title: A metabolite of equine estrogens, 4-hydroxyequilenin, induces DNA damage and apoptosis in breast cancer cell lines.
abstract: Estrogen replacement therapy has been correlated with an increased risk of developing breast or endometrial cancer. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin which is a minor component of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to quinoids which can consume reducing equivalents and molecular oxygen, are potent cytotoxins, and cause a variety of damage to DNA, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could contribute to the cytotoxic and genotoxic effects of equilenin in vivo. In the study presented here, we examined the relative toxicity of 4-OHEN in estrogen receptor (ER) positive cells (MCF-7 and S30) compared to that in breast cancer cells without the estrogen receptor (MDA-MB-231). The data showed that 4-OHEN was 4-fold more toxic to MCF-7 cells (LC(50) = 6.0 +/- 0. 2 microM) and 6-fold more toxic to S30 cells (LC(50) = 4.0 +/- 0.1 microM) than to MDA-MB-231 cells (LC(50) = 24 +/- 0.3 microM). Using the single-cell gel electrophoresis assay (comet assay) to assess DNA damage, we found that 4-OHEN causes concentration-dependent DNA single-strand cleavage in all three cell lines, and this effect could be enhanced by agents which catalyze redox cycling (NADH) or deplete cellular GSH (diethyl maleate). In addition, the ER(+) cell lines (MCF-7 and S30) were considerably more sensitive to induction of DNA damage by 4-OHEN than the ER(-) cells (MDA-MB-231). 4-OHEN also caused a concentration-dependent increase in the amount of mutagenic lesion 8-oxo-dG in the S30 cells as determined by LC/MS-MS. Cell morphology assays showed that 4-OHEN induces apoptosis in these cell lines. As observed with the toxicity assay and the comet assay, the ER(+) cells were more sensitive to induction of apoptosis by 4-OHEN than MDA-MB-231 cells. Finally, the endogenous catechol estrogen metabolite 4-hydroxyestrone (4-OHE) was considerably less effective at inducing DNA damage and apoptosis in breast cancer cell lines than 4-OHEN. Our data suggest that the cytotoxic effects of 4-OHEN may be related to its ability to induce DNA damage and apoptosis in hormone sensitive cells in vivo, and these effects may be potentiated by the estrogen receptor.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: E
authors.initials: E
authors.lastname: Pisha
authors.forename: A I
authors.initials: AI
authors.lastname: Constantinou
authors.forename: Y
authors.initials: Y
authors.lastname: Hua
authors.forename: L
authors.initials: L
authors.lastname: Shen
authors.forename: R B
authors.initials: RB
authors.lastname: van Breemen
authors.forename: E C
authors.initials: EC
authors.lastname: Elguindi
authors.forename: S Y
authors.initials: SY
authors.lastname: Blond
authors.forename: F
authors.initials: F
authors.lastname: Zhang
authors.forename: J L
authors.initials: JL
authors.lastname: Bolton
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology*; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects*; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology; D004855:Equilenin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D045166:Estradiol Congeners / Q000378:metabolism / Q000633:toxicity*; D002393:Estrogens, Catechol / Q000633:toxicity; D005260:Female; D006801:Humans; D006894:Hydroxyestrones / Q000633:toxicity; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D045166:Estradiol Congeners; D002393:Estrogens, Catechol; D006894:Hydroxyestrones; C026418:4-hydroxyestrone; C065250:4-hydroxy-equilenin; D004855:Equilenin