ROLE OF HUMAN HEPATIC CYTOCHROME-P450 1A2 AND 3A4 IN THE METABOLIC-ACTIVATION OF ESTRONE

The metabolic activation of estrone (E1), a potent estrogen was invest igated using: recombinant human cytochrome P450 enzymes, 1A2, 2B6, 2C8 , 2C9, 2C9(R144C), 2E1, 3A4, 3A5 and liver microsomes from 14 human or gan donors, At least five products of E1 were detected and quantitated by HPLC and gas chromatography-mass spectrometry (GC-MS). Among these metabolites, 16 alpha-OH-E1, 2-OH-E1 and 4-OH-E1, which are believed to be associated with estrogen carcinogenesis in animals, were definit ively identified. Of all P450s examined, 1A2 and 3A4 exhibited the hig hest activities with turnovers of 3.4 and 2.5 nmol/min/ nmol P450 for the total metabolism of E1, respectively, while 3A5, 2C9 and 2C9(R144C ) showed moderate activities, 2B6, 2E1 and 2C8 did not produce any sig nificant amount of products. 1A2 formed almost exclusively the 2-OH-E1 at a rate of 3.3 nmol/min/nmol but 3A4 preferentially formed the meta bolite X1 (an unknown hydroxylation product) and 16 alpha-OH-E1. Kinet ic characterization showed that the K-m values of 1A2, 3A4 and 3A5 wer e 14, 95 and 64 mu M and V-max were 5.43, 0.68 and 0.35 min(-1), respe ctively. All human liver microsomes were capable of metabolizing estro ne and a 4-fold variation was seen between individuals. The relative a mount of metabolites formed was generally 2-OH-E1 > metabolite X1 > 4- OH-E1 > 16 alpha-OH-E1 > metabolite X2, 3A4/5 enzyme complex was asses sed by inhibitory monoclonal antibody specific for 3A4/5 to contribute 60-88% to the formation of individual metabolites in human liver exce pt for 2-OH-E1 (3%). The formation of 2-OH-E1 and 16 alpha-OH-E1 by 14 human liver microsomes was significantly correlated with caffeine 3-d emethylation supported by 1A2 (r(2) = 0.87) and with testosterone 6 be ta-hydroxylation by 3A4 (r(2) = 0.66), respectively. Thus the metaboli c patterns exhibited by human liver are likely due to the combined act ivities of the P450 1A2 and 3A4 enzymes.