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: Y Chen, X Liu, E Pisha, A I Constantinou, Y Hua, L Shen, R B van Breemen, E C Elguindi, S Y Blond, F Zhang, J L Bolton
• Journal: Chemical research in toxicology (Chem Res Toxicol) Vol. 13 Issue 5 Pg. 342-50 (May 2000) ISSN: 0893-228X [Print] United States
• PMID: 10813650 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA, Neoplasm
• Estradiol Congeners
• Estrogens, Catechol
• Hydroxyestrones
• 4-hydroxyestrone
• 4-hydroxy-equilenin
• Equilenin

Topics

• Apoptosis (drug effects)
• Breast Neoplasms (drug therapy, pathology)
• Cell Survival (drug effects)
• Comet Assay
• DNA Damage (drug effects)
• DNA Fragmentation
• DNA, Neoplasm (drug effects)
• Dose-Response Relationship, Drug
• Epithelial Cells (drug effects, pathology)
• Equilenin (analogs & derivatives, metabolism, toxicity)
• Estradiol Congeners (metabolism, toxicity)
• Estrogens, Catechol (toxicity)
• Female
• Humans
• Hydroxyestrones (toxicity)
• Tumor Cells, Cultured