Excessive exposure to synthetic and endogenous
estrogens has been associated with the development of
cancer in several tissues.
4-Hydroxyequilenin (4-OHEN), a major metabolite of equine
estrogens present in
estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in
breast cancer cells, and cells that contain
estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to
estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in
8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (
NADH) or deplete GSH (
diethyl maleate). The role of the
estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist
tamoxifen, where decreases in
8-oxo-deoxyguanosine were observed. Another equine
estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce
reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of
estrogen-dependent
alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine
estrogen metabolites could involve oxidative stress and that the
estrogen receptor may play a role in this process.