Induction of Uterine Tumors in Mice by Catecholestrogens: Implications for Human Mammary and Uterine Oncogenesis
Induction of Uterine Tumors in Mice by Catecholestrogens: Implications for Human Mammary and Uterine Oncogenesis
By Joachim G. Liehr, PhD, and Retha Newbold
The carcinogenic activity of estrogenic hormones has been established in a variety of animal models.1 In humans, chronic, prolonged use of estrogen-containing medications, such as oral contraceptives or hormone replacement therapy, raises the risk for breast cancer. This risk is also increased by an elevated endogenous estrogen production, as indicated by positive correlations with higher plasma or urinary estrogen levels compared to controls.2
Background
Many mechanistic studies of tumor induction by estradiol have been carried out in the hamster kidney tumor model, because estrogens, including the natural hormones estradiol or estrone, induce renal tumors in this species with an incidence approaching 100% 6-8 months after initial administration.3 Moreover, estrogens are complete carcinogens in this system because no other carcinogen or co-carcinogen is needed for neoplastic development. There are only a few other animal models that are as convenient for mechanistic investigations yet provide unambiguous answers as to the direct and indirect effects of estrogens in oncogenesis.
The Dual Role of Estrogen
Based on such studies in the hamster kidney and also in other systems, a mechanism of tumor induction by estradiol has been proposed that emphasizes a dual role of estrogen (as hormone-stimulating cell proliferation and as a carcinogen inducing DNA damage and mutations).2 The hormonal property of estrogens (i.e., estrogen receptor-mediated stimulation of tumor cell proliferation) previously has been examined in many cell lines and in vivo and is widely accepted as a crucial event in the development of neoplasms. Less is known about the genotoxic and mutagenic activity of natural estrogens and their role in tumor induction. Specifically, estrogens are converted to catechol metabolites, 2- or 4-hydroxylated estrone, or estradiol, which may be further oxidized to semiquinone and quinone reactive intermediates. Metabolic redox cycling between quinone and hydroquinone (catechol) forms may generate potentially mutagenic free radicals.4,5 Alternatively, the quinone metabolites may bind to DNA and form carcinogenic DNA adducts.6
As part of an examination of the validity of this mechanistic proposal, the carcinogenic activities of the catechol estrogen metabolites have been examined in the hamster kidney tumor model. In two independent experiments, carried out in different laboratories by Liehr and colleagues and Li and Li, 4-hydroxyestradiol was as carcinogenic as estradiol (80-100% tumor incidence), whereas 2-hydroxyestradiol did not induce any tumors in this species.7,8 Li and Li also examined the carcinogenic activities of 2- and 4-hydroxyestrone and detected comparable results. 2-Hydroxyestrone did not induce any renal tumors, whereas 4-hydroxyestrone was carcinogenic, albeit at a lower tumor incidence (33%). Thus, the carcinogenicity data provide key evidence in support of the proposed mechanism of oncogenesis that 4-hydroxylated estrogens are procarcinogenic metabolites.
Nevertheless, the significance of these results for the genesis of human breast or uterine cancer has remained in question for several reasons. First, if this catechol metabolite mediated hormone-induced carcinogenesis, a tumor incidence exceeding that of the parent hormone has been expected for 4-hydroxyestradiol. This requirement is consistent with differences in tumor incidence data of several aromatic hydrocarbon carcinogens and their oxidized procarcinogenic metabolites. Secondly, the hamster kidney may serve as a mechanistic model but is not an organ model of human breast or uterine cancer. Therefore, an examination of the carcinogenic activities of estradiol and its catechol metabolites has been necessary in an animal model of human mammary or uterine adenocarcinoma. A significant induction of tumors by 4-hydroxylated estrogens in an animal model more closely related to human cancers would strengthen the applicability of this concept of catechols as procarcinogenic metabolites of estrogens to the human situation.
Tumor Induction by Catecholestrogens
The animal model chosen for the examination of the carcinogenic activities of catecholestrogens has been the estrogen-induced uterine adenocarcinoma of CD-1 mice, because this tumor resembles human endometrial neoplasms.9 Cancers are induced in this rodent species by administration of estrogen within the first five days of life (2 mcg of estrogen/neonate/d) and develop 12-18 months after this initial treatment without further experimental manipulation. With the synthetic estrogen diethylstilbestrol (DES), a close to 90% tumor incidence has been achieved in this system.9
Both catecholestrogens were carcinogenic in this mouse model.10
4-Hydroxyestradiol induced tumors in 23 of 35 animals (66%) and 2-hydroxyestradiol in five of 40 (12%), whereas no such neoplasms were detected in corn oil (vehicle)-treated controls (0 of 34 mice). The lesions were uterine adenocarcinomas of the type previously induced with other estrogens.9 Groups treated with estradiol or 17a-ethinylestradiol served as positive controls. Estradiol-induced tumors in one of 15 animals (7%), and 17a-ethinylestradiol in 18 of 42 mice (43%).
