16α-hydroxylation of estrone by human cytochrome P4503A4/5

The cytochrome P450 (P450) enzymes that catalyse metabolism of the estrogen, estrone (E-1), to the putative carcinogen 16 alpha-hydroxy E-1 (16 alpha-OHE1) in humans were determined, The potential of the most abundant circulating form of estrogen, estrone 3-sulfate (E1S), to be the substrate was also investigated, Human liver microsomal sulfatases convert E1S to E-1, an essential prerequisite for formation of 16 alpha-OHE1 from added E1S in this system. E-1 metabolism to 16 alpha-OHE1 in a panel of 15 human liver microsomal preparations correlated with total P450 concentrations (r(2) = 0.63) and with activities associated with P450 forms CYP3A4 and 3A5 (r(2) = 0.72), E-1 16 alpha-hydroxylase activity in human liver microsomes was inhibited by 75% by monoclonal anti human CYP3A4/5 antibodies at 4 mg antibody/nmol total P450, and by troleandomycin, a specific CYP3A4/5 inhibitor. Rates of E-1 metabolism to 16 alpha-OHE1 were 1.6-fold higher when E-1 was generated in situ from E1S than when E-1 was added, Microsomal preparations of cDNA expressed CYP3A4 or 3A5, with NADPH-P-450-reductase co-expressed, both metabolized E-1 to 16 alpha-OHE1, and added cytochrome b(5) increased the rates 5,1- and 7.5-fold, respectively. In these systems rates of E-1 metabolism to 16 alpha-OHE1 were 2,8-fold higher when E-1 was generated in situ from E1S than when E-1 was added, Kinetic values for E-1 metabolism to 16 alpha-OHE1 by human liver microsomes and far the expressed CYP3A4 system were K-m 154 and 172 mu M, respectively, and V-max 238 pmol/min/nmol total P450 and 1050 pmoymin/nmol CYP3A4, respectively. Thus, formation of the putative carcinogen 16 alpha-OHE1 is catalysed by CYP3A4 and 3A5 and stimulated by cytochrome b(5), E1S is not a substrate but formation of E-1 from E1S in situ stimulates formation of 16 alpha-OHE1, possibly because E1S is more water soluble and in situ generation of E-1 provides for facilitated exposure of E-1 to the P450 substrate binding sites. Blocking of the pathway of El to 16 alpha-OHE1 could provide a therapeutic approach for diminishing the risk of estrogen dependent breast cancer.