The
antiestrogen,
tamoxifen, has been extensively used in the treatment and prevention of
breast cancer. Although
tamoxifen showed benefits in the
chemotherapy and
chemoprevention of
breast cancer, epidemiological studies in both
tamoxifen-treated
breast cancer patients and healthy women indicated that treatment caused an increased risk of developing
endometrial cancer. These troubling side effects lead to concerns over long-term safety of the
drug. Therefore, it is important to fully understand the relationship between the antiestrogenic and the genotoxic mechanisms of
tamoxifen, other
antiestrogens, and their metabolites. Previously, we have shown that o-
quinone formation from
tamoxifen and its analogues,
droloxifene and
4-hydroxytoremifene, may not contribute to the cytotoxic effects of these
antiestrogens; however, these o-
quinones can form adducts with deoxynucleosides and this implies that the o-
quinone pathway could contribute to the genotoxicity of the
antiestrogens in vivo. To further investigate this potential genotoxic pathway, we were interested in the role of
estrogen receptor (ER)(1) alpha and beta since work with
catechol estrogens has shown that ERs seem to enhance DNA damage in
breast cancer cell lines. As a result, we investigated the binding affinities of 4-hydroxy and 3,4-dihydroxy derivatives of
tamoxifen and
toremifene to ER alpha and beta. The antiestrogenic activities of the metabolites using the Ishikawa cells were also investigated as well as their activity in
ERalpha and
ERbeta breast cancer cells using the transient transfection reporter,
estrogen response element-dependent
luciferase assay. The data showed that the antiestrogenic activities of these compounds in the
biological assays mimicked their activities in the ER binding assay. To determine if the compounds were toxic and if ERs played a role in this process, the cytotoxicity of these compounds in ERbeta41(2) (
ERbeta), S30 (
ERalpha), and MDA-MB-231 (ER(-)) cell lines was compared. The results showed that the cytotoxicity differences between the metabolites were modest. In addition, all of the metabolites showed similar toxicity patterns in both ER positive and negative cell lines, which means that the ER may not contribute to the cytotoxicity pathway. Finally, we compared the amount of DNA damage induced by these metabolites in these cell lines using the comet assay. The
catechols 3,4-dihydroxytoremifene and
3,4-dihydroxytamoxifen induced a greater amount of cellular single strand DNA cleavage as compared with the
phenols in all cell lines. The different amounts of DNA damage in ER positive and negative cell lines suggested that the ERs might play a role in this process. These data suggest that the formation of
catechols represents a minor role in cytotoxic and antiestrogenic effects in cells as compared with their
phenol analogues. However,
catechols induced more DNA damage at nontoxic doses in
breast cancer cells, which implies that o-
quinones formed from
catechols could contribute to genotoxicity in vivo, which is ER-dependent.