors.affiliation: Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan. nmiki@kenroku.kanazawa-u.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Nakajima
authors.forename: K
authors.initials: K
authors.lastname: Kobayashi
authors.forename: K
authors.initials: K
authors.lastname: Oshima
authors.forename: N
authors.initials: N
authors.lastname: Shimada
authors.forename: S
authors.initials: S
authors.lastname: Tokudome
authors.forename: K
authors.initials: K
authors.lastname: Chiba
authors.forename: T
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D001402:B-Lymphocytes / Q000378:metabolism; D001571:Benzoflavones / Q000494:pharmacology*; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D016666:Fluvoxamine / Q000494:pharmacology; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D001571:Benzoflavones; D011994:Recombinant Proteins; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C585710:CYP2A6 protein, human; C045793:CYP2C19 protein, human; C104464:CYP3A protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; C510163:CYP3A4 protein, human; D016666:Fluvoxamine; D007654:Ketoconazole; C023650:aniline