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.

Estrogen Receptor α Enhances the Rate of Oxidative DNA Damage by Targeting an Equine Estrogen Catechol Metabolite to the Nucleus

“…The equine estrogen equilenin is of special interest as a carcinogen because: 1) it is selectively oxidized to the more genotoxic 4-hydroxy catechol isomer (4-OHEN); and 2) 4-OHEN autoxidizes to a redox-cycling o-quinone without need for cytochrome P450 catalysis (27,28). 4-OHEN-o-quinone induces a variety of different types of DNA damage both in vitro and in vivo, including single strand breaks (29,30), oxidized bases (31,32), apurinic sites, and formation of cyclic DNA adducts (20,33,34).…”

4-Hydroxyequilenin-Adenine Lesions in DNA Duplexes: Stereochemistry, Damage Site, and Structure

“…It appears that the hydrogen bond between the modified adenine N7 and its partner T 19 limits the bending in A2 and A4. The major groove width around the lesion site is generally enlarged due to accommodation of the equilenin rings (Figure 4), although only modestly in the case of 4-OHEN-A3 because of a hydrogen bond between the carbonyl group of the equilenin with N4H of C 16 . The dimensions of the minor groove in the syn 4-OHEN-A adducts fluctuate around those of the unmodified duplex.…”

Detection of estrogen DNA-adducts in human breast tumor tissue and healthy tissue by combined nano LC-nano ES tandem mass spectrometry

“…Equilenin metabolite and its adducts were prepared according to the method Shen et al [19], also described by us in an earlier paper [33]. Aliquots of 1 mL of sample were dried under reduced pressure and dissolved in 1 mL of methanol.…”