Inhibition of the Catechol-O-Methyltransferase-Catalyzed O-Methylation of 2- and 4-Hydroxyestradiol by Catecholamines: Implications for the Mechanism of Estrogen-Induced Carcinogenesis

Catechol estrogens have been postulated to mediate estrogen-induced carcinogenesis. As part of our examination of this hypothesis, we studied the catechol-O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol and the inhibition of this reaction by catecholamines. Epinephrine, norepinephrine, or dopamine (2 μM) inhibited the methylation of 2- and 4-hydroxyestradiol (20 nM) catalyzed by porcine liver catechol-O-methyltransferase by approximately 28-46% and 37-57%, respectively. One millimolar concentrations of catecholamines also inhibited the methylation of 5 μM 2-and 4-hydroxyestradiol by hamster kidney cytosol catechol-O-methyltransferase by approximately 27-31% and 19-33%, respectively. At a 15 μM 4-hydroxyestradiol concentration, the IC50 values for epinephrine and for dopamine were approximately 1200 and 3000 μM, respectively. Kinetic analyses of the methylation of 4-hydroxyestradiol in the presence of epinephrine, norepinephrine, or dopamine all revealed a competitive mechanism of inhibition. In contrast, the methylation of 160 μM 2-hydroxyestradiol was enhanced by approximately 75% in the presence of 1600 μM epinephrine or 2400 μM norepinephrine, likely due to a strong positive allosteric effect. An analysis of the substrate concentration dependence of O-methylation of 2-hydroxyestradiol revealed that at low concentrations (<15 μM) this reaction was inhibited by epinephrine or norepinephrine, whereas it was significantly increased by approximately 50-100% at high substrate concentrations (50-200 μM). In contrast, dopamine competitively inhibited the methylation of all concentrations of 2-hydroxyestradiol (5-160 μM) tested. High levels of catecholamines were measured in hamster kidney or mouse uterus (1041 ± 204 or 882 ± 214 ng norepinephrine/g wet tissue, respectively) and in Fisher 344 rat pituitary (9.4 ± 1.6 ng dopamine/mg protein), target organs of estrogen-induced carcinogenesis. Values were much lower in other organs of the same animals or in kidney, uterus, or pituitary of other rodent strains or species, which do not develop tumors under these conditions. High levels of catecholamines in target organs of hormonal cancer, the inhibition of O-methylation of 4-hydroxyestradiol, and the differential regulation of O-methylation of 2-hydroxyestradiol by catecholamines all support a role of 4-hydroxyestrogen metabolites in estrogen-induced carcinogenesis.