A metabolite of equine estrogens, 4-hydroxyequilenin, induces DNA damage and apoptosis in breast cancer cell lines

Estrogen replacement therapy has been correlated with an increased risk of developing breast or endometrial cancer. 4-Hydroxyequilenin (4-OHEN) is a c atechol 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 consum e reducing equivalents and molecular oxygen, are potent cytotoxins, and cau se a variety of damage to DNA, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/p yrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Ch em. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could co ntribute to the cytotoxic and genotoxic effects of equilenin in vivo. In th e study presented here, we examined the relative toxicity of 4-OHEN in estr ogen receptor (ER) positive cells (MCF-7 and S30) compared to that in breas t 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 mu M) and 6-fold more toxic to S30 cells (LC50 = 4.0 +/- 0.1 mu M) than to MDA-MB -231 cells (LC50 = 24 +/- 0.3 mu M). Using the single-cell gel electrophore sis assay (comet assay) to assess DNA damage, we found that 4-OHEN causes c oncentration-dependent DNA single-strand cleavage in all three cell lines, and this effect could be enhanced by agents which catalyze redox cycling (N ADH) or deplete cellular GSH (diethyl maleate). In addition, the ER+ cell l ines (MCF-7 and S30) were considerably more sensitive to induction of DNA d amage by 4-OHEN than the ER- cells (MDA-MB-231). 4-OHEN also caused a conce ntration-dependent increase in the amount of mutagenic lesion 8-oxo-dG in t he 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 catec hol estrogen metabolite 4-hydroxyestrone (4-OHE) was considerably less effe ctive 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 relat ed to its ability to induce DNA damage and apoptosis in hormone sensitive c ells in vivo, and these effects may be potentiated by the estrogen receptor .