title: Bioactivation of estrone and its catechol metabolites to quinoid-glutathione conjugates in rat liver microsomes.
abstract: Although the carcinogenic effects of estrogens have been mainly attributed to hormonal properties, there is interest in estrogens acting as chemical carcinogens by binding to cellular macromolecules. In the present study, we explored factors which influence the rate of P450-catalyzed 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 were trapped as their GSH conjugates which were separated and characterized 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 had added twice to the molecule producing a di-GSH conjugate. 4-OHE-o-quinone 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-quinone 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 suggest that the o-quinones of both 2-OHE and 4-OHE could isomerize to quinone methides (4-alkyl-2,5-cyclohexadien-1-ones, QMs). The o-quinones 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 combined with GSH. From the pseudo-first-order rate of disappearance of the o-quinone GSH adducts, the half-lives of the o-quinones were determined. The o-quinone from 2-OHE has a half-life of 42 +/- 3 s at 37 degrees 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 analogs of the catechol estrogens (3,4-dihydroxy-5,6,7,8-tetrahydronaphthalene and 1,2-dihydroxy-5,6,7,8-tetrahydronaphthalene) isomerize to QMs, suggesting that a similar reaction pathway could occur with the o-quinones from catechol estrogens. In support of this, oxidation of 4-OHE and quenching with GSH after 70 min produced 9-dehydro-4-hydroxyestrone (3-hydroxy-1,3,5-(10),9(11)-estratetraen-17-one), a product which could result from either the QM hydrolysis product or the QM--glutathione conjugate, both of which could eliminate to give the conjugated alkene 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 direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago 60612-7231, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Iverson
authors.forename: L
authors.initials: L
authors.lastname: Shen
authors.forename: N
authors.initials: N
authors.lastname: Anlar
authors.forename: J L
authors.initials: JL
authors.lastname: Bolton
mesh_terms: D000818:Animals; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology; D004970:Estrone / Q000031:analogs & derivatives* / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006894:Hydroxyestrones / Q000378:metabolism*; D045563:Indolequinones; D007211:Indoles / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000502:physiology*; D011485:Protein Binding; D011809:Quinones / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D006894:Hydroxyestrones; D045563:Indolequinones; D007211:Indoles; D011809:Quinones; C068040:quinone methide; D004970:Estrone; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione