Response of human mammary epithelial cells to DNA damage induced by 4-hydroxyequilenin: Lack of p53-mediated G1 arrest.
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Long-term exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. One potential mechanism of estrogen carcinogenesis involves catechol formation and these catechols are further oxidized to electrophilic/redox active o-quinones, which have the potential to both initiate and promote the carcinogenic process. 4-Hydroxyequilenin (4-OHEN), a major phase I metabolite of several estrogens present in Premarin, is considerably more cytotoxic, carcinogenic, and mutagenic as compared to the catechol estrogen metabolites of endogenous estrogens. Previously, we showed that 4-OHEN autoxidized to an o-quinone and caused a variety of damage to DNA. Allowing more time between the induction of DNA damage and the entry of a damaged cell into the DNA synthetic phase of the cell cycle protects that cell from mutagenesis. Central to this response is the establishment of a G1 checkpoint. This checkpoint is mediated by the cyclin-dependent kinase inhibitor p21WAF1, a direct downstream target for transcriptional activation by p53. In this study, we investigated this signaling pathway. Surprisingly, exposure of the human MCF-10A immortalized nontransformed mammary epithelial cell line to 4-OHEN did not induce a p53-induced G1 arrest. A 24 h treatment with 4-OHEN significantly induced p53 and p21WAF1 protein expression at 10 and 20 microM, as well as significantly induced the transactivation of a p53-luciferase reporter gene at 20 microM. Significant decreases in cell proliferation were also observed with concentrations of 5 microM and higher of 4-OHEN. However, 4-OHEN did not induce a G1 checkpoint and cells with damaged DNA accumulated in the S phase. This S phase delay could be beneficial for the survival of the damaged cells which could contribute to the carcinogenic process. (+info)
4-hydroxyequilenin-adenine lesions in DNA duplexes: stereochemistry, damage site, and structure.
(10/29)
The equine estrogens, equilin and equilenin, are major components of the drug Premarin, the most widely used formula for hormone replacement therapy. The derivative 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react in vitro and in vivo with cytosine and adenine in DNA. Unique cyclic adducts containing the same bicyclo[3.3.1]nonane-type connection ring are produced. Each base adduct has four stereoisomers. In order to elucidate the structural effects of A versus C modification, we have carried out molecular dynamics simulations of the stereoisomeric 4-OHEN-A adducts in DNA 11-mer duplexes and compared results with an earlier study of the C adducts (Ding, S., Shapiro, R., Geacintov, N.E., and Broyde, S. (2005) Equilenin-Derived DNA Adducts to Cytosine in DNA Duplexes: Structures and Thermodynamics, Biochemistry 44, 14565-14576). Similar stereochemical principles govern the orientations in DNA duplexes of the 4-OHEN-A adducts as for the analogous C adducts, with opposite orientations of the equilenin rings in stereoisomeric pairs of adducts characterized by near-mirror image circular dichroism (CD) spectra. However, the larger purine adducts have unique structural properties in the duplexes that distinguish their characteristics from those of the pyrimidine adducts. Significant differences are observed in terms of hydrogen bonding, stacking, bending, groove dimensions, solvent exposure, and hydrophobic interactions; also, each of the four stereoisomeric 4-OHEN-A adducts exhibit distinct structural features. Each base adduct and stereoisomer distorts the structure of the DNA duplex differently. These characteristics may manifest themselves in terms of differential nucleotide excision repair susceptibilities and mutagenic activities of the 4-OHEN-A and C adducts. (+info)
Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases.
(11/29)
4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy. (+info)
Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry.
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Sulfonyl chlorides substituted with functional groups having high proton affinity can serve as derivatization reagents to enhance the sensitivity for steroidal estrogens in liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The most commonly used reagent for derivatization of estrogens for LC-ESI-MS/MS is dansyl chloride. In this study, we compared dansyl chloride, 1,2-dimethylimidazole-4-sulfonyl (DMIS) chloride, pyridine-3-sulfonyl (PS) chloride, and 4-(1H-pyrazol-1-yl)benzenesulfonyl (PBS) chloride for derivatization of 17beta-estradiol (E2) prior to LC-ESI-MS/MS. The product ion spectra of the dansyl and DMIS derivatives were dominated by ions representing derivatization reagent moieties. In contrast, the product ion spectrum of the PS derivative of E2 and, to a lesser extent, the PBS derivative, showed analyte-specific fragment ions. Derivatization with PS chloride was therefore chosen for further investigation. The product ion spectrum of the PS derivative of E2 showed intense ions at m/z 272, assigned to the radical E2 cation, and at m/z 350, attributed to the loss of SO(2) from the [M+H](+) ion. Third-stage mass spectrometry of the PS derivative of E2 with isolation and collisional activation of the m/z 272 ion resulted in steroid C and D ring cleavages analogous to those observed in electron ionization mass spectrometry. The product ion spectra of the PS derivatives of estrone, 17alpha-ethinylestradiol, equilin, and equilenin showed similar estrogen-specific ions. Using derivatization with PS chloride, we developed an LC-ESI-MS/MS method with multiple reaction monitoring of primary and confirmatory precursor-to-product ion transitions for the determination of E2 in serum. (+info)
Conformational properties of equilenin-DNA adducts: stereoisomer and base effects.
(13/29)
Determination of absolute configurations of 4-hydroxyequilenin-cytosine and -adenine adducts by optical rotatory dispersion, electronic circular dichroism, density functional theory calculations, and mass spectrometry.
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Development of a liquid chromatography electrospray ionization tandem mass spectrometry method for analysis of stable 4-hydroxyequilenin-DNA adducts in human breast cancer cells.
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