Alkylation of 2'-Deoxynucleosides and DNA by the Premarin Metabolite 4-Hydroxyequilenin Semiquinone Radical

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• 作者：Li Shen ; Shengxiang Qiu ; Yumei Chen ; Fagen Zhang ; Richard B. van Breemen ; Dejan Nikolic ; and Judy L. Bolton
• 刊名：Chemical Research in Toxicology
• 出版年：1998
• 出版时间：February 1998
• 年：1998
• 卷：11
• 期：2
• 页码：94 - 101
• 全文大小：189K
• 年卷期：v.11,no.2(February 1998)
• ISSN：1520-5010

文摘

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment ofchoice and continues tobe one of the most widely dispensed prescriptions in the United States.In addition toendogenous estrogens, Premarin contains unsaturated estrogens includingequilenin. Wesynthesized the catechol metabolite of equilenin, 4-hydroxyequilenin(4-OHEN), and foundthat the semiquinone radical of 4-OHEN reacted with 2'-deoxynucleosidesgenerating veryunusual adducts. 2'-Deoxyguanosine (dG), 2'-deoxyadenosine (dA),or 2'-deoxycytosine (dC)all gave four isomers, but no product was observed for thymidine undersimilar physiologicalconditions. The structures of these adducts were determined byelectrospray mass spectrometryand NMR experiments including ¹H, ¹³C,DQF-COSY, ROESY, HOHAHA, HMQC, and HMBC.The spectral data show that dG forms a cyclic adduct with the4-OHEN producing2-N¹,3-N²-deoxyguanosyl-1,3-dihydroxy-5,7,9(10)-estratriene-4,17-dione.Similarly, reaction with dAproduced1-N⁶,3-C²-deoxyadenosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-dione,and incubations with dC resulted in1-N³,3-N⁴-deoxycytosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-dione.We found that care needed to be taken during the isolation of thedA adducts in particular, asany exposure to acidic environments caused hydrolysis of the sugarmoiety leaving alkylatedadenine. In mixtures of the deoxynucleosides treated with 4-OHEN,reaction occurred primarilywith dG followed by dC and dA. With DNA significant apurinic siteswere produced as4-OHEN-adenine adducts were detected in the ethanol wash prior tohydrolysis. When theDNA was hydrolyzed to deoxynucleosides and analyzed by electrospraymass spectrometry,only one isomer of 4-OHEN-dG and one isomer of 4-OHEN-dC were observed.Our data suggestthat several different types of DNA lesions could be expected from4-OHEN including apurinicsites and bulky stable adducts, in addition to the published oxidizeddamage to DNA causedby 4-OHEN. The production of these semiquinone radical-derived DNAadducts could play arole in the carcinogenic effects of Premarin estrogens.