Base selectivity and effects of sequence and DNA secondary structure on the formation of covalent adducts derived from the equine estrogen metabolite 4-hydroxyequilenin

Alexander Kolbanovskiy, Vladimir Kuzmin, Anant Shastry, Marina Kolbanovskaya, Dandan Chen, Minsun Chang, Judith L. Bolton, Nicholas E. Geacintov

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Equilenin, an important component of a widely prescribed hormone replacement formualtion for postmenopausal women, is metabolized by mammalian P450 enzymes to the catechol 4-hydroxyequilenin (4-OHEN). The oxidized o-quinone derivative of 4-OHEN is known to form cyclic covalent adducts with DNA [Bolton, J. (1998) Chem. Res. Toxicol. 11, 1113] in vitro and in vivo. The characteristics of 4-OHEN-DNA adduct formation were investigated with the oligonucleotides 5′-d(CCATCGCTACC) (I), its complementary strand 5′-d(GGTAGCGATGG) (II), one rich in C and the other in G, and the duplexes I·II. The identities of the modified bases were elucidated in terms of four stereoisomeric 4-OHEN-2′-deoxynucleoside standards described earlier [Shen et al. (2001) Chem. Res. Toxicol. 11, 94; Embrechts et al. J. Mass Spectrom. 36, 317]. The reactions of 4-OHEN with C are favored overwhelmingly in both single-stranded I and II with no guanine adducts observed in either case, and only minor proportions of A adducts were detected in sequence II. However, guanine adducts are observed in oligonucleotides that contain only G and unreactive T residues. The relative levels of cyclic covalent adducts observed in single-stranded I, II, and duplex I·II are ∼54:21:5, with only the end C groups in I modified in the I·II duplex. When 4-OHEN is reacted with calf thymus DNA, the reaction yield of cyclic adducts is more than ∼103-fold lower than in I. The cyclic 4-OHEN adducts lead to a pronounced thermal destabilization of duplexes I·II. Overall, cyclic adduct formation is markedly dependent on the sequence context and secondary structure of the DNA. The latter effect is attributed to the poor accessibilities of 4-OHEN to the reactive nucleotide Watson-Crick hydrogen-bonding interface in the interior of the duplex. In the single-stranded oligonucleotides I and II, the strikingly different selectivities of adduct formation are attributed to the formation of noncovalent preassociation complexes that favor reaction geometries with C, rather than with A or G. Finally, the levels of several typical biomarkers of oxidative DNA damage (including 8-oxo-2′-deoxyguanosine) are formed in I in aqueous solutions with a yield at least 10 times smaller than the yield of cyclic 4-OHEN-dC adducts under identical reaction conditions.

Original languageEnglish (US)
Pages (from-to)1737-1747
Number of pages11
JournalChemical research in toxicology
Issue number11
StatePublished - Nov 2005

ASJC Scopus subject areas

  • Toxicology


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