toremifene and quinone-methide

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 sin

Topics: Binding, Competitive; Breast Neoplasms; Catechols; Cell Line, Tumor; Cell Survival; Comet Assay; DNA Damage; Drug Screening Assays, Antitumor; Estradiol; Estrogen Receptor Modulators; Female; Humans; Indolequinones; Quinones; Receptors, Estrogen; Tamoxifen; Toremifene

Tamoxifen remains the endocrine therapy of choice in the treatment of all stages of hormone-dependent breast cancer. However, tamoxifen has been shown to increase the risk of endometrial cancer which has stimulated research for new effective antiestrogens, such as droloxifene and toremifene. In this study, the potential for these compounds to cause cytotoxic effects was investigated. One potential cytotoxic mechanism could involve metabolism of droloxifene and toremifene to catechols, followed by oxidation to reactive o-quinones. Another cytotoxic pathway could involve the oxidation of 4-hydroxytoremifene to an electrophilic quinone methide. Comparison of the amounts of GSH conjugates formed from 4-hydroxytamoxifen, droloxifene, and 4-hydroxytoremifene suggested that 4-hydroxytoremifene is more effective at formation of a quinone methide. However, all three substrates formed similar amounts of o-quinones. Both the tamoxifen-o-quinone and toremifene-o-quinone reacted with deoxynucleosides to give corresponding adducts. However, the toremifene-o-quinone was shown to be considerably more reactive than the tamoxifen-o-quinone in terms of both kinetic data as well as the yield and type of deoxynucleoside adducts formed. Since thymidine formed the most abundant adducts with the toremifene-o-quinone, sufficient material was obtained for characterization by (1)H NMR, COSY-NMR, DEPT-NMR, and tandem mass spectrometry. Cytotoxicity studies with tamoxifen, droloxifene, 4-hydroxytamoxifen, 4-hydroxytoremifene, and their catechol metabolites were carried out in the human breast cancer cell lines S30 and MDA-MB-231. All of the metabolites tested showed cytotoxic effects that were similar to the parent antiestrogens which suggests that o-quinone formation from tamoxifen, droloxifene, and 4-hydroxytoremifene is unlikely to contribute to their cytotoxicity. However, the fact that the o-quinones formed adducts with deoxynucleosides in vitro implies that the o-quinone pathway might contribute to the genotoxicity of the antiestrogens in vivo.

Topics: Animals; Antineoplastic Agents; Benzoquinones; Breast Neoplasms; Cell Survival; Deoxyribonucleosides; DNA Adducts; Female; Glutathione; Indolequinones; Indoles; Microsomes, Liver; Quinones; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization; Tamoxifen; Toremifene; Tumor Cells, Cultured

Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and it has recently been approved by the Food and Drug Administration for the chemoprevention of this disease. However, long-term usage of tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe the chemical reactivity and toxicity of such an electrophilic species, we have prepared the 4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity under physiological conditions, and quantified its reactivity with GSH. Interestingly, this quinone methide is unusually stable; its half-life under physiological conditions is approximately 3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between 4-hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the quinone methide GSH conjugates slowly decompose over time, regenerating the quinone methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in microsomal incubations with 4-hydroxytamoxifen; however, none were observed in breast cancer cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give 4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The toremifene quinone methide has a half-life of approximately 1 h under physiological conditions, and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two molecules of GSH and loses chlorine to give the corresponding di-GSH conjugates. The reaction mechanism likely involves an episulfonium ion intermediate which may contribute to the potential cytotoxic effects of toremifene. Similar to what was observed with 4-hydroxytamoxifen, 4-hydroxytoremifene was metabolized to di-GSH conjugates in microsomal incubations at about 3 times the rate of 4-hydroxytamoxifen, although no conjugates were detected with MCF-7 cells. Finally, these data suggest that quinone

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cytochrome P-450 Enzyme System; Estrogen Receptor Modulators; Female; Glutathione; Humans; Hydroxylation; Indolequinones; Indoles; Mass Spectrometry; Oxidation-Reduction; Quinones; Rats; Rats, Sprague-Dawley; Tamoxifen; Toremifene; Tumor Cells, Cultured