Activation of phenacetin O-deethylase activity by α-naphthoflavone in human liver microsomes

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 mu M were inhibited by 0.1 and 1 mu M alpha-naphthoflavone and activated by 10 and 100 mu M alpha-naphthoflavone. The activation of POD activity in human liver microsomes by alpha-naphthoflavone was inhibited by 100 mu M aniline, anti-CYP2E1 antibody, 1 mu M ketoconazole and anti-CYP3A4 antibody. 2. In recombinant CYP from human B-lymphoblast cells, POD activities at phenacetin concentration of 500 mu M 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 b(5) (b(5)) for POD activities catalysed by CYPZEI and CYP3A4 was observed. The activation of POD activity by alpha-naphthoflavone was observed for GYP3A4, but not for CYP2E1. Co-expression of b(5) 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 mu M phenacetin was significantly inhibited (p < 0.0001) by 10 mu M fluvoxamine, but not by 1 mu M ketoconazole. In the presence of alpha-naphthoflavone, the activity was significantly inhibited (p < 0.0001) by 1 mu M ketoconazole, but not by 10 mu M 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.