7,12-Dimethylbenz[a]anthracene-Induced Malignancies in a Mouse Model of Menopause

Ovarian cancer has a high mortality rate because there are few symptoms in early disease development. The incidence of ovarian cancer increases in women after menopause. Understanding early events in this disease can best be achieved by using animal models. Therefore, the objective of this study was to develop and track the onset of ovarian tumorigenesis in mice mimicking characteristics of postmenopausal epithelial cancer in women. Female B6C3F1 mice (age, 28 d) received 4-vinylcyclohexene diepoxide (VCD, 160 mg/kg IV daily for 20 d) to cause ovarian failure. Four months after VCD treatment, via surgical intervention, each mouse received a single injection of 7,12-dimethylbenz[a]anthracene (DMBA) or vehicle control (sesame oil) under the bursa of the right ovary to cause ovarian neoplasms. The experimental groups were untreated controls (Con–Con), DMBA-treatment only (Con–DMBA), VCD treatment only (VCD–Con), and VCD+DMBA-treated (VCD+DMBA) mice. At 3, 5, 7, and 9 mo after DMBA injection, ovaries were collected for histologic and immunohistochemical evaluation. No tumors developed in Con–Con mice. All VCD-treated mice (with or without DMBA) exhibited ovarian failure. Mice that received both VCD and DMBA exhibited tumors at 3 mo (50%), 5 mo (14%), 7 mo (90%), and 9 mo (57%) after DMBA treatment; 31% of the tumors were epithelial in origin. Our findings confirm that inducing ovarian tumors in mice by chemical means is an effective method for studying early stages of tumor development that may be relevant to epithelial ovarian cancers that arise in postmenopausal women.Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; VCD, 4-vinylcyclohexene diepoxideOvarian cancer, the most deadly female reproductive malignancy, has a high mortality rate because high-grade cancers are thought to metastasize early prior to the development of symptoms in early stages of disease.^4,27,28 The risk of contracting ovarian cancer over a lifetime is about 1 in 70, so it is a relatively rare cancer.²⁸ Although more than 20 types of ovarian malignancies exist, about 90% of human ovarian cancers are epithelial in origin.²⁸ Most cases are diagnosed at stages when the disease has metastasized outside the ovary, hindering efforts to treat or cure the disease. In addition, few reliable detection methods exist for early diagnosis of this disease. The incidence of ovarian cancer increases 8- to 10-fold among women in the peri- to postmenopausal period when compared with younger women.²⁸ The generation of animal models of ovarian cancer has been attempted for decades. These models have included whole-body irradiation,^5-7 chemical induction,^13,15,17,21 genetic manipulation,^18,25 and xenograph development.^9,23 It was observed as early as 1936 that the removal of all follicles from a mouse ovary was followed by the appearance of benign tubular adenomas in the residual ovarian tissue.^6,7,21,27 These adenomas appear to originate at the surface epithelium and proceed to invaginate and spread throughout the ovary.As women transition from peri- to postmenopause, circulating levels of estrogen and progesterone decrease, and the relative ratio of estrogens to androgens decreases in response to the decline of estrogen. In addition, gonadotropin (follicle stimulating hormone, luteinizing hormone) levels rise due to loss of negative feedback on the anterior pituitary and, thereafter, remain elevated.²⁶ One theory of ovarian carcinogenesis proposes that increased circulating gonadotropin levels after menopause contribute to the development of ovarian epithelial cancers by stimulating surface epithelium proliferation.¹⁸ Women who have undergone a natural progression to menopause have lost ovarian function but retain residual ovarian tissue. Therefore, because ovarian cancers in women arise more frequently after than before menopause, models developed in animals that have undergone ovarian failure and retain residual ovarian tissue likely most closely resemble the disease in postmenopausal women.Repeated daily dosing of mice with the ovotoxic chemical 4-vinylcyclohexene diepoxide (VCD) results in a gradual onset of ovarian failure.²⁴ Because VCD selectively targets primordial and primary follicles,²² larger follicles remain and develop toward ovulation.⁸ With the depletion of primordial and primary follicles, recruitment into the larger follicle pool eventually ceases, and a gradual onset of ovarian failure results. In VCD-treated mice, estrogen and progesterone concentrations decline and follicle-stimulating hormone levels rise after follicle depletion, similar to the scenario in postmenopausal women.¹⁹ A recent mouse model that combined virally induced changes in genes within the ovary and treatment with VCD resulted in ovarian failure along with induction of tumors characterized as undifferentiated tumors with mixed epithelial and stromal components along with some features of sex cord stromal tumors.¹⁸In a previous study, female Fisher 344 rats with VCD-induced ovarian failure developed ovarian tumors after treatment with 7, 12-dimethylbenz[a]anthracene (DMBA).¹¹ Specifically, 57% of the VCD+DMBA-treated rats developed ovarian tumors within 5 months after DMBA treatment. However, the tumors were all classified as Sertoli–Leydig cell type lesions, which are rare ovarian neoplasms in women and often much less aggressive than are their epithelial counterpart.^27,28 In another study,³ female B₆C₃F₁ mice were treated in the same way as in the Fisher 344 rat study.⁹ Similarly, all tumors that developed within 5 mo in treated mice (28%) were also Sertoli–Leydig cell type masses. Therefore, the present study was undertaken in B₆C₃F₁ mice to observe and classify DMBA-induced ovarian tumor development at later time points (7 and 9 mo after DMBA exposure) to determine whether epithelial tumors would develop and, if so, when.