A Metabolite of Equine Estrogens, 4-Hydroxyequilenin, Induces DNA Damage and Apoptosis in Breast Cancer Cell Lines
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- 作者：Yumei Chen ; Xuemei Liu ; Emily Pisha ; Andreas I. Constantinou ; Yousheng Hua ; Lixin Shen ; Richard B. van Breemen ; Ebrahim C. Elguindi ; Sylvie Y. Blond ; Fagen Zhang ; and Judy L. Bolton
- 刊名：Chemical Research in Toxicology
- 出版年：2000
- 出版时间：May 2000
- 年：2000
- 卷：13
- 期：5
- 页码：342 - 350
- 全文大小：185K
- 年卷期：v.13,no.5(May 2000)
- ISSN：1520-5010
文摘
Estrogen replacement therapy has been correlated with an increased risk of developing breastor endometrial cancer. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin whichis a minor component of the estrogen replacement formulation marketed under the name ofPremarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to quinoids whichcan consume reducing equivalents and molecular oxygen, are potent cytotoxins, and cause avariety of damage to DNA, including formation of bulky stable adducts, apurinic sites, andoxidation 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 deleteriouseffects could contribute to the cytotoxic and genotoxic effects of equilenin in vivo. In the studypresented here, we examined the relative toxicity of 4-OHEN in estrogen receptor (ER) positivecells (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 (LC50 =6.0 ± 0.2 
M) and 6-fold more toxic to S30 cells (LC50 = 4.0 ± 0.1 M) than to MDA-MB-231cells (LC50 = 24 ± 0.3 M). Using the single-cell gel electrophoresis assay (comet assay) toassess 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 catalyzeredox 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-OHENthan the ER- cells (MDA-MB-231). 4-OHEN also caused a concentration-dependent increasein 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 observedwith the toxicity assay and the comet assay, the ER+ cells were more sensitive to induction ofapoptosis by 4-OHEN than MDA-MB-231 cells. Finally, the endogenous catechol estrogenmetabolite 4-hydroxyestrone (4-OHE) was considerably less effective at inducing DNA damageand apoptosis in breast cancer cell lines than 4-OHEN. Our data suggest that the cytotoxiceffects of 4-OHEN may be related to its ability to induce DNA damage and apoptosis in hormonesensitive cells in vivo, and these effects may be potentiated by the estrogen receptor.
M) and 6-fold more toxic to S30 cells (LC50 = 4.0 ± 0.1 M) than to MDA-MB-231cells (LC50 = 24 ± 0.3 M). Using the single-cell gel electrophoresis assay (comet assay) toassess 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 catalyzeredox 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-OHENthan the ER- cells (MDA-MB-231). 4-OHEN also caused a concentration-dependent increasein 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 observedwith the toxicity assay and the comet assay, the ER+ cells were more sensitive to induction ofapoptosis by 4-OHEN than MDA-MB-231 cells. Finally, the endogenous catechol estrogenmetabolite 4-hydroxyestrone (4-OHE) was considerably less effective at inducing DNA damageand apoptosis in breast cancer cell lines than 4-OHEN. Our data suggest that the cytotoxiceffects of 4-OHEN may be related to its ability to induce DNA damage and apoptosis in hormonesensitive cells in vivo, and these effects may be potentiated by the estrogen receptor.