BIOACTIVATION OF ESTRONE AND ITS CATECHOL METABOLITES TO QUINOID-GLUTATHIONE CONJUGATES IN RAT-LIVER MICROSOMES

Although the carcinogenic effects of estrogens have been mainly attrib uted to hormonal properties, there is interest in estrogens acting as chemical carcinogens by binding to cellular macromolecules. In the pre sent study, are explored factors which influence the rate of P450-cata lyzed formation of the o-quinones (3,5-cyclohexadiene-1,2-diones) from 2-hydroxyestrone (2-OHE) and 4-hydroxyestrone (4-OHE) as well as from estrone in rat liver microsomes. The initially formed o-quinones mere trapped as their GSH conjugates which were separated and characterize d by HPLC with electrospray-MS detection. Two mono-GSH conjugates were observed from the 2-OHE-o-quinone as well as a conjugate where GSH ha d added twice to the molecule producing a di-GSH conjugate. 4-OHE-o-qu inone gave only one mono-GSH adduct as well as a di-GSH adduct. Both 2 -OHE and 4-OHE were excellent substrates for P450, generating o-quinon e GSH adducts at 94 and 40 times, respectively, the rate of estrone. 2 -OHE but not 4-OHE saturated P450 at unusually low concentrations (0.2 nmol of P450/mL) perhaps due to differences in the stability of the o -quinones formed in the active site of the enzyme. Preliminary data su ggest that the o-quinones of both 2-OHE and 4-OHE could isomerize to q uinone methides (4-alkyl-2,5-cyclohezadien-1-ones, QMs). The o-quinone s of the catechol estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combin ed with GSH. From the pseudo-first-order rate of disappearance of the o-quinone GSH adducts, the half-lives of the o-quinones were determine d. The o-quinone from 2-OHE has a half-life of 42 +/- 3 s at 37 degree s C (pH 7.4), and the o-quinone from 4-OHE has a half-life of 12.2 +/- 0.4 min under identical conditions, The o-quinones of the AB ring ana logs of the catechol estrogens (3,4-dihydroxy-5,6,7,8-tetrahydronaphth alene and 1,2-dihydroxy-5,6,7,8-tetrahydronaphthalene) isomerize to QM s, suggesting that a similar reaction pathway could occur with the o-q uinones from catechol estrogens. In support of this, oxidation of 4-OH E and quenching with GSH after 70 min produced 9-dehydro-4-hydroxyestr one (3-hydroxy-1,3,5-(10),9(1 1)-estratetraen-17-one), a product which could result from either the QM hydrolysis product or the QM-glutathi one conjugate, both of which could eliminate to give the conjugated al kene of 4-OHE. The implications of the o-quinone/QM pathway to the in vivo effects of catechol estrogens are not known; however, given the d irect link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additio nal reactive intermediates needs to be explored.