title: Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis.
abstract: Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.
authors: Department of Internal Medicine, University of Virginia Health Sciences System, PO Box 801416, Charlottesville, VA 22903, USA. wy9c@virginia.edu
authors: W
authors: W
authors: Yue
authors: R J
authors: RJ
authors: Santen
authors: J-P
authors: JP
authors: Wang
authors: Y
authors: Y
authors: Li
authors: M F
authors: MF
authors: Verderame
authors: W P
authors: WP
authors: Bocchinfuso
authors: K S
authors: KS
authors: Korach
authors: P
authors: P
authors: Devanesan
authors: R
authors: R
authors: Todorovic
authors: E G
authors: EG
authors: Rogan
authors: E L
authors: EL
authors: Cavalieri
mesh_terms: D000818:Animals; D001141:Aromatase / Q000235:genetics / Q000378:metabolism; D001943:Breast Neoplasms / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity*; D002394:Catechol O-Methyltransferase / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D004958:Estradiol / Q000378:metabolism* / Q000633:toxicity*; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D015674:Mammary Neoplasms, Animal / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011110:Polymorphism, Genetic; D012307:Risk Factors
chemical_list: D002273:Carcinogens; D004970:Estrone; D004958:Estradiol; D001141:Aromatase; D002394:Catechol O-Methyltransferase