Uterine wet weight increases also were assayed on day 5 after birth in neonatal mice treated with these estrogens on days 1-4 of neonatal life (2 mcg estrogen/neonate/d), whereas controls received only corn oil vehicle.10 Uterine weights of animals treated with 4-hydroxyestradiol were double the control values. Treatment with 2-hydroxyestradiol, estradiol, and 17a-ethinylestradiol resulted in uterine wet weight values that were slightly elevated but not significantly different from controls.
Significance
The high carcinogenic activity of 4-hydroxyestradiol in this animal model demonstrates that this catecholestrogen is carcinogenic. These data are consistent with previous results obtained in the hamster kidney system, which have identified this estrogen metabolite as a carcinogen.7,8 The more recent data provide an important link to human cancer, because the tumors have been induced by this catechol in an organ model of human uterine cancer and closely resemble human uterine adenocarcinoma. Estradiol also induces these tumors in mice (albeit at a lower incidence).10 By analogy, estrogen exposure unopposed by progestin is a risk factor for human uterine adenocarcinoma.11 Moreover, human and mouse uterus expresses a specific estrogen 4-hydroxylase (cytochrome P450 1B1), which converts the parent hormone to 4-hydroxyestradiol.12-14
The nine-fold higher tumor incidence by 4-hydroxyestradiol in mice compared to that of the parent hormone estradiol provides strong support for the concept of a dual role of estrogen in oncogenesis functioning as a hormone and as a carcinogen.2 It is unlikely that the hormonal potency of 4-hydroxyestradiol evident from a 60% higher uterine wet weight gain compared to estradiol by itself suffices to increase the tumor incidence nine-fold over values obtained with estradiol.10 More likely, it is a complex mechanism of hormone receptor-mediated proliferation of cells altered by 4-hydroxyestradiol-induced DNA damage and mutations.2 4-Hydroxyestradiol may be further oxidized to a quinone reactive intermediate.4 Metabolic redox cycling between this quinone and the hydroquinone (4-hydroxyestradiol) potentially may produce mutagenic free radicals.4,5 This concept of metabolic activation is supported by multiple types of DNA damage generated by 4-hydroxyestradiol or its corresponding quinone in cell-free systems, cells in culture, or in vivo.2 Recently, 4-hydroxyestrone and 4-hydroxyestradiol have been shown to induce hprt gene mutations in Syrian hamster embryo cells, whereas estradiol or other estrogen metabolites were negative in that gene mutation assay.15
The induction of uterine tumors in mice by 2-hydroxyestradiol is the first demonstration of carcinogenic activity of this estrogen metabolite in any system, because this catechol has not induced renal tumors in the hamster. Both catechol estrogen metabolites, 2- and 4-hydroxyestradiol are capable of metabolic redox cycling between hydroquinone (catechol) and quinone forms.5 Consistent with this metabolic activation, 2-hydroxyestradiol induces various forms of DNA damage in cell-free test systems and structural chromosomal aberrations in Syrian hamster embryo cells in culture.7,15,16 These characteristics of 2-hydroxyestradiol point to a cell damaging potential of this estrogen metabolite. They are inconsistent with a characterization of 2-hydroxyestrogens as "good estrogens" or antiestrogens.17,18 2-Hydroxyestrogens may have failed to induce renal tumors in Syrian hamsters because these estrogen metabolites are rapidly inactivated by catechol-O-methyltransferase and converted to inactive methoxyestrogens. This conjugating enzyme activity may not have been fully expressed in neonatal mice, whereas the hamsters have been exposed at an adult age to estrogens and may thus have been protected.
17a-Ethinylestradiol has been six-fold more carcinogenic in CD-1 mice than estradiol, although their hormonal activities (uterine wet weight gain) were not significantly different.10 In contrast, 17a-ethinylestradiol is a poor carcinogen (10% incidence) in the hamster kidney system.3,8 These differential carcinogenic activities of this synthetic estrogen may be due to species differences in metabolic activation. Nevertheless, the values obtained in the mouse model probably are more relevant to the human situation, because 17a-ethinylestradiol is the estrogen used in oral contraceptives, which are known to raise the risk of breast cancer in humans.19 In line with human mammary tumor induction by estrogens, specific estrogen 4-hydroxylases of normal mammary tissue and tumors convert estradiol to the 4-hydroxylated catechol metabolite.20 In tumor tissue, this process predominates over 2-hydroxylation.
In summary, the study by Newbold and Liehr demonstrates a higher carcinogenic activity of 4-hydroxyestradiol than of estradiol in the mouse uterus.10 It also provides the first evidence in any model for the carcinogenic activity of 2-hydroxyestradiol. This potent carcinogenicity of the 4-hydroxylated catechol estrogen supports the concept that this metabolite mediates hormone-induced cancer by inducing DNA damage and genetic lesions. Thus, estrogens may induce tumors including human breast and uterine cancers by a dual action as carcinogen inducing DNA damage and genetic lesions and as hormone stimulating the proliferation of cells damaged by such processes. (Dr. Liehr is Chief Pharmacologist, The Stehlin Foundation for Cancer Research, Houston, TX; and Retha Newbold is Head, Developmental Endocrinology Section, Laboratory of Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC.)
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