Cytochrome P450 3A activities and their modulation by α-naphthoflavone in vitro are dictated by the efficiencies of model experimental systems

The knowledge on efficiencies of different in vitro systems containing cytochromes P450 (CYP) of a 3A subfamily is crucial to screen potential substrates of these CYPs. We evaluated and compared efficiencies of several in vitro CYP3A enzymatic systems to oxidize the model substrates, α-NF and testosterone, under the standardized experimental conditions. Five CYP3A systems were tested: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (SupersomesTM), (iv) membranes isolated from Escherichia coli, containing recombinant human CYP3A4, NADPH:CYP reductase and cytochrome b5, and (v) human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. All systems oxidize testosterone to its 6β-hydroxylated metabolite and α-NF to trans-7,8-dihydrodiol and 5,6-epoxide. The most efficient systems oxidizing both compounds were CYP3A4-SupersomesTM containing cytochrome b5, followed by human hepatic microsomes. This finding suggests these systems to be suitable for general evaluating a variety of compounds as potential substrates of CYP3A4. The lowest efficiencies to oxidize α-NF and testosterone were found for CYP3A4 expressed in membranes of E. coli, and for reconstituted CYP3A4 or CYP3A6. Utilizing the tested enzymatic systems, we also explain here the discrepancies, which showed previously the controversial effects of α-NF on CYP3A-mediated reactions. We demonstrate that inhibition or stimulation of the CYP3A-mediated testosterone hydroxylation by α-NF is dictated by efficiencies of individual enzymatic systems to oxidize the CYP3A substrates.