The O⁶-methylation of 4-hydroxyestradiol is inhibited by 2-hydroxyestradiol: Implications for estrogen-induced carcinogenesis

O-Methylation of catecholestrogens catalyzed by catechol-O-methyltransferase provides a major route for the rapid metabolic clearance of these steroids. However, the metabolic clearance rate of 4-hydroxyestradiol (4-OH-E₂) is considerably lower than that of 2-hydroxyestradiol, although 2- and 4-hydroxycatecholestrogens (2- and 4-OH-CE) have similar apparent affinities for the enzyme. To determine the reason for this apparent paradox we have examined whether the efficiency of O-methylation of 4-OH-E₂ could be affected by other catecholestrogens or their O-methyl ethers. The ratio of 4-methoxyestradiol:4-hydroxyestradiol 3-methyl ether was 2.6 at pH 8.5, the pH optimum for the reaction. The O-methylation of 4-OH-E₂ (apparent K_m 10 μM) was inhibited by 2-hydroxyestradiol (2-OH-E₂) but not by 2- or 4-methoxyestrogens. The values for K_m, V_max as well as the slope for the methylation of 4-OH-E₂ were altered by 2-OH-E₂ indicating a mixed inhibition. The inhibition constant for the intercept 1/V'_max versus 2-OH-E₂ concentrations and the inhibition constant for the slope versus 2-OH-E₂ concentrations were 35 and 5.7 μM, respectively. The inhibition of O-methylation of 4-OH-E₂ by 2-OH-E₂ increased with the pH. In target tissues of the carcinogenic action of estrogens such as the rat pituitary, hamster kidney, or mouse uterus in which 2- and 4-OH-CE are both generated in almost equal amounts, the inactivation of 4-OH-CE by O-methylation may be impeded. Consequently, 4-OH-E₂ would remain available as substrate for redox cycling, generation of active radicals and DNA damage.