Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes.

Previous results demonstrating homotropic activation of human UDP-glucuronosyltransferase (UGT) 1A1-catalyzed estradiol-3-glucuronidation led us to investigate the effects of 16 compounds on estradiol glucuronidation by human liver microsomes (HLM). In confirmation of previous work using alamethicin-treated HLM pooled from four livers, UGT1A1-catalyzed estradiol-3-glucuronidation demonstrated homotropic activation kinetics (S(50) = 22 microM, Hill coefficient, n = 1.9) whereas estradiol-17-glucuronidation (catalyzed by other UGT enzymes) followed Michaelis-Menten kinetics (K(m) = 7 microM). Modulatory effects of the following compounds were investigated: bilirubin, eight flavonoids, 17alpha-ethynylestradiol (17alpha-EE), estriol, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), anthraflavic acid, retinoic acid, morphine, and ibuprofen. Although the classic UGT1A1 substrate bilirubin was a weak competitive inhibitor of estradiol-3-glucuronidation, the estrogens and anthraflavic acid activated or inhibited estradiol-3-glucuronidation dependent on substrate and effector concentrations. For example, at substrate concentrations of 5 and 10 microM, estradiol-3-glucuronidation activity was stimulated by as much as 80% by low concentrations of 17alpha-EE but was unaltered by flavanone. However, at higher substrate concentrations (25-100 microM) estradiol-3-glucuronidation was inhibited by about 55% by both compounds. Anthraflavic acid and PhIP were also stimulators of estradiol 3-glucuronidation at low substrate concentrations. The most potent inhibitor of estradiol 3-glucuronidation was the flavonoid tangeretin. The UGT2B7 substrates morphine and ibuprofen had no effect on estradiol 3-glucuronidation, whereas retinoic acid was slightly inhibitory. Estradiol-17-glucuronidation was inhibited by 17alpha-EE, estriol, and naringenin but was not activated by any compound. This study demonstrates that the interactions of substrates and inhibitors at the active site of UGT1A1 are complex, yielding both activation and competitive inhibition kinetics.