4-hydroxyestrone and 4-hydroxy-equilenin

Metabolic activation of estrogens to catechols and further oxidation to highly reactive o-quinones generates DNA damage including apurinic/apyrimidinic (AP) sites. 4-Hydroxyequilenin (4-OHEN) is the major catechol metabolite of equine estrogens present in estrogen replacement formulations, known to cause DNA strand breaks, oxidized bases, and stable and depurinating adducts. However, the direct formation of AP sites by 4-OHEN has not been characterized. In the present study, the induction of AP sites in vitro by 4-OHEN and the endogenous catechol estrogen metabolite, 4-hydroxyestrone (4-OHE), was examined by an aldehyde reactive probe assay. Both 4-OHEN and 4-OHE can significantly enhance the levels of AP sites in calf thymus DNA in the presence of the redox cycling agents, copper ion and NADPH. The B-ring unsaturated catechol 4-OHEN induced AP sites without added copper, whereas 4-OHE required copper. AP sites were also generated much more rapidly by 4-OHEN. For both catechol estrogens, the levels of AP sites correlated linearly with 8-oxo-dG levels, implying that depuriniation resulted from reactive oxygen species (ROS) rather than depurination of estrogen-DNA adducts. ROS modulators such as catalase, which scavenges hydrogen peroxide and a Cu(I) chelator, blocked the formation of AP sites. In MCF-7 breast cancer cells, 4-OHEN significantly enhanced the formation of AP sites with added NADH. In contrast, no significant induction of AP sites was detected in 4-OHE-treated cells. The greater redox activity of the equine catechol estrogen produces rapid oxidative DNA damage via ROS, which is enhanced by redox cycling agents and interestingly by NADPH-dependent quinone oxidoreductase.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cattle; Cell Line, Tumor; Chelating Agents; Copper; Deoxyguanosine; DNA; DNA Damage; DNA, Neoplasm; Equilenin; Estrogens, Catechol; Free Radical Scavengers; Horses; Humans; Hydrogen Peroxide; Hydroxyestrones; Molecular Structure; NADP; Oxidation-Reduction; Reactive Oxygen Species; Structure-Activity Relationship

Estrogen replacement therapy has been correlated with an increased risk of developing hormone-dependent cancers. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin and equilin which are components of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to potent cytotoxic quinoids which can consume reducing equivalents and molecular oxygen, and cause a variety of DNA lesions, 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/genotoxic effects of equine estrogens in vivo. In the study presented here, we studied the oxidative and carcinogenic potential of 4-OHEN and the catechol metabolite of the endogenous estrogen, 4-hydroxyestrone (4-OHE), in the JB6 clone 41 5a and C3H 10T(1/2) murine fibroblast cells. The relative ability of 4-OHEN and 4-OHE to induce oxidative stress was measured in these cells by oxidative cleavage of 2',7'-dichlorodiacylfluorosceindiacetate to dichlorofluoroscein. 4-OHEN (1 microM) displayed an increase in the level of reactive oxygen species comparable to that observed with 100 microM H(2)O(2). In contrast, 4-OHE demonstrated antioxidant capabilities in the 5-50 microM range. With both cell lines, we assessed single-strand DNA cleavage using the comet assay and the formation of oxidized DNA bases, such as 8-oxodeoxyguanosine, utilizing the Trevigen Fpg comet assay. 4-OHEN caused single-strand breaks and oxidized bases in a dose-dependent manner in both cell lines, whereas 4-OHE did not induce DNA damage. Since oxidative stress has been implicated in cellular transformation, we used the JB6 clone 41 5a anchorage independence assay to ascertain the relative ability of 4-OHEN and 4-OHE to act as tumor promoters. 4-OHEN caused a slight but significant increase in the extent of cellular transformation at the 100 nM dose; however, in the presence of NADH, which catalyzes redox cycling of 4-OHEN, the transformation ability of 4-OHEN was dramatically increased. 4-OHE did not induce transformation of the JB6 clone 41 5a in the 0.1-10 microM range. The initiation, promotion, and complete carcinogenic transformation potentials of both metabolites were measured in the C3H 10T(1/2) cells. 4-OHEN demonstrated

Topics: Animals; Carcinogens; Cell Line; Cell Transformation, Neoplastic; DNA Damage; Epidermal Cells; Epidermis; Equilenin; Estradiol Congeners; Fibroblasts; Horses; Hydroxyestrones; Mice; Mice, Inbred C3H; Reactive Oxygen Species

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.

Topics: Apoptosis; Breast Neoplasms; Cell Survival; Comet Assay; DNA Damage; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Epithelial Cells; Equilenin; Estradiol Congeners; Estrogens, Catechol; Female; Humans; Hydroxyestrones; Tumor Cells, Cultured