ALKYLATION OF 2'-DEOXYNUCLEOSIDES AND DNA BY THE PREMARIN METABOLITE 4-HYDROXYEQUILENIN SEMIQUINONE RADICAL

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choic e and continues to be one of the most widely dispensed prescriptions i n the United States. In addition to endogenous estrogens, Premarin con tains unsaturated estrogens including equilenin. We synthesized the ca techol metabolite of equilenin, 4-hydroxyequilenin (4-OHEN), and found that the semiquinone radical of 4-OHEN reacted with 2'-deoxynucleosid es generating very unusual adducts. 2'-Deoxyguanosine (dG), 2'-deoxyad enosine (dA), or 2'-deoxycytosine (dC) all gave four isomers, but no p roduct was observed for thymidine under similar physiological conditio ns. The structures of these adducts were determined by electrospray ma ss spectrometry and NMR experiments including H-1, C-13, DQF-COSY, ROE SY, HOHAHA, HMQC and HMBC. The spectral data show that dG forms a cycl ic adduct with the 4-OHEN producing oxyguanosyl-1,3-dihydroxy-5,7,9(10 )-estratriene-4, 17-dione. Similarly, reaction with dA produced oxyade nosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4, 17-dione, and incubations with dC resulted in deoxycytosyl-2,3-dihydroxy-5,7,9(10)-estratiene-4 , 17-dione. We found that care needed to be taken during the isolation of the dA adducts in particular, as any exposure to acidic environmen ts caused hydrolysis of the sugar moiety leaving alkylated adenine. In mixtures of the deoxynucleosides treated with 4-OHEN, reaction occurr ed primarily with dG followed by dC and dA. With DNA significant apuri nic sites were produced as 4-OHEN-adenine adducts were detected in the ethanol wash prior to hydrolysis. When the DNA was hydrolyzed to deox ynucleosides and analyzed by electrospray mass spectrometry, only one isomer of 4-OHEN-dG and one isomer of 4-OHEN-dC were observed. Our dat a suggest that several different types of DNA lesions could be expecte d from 4-OHEN including apurinic sites and bulky stable adducts, in ad dition to the published oxidized damage to DNA caused by 4-OHEN. The p roduction of these semiquinone radical-derived DNA adducts could play a role in the carcinogenic effects of Premarin estrogens.