title: Alkylation of 2'-deoxynucleosides and DNA by the Premarin metabolite 4-hydroxyequilenin semiquinone radical.
abstract: Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in the United States. In addition to endogenous estrogens, Premarin contains unsaturated estrogens including equilenin. We synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin (4-OHEN), and found that the semiquinone radical of 4-OHEN reacted with 2'-deoxynucleosides generating very unusual adducts. 2'-Deoxyguanosine (dG), 2'-deoxyadenosine (dA), or 2'-deoxycytosine (dC) all gave four isomers, but no product was observed for thymidine under similar physiological conditions. The structures of these adducts were determined by electrospray mass spectrometry and NMR experiments including 1H, 13C, DQF-COSY, ROESY, HOHAHA, HMQC, and HMBC. The spectral data show that dG forms a cyclic adduct with the 4-OHEN producing 2-N1,3-N2-deoxyguanosyl-1,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione. Similarly, reaction with dA produced 1-N6,3-C2-deoxyadenosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione, and incubations with dC resulted in 1-N3,3-N4-deoxycytosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-di one. We found that care needed to be taken during the isolation of the dA adducts in particular, as any exposure to acidic environments caused hydrolysis of the sugar moiety leaving alkylated adenine. In mixtures of the deoxynucleosides treated with 4-OHEN, reaction occurred primarily with dG followed by dC and dA. With DNA significant apurinic sites were produced as 4-OHEN-adenine adducts were detected in the ethanol wash prior to hydrolysis. When the DNA was hydrolyzed to deoxynucleosides and analyzed by electrospray mass spectrometry, only one isomer of 4-OHEN-dG and one isomer of 4-OHEN-dC were observed. Our data suggest that several different types of DNA lesions could be expected from 4-OHEN including apurinic sites and bulky stable adducts, in addition to the published oxidized damage to DNA caused by 4-OHEN. The production of these semiquinone radical-derived DNA adducts could play a role in the carcinogenic effects of Premarin estrogens.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 60612-7231, USA.
authors.forename: L
authors.initials: L
authors.lastname: Shen
authors.forename: S
authors.initials: S
authors.lastname: Qiu
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: F
authors.initials: F
authors.lastname: Zhang
authors.forename: R B
authors.initials: RB
authors.lastname: van Breemen
authors.forename: D
authors.initials: D
authors.lastname: Nikolic
authors.forename: J L
authors.initials: JL
authors.lastname: Bolton
mesh_terms: D000478:Alkylation; D002273:Carcinogens / Q000378:metabolism; D003596:Cytosine / Q000031:analogs & derivatives / Q000096:biosynthesis; D004247:DNA / Q000737:chemistry; D018736:DNA Adducts / Q000096:biosynthesis* / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects*; D003839:Deoxyadenosines / Q000096:biosynthesis; D003849:Deoxyguanosine / Q000096:biosynthesis; D004855:Equilenin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004966:Estrogens, Conjugated (USP) / Q000494:pharmacology*; D005609:Free Radicals / Q000009:adverse effects; D006801:Humans; D018384:Oxidative Stress
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D003839:Deoxyadenosines; D004966:Estrogens, Conjugated (USP); D005609:Free Radicals; C085540:2'-deoxycytosine; C065250:4-hydroxy-equilenin; D003596:Cytosine; D004247:DNA; D003849:Deoxyguanosine; D004855:Equilenin