Activation of phenacetinO-deethylase activity byα-naphthoflavone in human liver microsomes

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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 μM were inhibited by 0.1 and 1 μM α-naphthoflavone and activated by 10 and 100 μM α-naphthoflavone. The activation of POD activity in human liver microsomes by α-naphthoflavone was inhibited by 100 μM aniline, anti-CYP2E1 antibody, 1 μM ketoconazole and anti-CYP3A4 antibody. 2. In recombinant CYP from human B-lymphoblast cells, POD activities at a phenacetin concentration of 500 μM 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 α-naphthoflavone was observed for CYP3A4, but not for CYP2E1. Co-expression of b5 with CYP3A4 enhanced the activation of POD activity by α-naphthoflavone. 3. In the absence of α-naphthoflavone, the POD activity in pooled human liver microsomes at 500 μM phenacetin was significantly inhibited (p < 0.0001) by 10 μM fluvoxamine, but not by 1 μM ketoconazole. In the presence of α-naphthoflavone, the activity was significantly inhibited (p < 0.0001) by 1 μM ketoconazole, but not by 10 μM fluvoxamine. 4. Inter-individual differences in the effects of α-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.