Base Selectivity and Effects of Sequence and DNA Secondary Structure on the Formation of Covalent Adducts Derived from the Equine Estrogen Metabolite 4-Hydroxyequilenin

Abstract

Equilenin, an important component of a widely prescribed hormone replacement formulation 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 approximately 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 approximately 10(3)-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.

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

“…Near mirror image conformations in stereoisomer adduct pairs, C1/C2 (A1/A2), and C3/C4 (A3/A4), were found. This is consistent with near-mirror image CD spectra of each stereoisomeric pair observed experimentally (21).…”

“…We employed the sequence of Figure 1, which is also under experimental investigation (21). This duplex is the same as the one employed in our study of the 4-OHEN-C adducts, where modification was at C 18 of the complementary strand.…”

Translesion Synthesis Past Equine Estrogen-Derived 2‘-Deoxyadenosine DNA Adducts by Human DNA Polymerases η and κ

“…Therefore, Pk-1 may contain either fr-1 or fr-2 of monomeric 4-OHEN-dA or both. The yield of each diastereoisomers may be influenced by the sequence context of oligomer, as reported recently (39). To explore the miscoding potential, the major 4-OHEN-dA-modified oligodeoxynucleotides were used for preparation of DNA template that can be used for primer extension reactions.…”

DNA Helix Destabilization by Alkylating Agents: From Covalent Bonding to DNA Repair

“…The hormone-derived genotoxic compound, 4-OHEN, derives from equilin and equilenin, two equine oestrogens present in hormone substitution therapies used to prevent the uncomfortable effects of menopauses but are also thought to increase breast cancer incidence in the population of hormonally-treated women (Rossouw et al, 2002). Its orthoquinone form is cytotoxic and genotoxic (Pisha et al, 2001) through the formation of bulky DNA lesions at dA, dC and dG (but not at T residues) (Kolbanovskiy et al, 2005) which were detected in both cell culture and breast cancer biopsies from patients treated with hormone substitution therapies (Embrechts et al, 2003). 4-OHEN derived from the intermediate catechol 4-hydroxyequilenin which was generated from a rapid conversion of both equilin and equilenin in the organism to four stereo-isomers differently affecting the 3D-structure of the DNA helix (Ding et al, 2007).…”

“…Interestingly, alkylation at dA or dC residues is associated with a strong www.intechopen.com decrease in the melting temperature (Tm) of a 11-bp oligonucleotide, with the magnitude of the negative Tm values being lower when the adduct is located at 1 or 2-bp from the end of the 11-bp DNA (-6 to -9°C) then when it is located in its medium part (positions 4 to 8) with up to a -21 to -27°C decrease of Tm. Similarly, the stereoisomeric orientation of the 4-OHEN adduct affects the base-stacking, groove sizes and subsequent distortions and is also crucial for the extent of DNA destabilization (Kolbanovskiy et al, 2005).…”