A new selective and potent inhibitor of human cytochrome P4501B1 and its application to antimutagenesis

Human cytochrome P450 (P450) 1B1 is found mainly in extrahepatic tissues an d is overexpressed in a variety of human tumors. Metabolic activation of 17 beta -estradiol (E-2) to 4-hydroxy E-2 by P450 1B1 has been postulated to be a factor in mammary carcinogenesis. The inhibition of recombinant human P450 1B1 by 2,4,3',5'-tetramethoxystilbene (TMS) was investigated using eit her bacterial membranes from a human P450/NADPH-P450 reductase bicistronic expression system or using purified enzymes. TMS showed potent and selectiv e inhibition of the ethoxyresorufin O-deethylation (EROD) activity of P450 1B1 with an IC50 value of 6 nm. TMS exhibited 50-fold selectivity for P450 1B1 over P450 1A1 (IC50 = 300 nm) and 500-fold selectivity for P450 1B1 ove r P450 1A2 (IC50 = 3 muM). The inhibitory effects of TMS on EROD activity o f human liver microsomes were determined. TMS inhibited EROD activity of hu man liver microsomes at the same concentration as with recombinant human P4 50 1A2. TMS also strongly inhibited 4- and 2-hydroxylation of E-2 by P450 1 B1-expressing membranes or purified P450 1B1. TMS was a competitive inhibit or of P450 1B1 with a K-i of 3 nM. The inhibition by TMS was not mechanism- based, and the loss of activity was not blocked by the trapping agents glut athione, N-acetylcysteine, or dithiothreitol. Using purified histidine-tagg ed P450 1B1, the binding kinetic analysis was performed with TMS, yielding a K-d of 3 muM. The activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoli ne in an Escherichia coli lac-based mutagenicity tester system containing f unctional human P450 1B1 was strongly inhibited by TMS. Our results indicat e that TMS is a very selective and potent competitive inhibitor of P450 1B1 . TMS is selective for inhibiting P450 1B1 among other human P450s includin g 1A1, 1A2, and 3A4 and warrants consideration as a candidate for preventin g mammary tumor formation by E-2 in humans.