Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPHdependent activity, hamster kidney microsomes exhibited high levels of both E2-2-and E2hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2-and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with El and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of EY from control and Es-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein. min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPHdependent 4-hydroxylation of El in controls approached and in EZtreated hamsters exceeded 2-hydroxylation of E, (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and EBtreated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P < 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dosedependent decrease in 2-hydroxylation of E, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:O.g and 1:l.O in control and E2-treated hamsters, respectively). Thus, this study estahlishes that CE can he generated in the same tissue hy three different pathways, i.e. NADPH-dependent Es-2-hydroxylase, NADPH-dependent Ez-4-hydroxylase, and organic hydroperoxide-dependent E, -2/4- hydroxylase activities. We also show that these three activities can he regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could he a factor in this tissue’s vulnerability to estrogen-induced carcinogenesis.
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