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

The cytochrome P450 (P450) enzymes that catalyse metabolism of theestrogen, estrone (E1), to the putative carcinogen 16alpha-hydroxy E1(16alpha-OHE1) in humans were determined. The potential of the mostabundant circulating form of estrogen, estrone 3-sulfate (E1S), to be thesubstrate was also investigated. Human liver microsomal sulfatases convertE1S to E1, an essential prerequisite for formation of 16alpha- OHE1 fromadded E1S in this system. E1 metabolism to 16alpha-OHE1 in a panel of 15human liver microsomal preparations correlated with total P450concentrations (r2 = 0.63) and with activities associated with P450 formsCYP3A4 and 3A5 (r2 = 0.72). E1 16alpha-hydroxylase activity in human livermicrosomes was inhibited by 75% by monoclonal anti human CYP3A4/5antibodies at 4 mg antibody/nmol total P450, and by troleandomycin, aspecific CYP3A4/5 inhibitor. Rates of E1 metabolism to 16alpha-OHE1 were1.6-fold higher when E1 was generated in situ from E1S than when E1 wasadded. Microsomal preparations of cDNA expressed CYP3A4 or 3A5, withNADPH-P450-reductase co-expressed, both metabolized E1 to 16alpha-OHE1, andadded cytochrome b5 increased the rates 5.1- and 7.5-fold, respectively. Inthese systems rates of E1 metabolism to 16alpha-OHE1 were 2.8-fold higherwhen E1 was generated in situ from E1S than when E1 was added. Kineticvalues for E1 metabolism to 16alpha- OHE1 by human liver microsomes and forthe expressed CYP3A4 system were Km 154 and 172 microM, respectively, andVmax 238 pmol/min/nmol total P450 and 1050 pmol/min/nmol CYP3A4,respectively. Thus, formation of the putative carcinogen 16alpha-OHE1 iscatalysed by CYP3A4 and 3A5 and stimulated by cytochrome b5. E1S is not asubstrate but formation of E1 from E1S in situ stimulates formation of16alpha-OHE1, possibly because E1S is more water soluble and in situgeneration of E1 provides for facilitated exposure of E1 to the P450substrate binding sites. Blocking of the pathway of E1 to 16alpha-OHE1could provide a therapeutic approach for diminishing the risk of estrogendependent breast cancer.