Ovarian neoplasm development by 7,12-dimethylbenz[a]anthracene (DMBA) in a chemically-induced rat model of ovarian failure
Introduction
Ovarian cancer is the most lethal of gynecologic malignancies [1]. The incidence of ovarian cancer increases by about ten-fold in women during the peri- to post-menopausal period, when compared to younger women [2]. This increase is attributed, in part, to three major factors associated with ovarian senescence, depletion of oocytes, loss of ovarian steroid production, and increased circulating gonadotropic hormones resulting from loss of negative feedback from ovarian hormones on the pituitary [2]. Thus, development of an animal model for ovarian cancer in which ovarian failure has been induced and the animal is follicle depleted, but retains residual ovarian tissue would provide a model with improved physiological relevance compared with a cycling animal.
Previous studies in rats and mice have shown that the occupational chemical, 4-vinylcyclohexene diepoxide (VCD) specifically targets and destroys primordial and primary follicles in rats and mice while leaving large pre-antral (secondary) and antral follicles unaffected [3], [4]. Mechanistic studies have determined that this selective follicle loss is due to enhancement of the natural process of atresia (apoptosis) [4], [5], [6]. Therefore, VCD has been used in mice to accelerate ovarian failure and generate an animal model for peri- and post-menopause [7]. Extensive investigation has determined that, whereas, VCD destroys small pre-antral follicles, it does not produce effects on larger follicles or any other tissue [3], [8], thus ovarian failure results only after secondary and antral follicles have become depleted via ovulation or atresia. Therefore, compared with ovariectomized animals more commonly used for modeling menopause, the VCD-induced ovarian failure model is more relevant to the study of post-menopause because the animal retains residual ovarian tissue. Furthermore, unlike the ovariectomized animal, in the VCD-treated animal, onset of ovarian failure is gradual, providing a model for the peri-menopausal transition [9]. Thus, adaptation of the VCD-treated animal to a relevant model for ovarian cancer would represent an important advancement and provide a model useful for developing diagnostic, therapeutic and preventative strategies.
In modeling ovarian cancer, recent approaches have induced ovarian tumors in rodents using carcinogens. One particularly promising approach for inducing epithelial ovarian cancer in rats has utilized direct application of 7,12-dimethylbenz[a]anthracene (DMBA) to the ovary [10], [11], [12], [13], [14], [15], [16], [17].
The present study was designed to determine the time-course for impending VCD-induced ovarian failure in rats and apply the DMBA approach in those animals for the development of ovarian neoplasms. This will provide a more physiologically relevant animal model for ovarian cancer in peri/post-menopausal women when compared with that in cycling rats. The hypothesis being tested is that ovarian neoplasms can be induced more readily in animals that have undergone chemical-induced ovarian failure.
Section snippets
Animals
Female Fisher-344 rats (age d21) were purchased from Harlan, and housed and used in accordance with NIH guidelines and the policies of the University of Arizona Institutional Animal Care and Use Committee. Temperature, humidity, and photoperiod were constant (12 h light, 12 h dark at 22 °C). Animals were allowed access to food and water ad libitum. Rats were allowed to acclimate 7 days before the experiment began.
Reagents
4-vinylcyclohexene diepoxide (VCD), 7,12-Dimethylbenz[a]anthracene (DMBA) and
Dosing
Day 28 old rats were injected (i.p.) daily (25 days) with sesame oil vehicle (2.5 ml/kg) or VCD (160 mg/kg, 1.14 mmol/kg). The day 25 time of dosing was chosen based on the results of a preliminary study comparing ovotoxicity in F344 rats following 25 or 30 days of repeated daily dosing with VCD (160 mg/kg; unpublished). Following 30 days of dosing, rats began to show adverse effects such as weight loss. There was no difference (p > 0.05) in primordial/primary follicle loss between the 2 days.
Results
VCD-induced follicle depletion was performed in F344 female rats to determine an optimal time of impending ovarian failure in which DMBA could directly be applied to the ovary (Table 1, Fig. 1). 3 months following the end of the 25 days of VCD dosing there was a reduction (p < 0.05) in follicles of all sizes, relative to age-matched (vehicle-treated) controls, whereas, by 6 months, no primordial or primary follicles remained and only a few secondary and antral follicles were observed (Table 1).
Discussion
In the mouse model for VCD-induced ovarian failure, repeated daily dosing of mice with VCD (15 days) resulted in the onset of ovarian failure at 59 days following the onset of dosing (1.5 months after dosing had stopped) [7]. Mice are known to be more susceptible to ovotoxic effects of VCD than rats [20]. Thus, in rats, a longer period of dosing (25 days) was required and a longer time to onset of ovarian failure resulted (> 6 months after dosing had stopped). The three to six month range of
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
These studies were supported by R01s AG021948 (PBH) and CA119200 (JKB) and a center grant, ES06694. We wish to thank Andrea Grantham for preparation of tissues for histological evaluation, Doug Cromey for help with the micrographs, and Nivedita Sen for help with the figures.
References (24)
- et al.
Early effects of ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats and mice
Reprod Toxicol
(1999) - et al.
Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats
Toxicol Appl Pharmicol
(1996) - et al.
Lack of modifying effects of an estrogenic compound atrazine on 7,12-dimethylbenz[a]anthracene-induced ovarian carcinogenesis in rats
Cancer Lett
(2004) - et al.
Inhibitory effects of estrogenic compounds, 4-nonylphenol and genistein, on 7,12-dimethylbenz[a]anthracene-induced ovarian carcinogenesis in rats
Ecotoxicol and Environ Safety
(2002) - et al.
Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide-induced ovotoxicity in rats
Reprod toxicol
(1994) - et al.
The role of epoxidation in 4-vinylcyclohexene-induced ovarian toxicity
Toxicol Appl Pharmicol
(1990) - et al.
Ovarian sex cord-stromal tumors: an immunohistochemical study including a comparison of calretinin and inhibin
Mod Pathol
(2003) - et al.
Rodent models for ovarian cancer research
Int J Gynecol Cancer
(2003) Loss of ovarian function and the risk of ovarian cancer
Cell Tiss Res
(2005)- et al.
Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades
Biol Reprod
(2001)
Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats
Biol Reprod
The follicle-depleted mouse ovary produces androgen
Biol Reprod
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