Female Noble Rats Research Articles

Noble Rats Display Decreased Weight Gain and Visceral Adiposity via Lifelong Exposure to an Isoflavone-Rich Diet

Obesity and cancer represent two of the greatest public health concerns. The relationship between obesity and cancer suggests an important role for visceral adipose tissue (VAT) versus generalized body fat. Male and female Noble rats develop a relatively high incidence of spontaneous and hormone-induced carcinomas. The main purpose of this study was to make an initial assessment of whether a high soy-isoflavonoid diet could decrease VAT and reduce overall body weight gain in Noble rats. The male and female rats were exposed solely to either a high soy-isoflavonoid diet (High-Iso) or a low-isoflavonoid diet (Low-Iso) from conception to adulthood. At the end of the experiment, at 145 days of age, overall weight gain and visceral fat deposition were recorded along with quantification of serum isoflavone levels (genistein, daidzein and equol). Serum isoflavone levels in the High-Iso treatment groups were significantly higher (by approximately 15-fold) compared to animals in the LowIso diet groups. Male and female Nobel rats fed the High-Iso diet displayed significantly lower body weight gain (by approximately 10%) compared to animals fed the Low-Iso diet. Notably, VAT deposition was reduced by 61% in females but 21% in males fed the High-Iso diet versus the Low-Iso diet. In summary, consumption of a diet high in isoflavones significantly lowered body weight and VAT deposition and a hypothesis may be proposed as to how soy-derived isoflavones may decrease VAT storage areas to potentially improve obesity and cancer outcomes.

An expression study of hormone receptors in spontaneously developed, carcinogen-induced and hormone-induced mammary tumors in female Noble rats

The Noble (Nb) rat model has been used in the study of hormonal carcinogenesis of mammary and prostate glands, as this rat strain is susceptible to tumor induction in these glands by hormonal treatments. Recently, we demonstrated that this rat strain can develop spontaneously mammary tumors at high incidence in aged animals and also show high sensitivity to chemical carcinogens (DMBA and MNU) and combined treatments with sex hormones in mammary tumor induction. In the present study, we examined and compared the expression of hormone receptors [including estrogen receptors (ERalpha and ERbeta), androgen receptor (AR), progesterone receptor (PR), prolactin receptor (PRLR)] and prolactin (PRL) by immunohistochemistry, Western blotting and RT-PCR in spontaneous mammary tumors, and mammary tumors induced by sex hormones (T+E2 and T+DES for 8-10 months) and DMBA in Nb rat model. Immunohistochemistry and Western blotting showed that both the spontaneously developed and hormone-induced carcinomas exhibited strong immunoreactivity of ERalpha, ERbeta, AR, PR and PRLR, while the spontaneous fibroadenomas showed weak to moderate immunoreactivity of ERalpha and PRLR, whereas the DMBA-induced carcinomas exhibited weak to moderate immunoreactivity of ERalpha, AR, PR and PRLR, and sporadic weak ERbeta immunoreactivity. RT-PCR analyses showed that mRNA expression pattern of these markers resembled that of proteins. In addition, weak mRNA expression of PRL was detected in spontaneous carcinomas and carcinomas induced by DMBA and hormones, suggesting that PRL could be produced locally within the tumors. The results showed that the expression status of hormone receptors and PRL was different in spontaneous mammary tumors and tumors induced by carcinogen or hormones, suggesting that the extent of involvement of steroid hormones and their receptors in the spontaneous, carcinogen- or hormone-induced mammary carcinogenesis might be different.

Sex hormone-induced mammary carcinogenesis in female Noble rats: detection of differentially expressed genes.

Breast cancer is the most common cancer and the second most frequent cause of cancer death in women. Epidemiological data has recognized that an increased cumulative exposure to estrogen is the common tie linking most of the established risk factors for breast cancer. Sex hormone-induced mammary gland carcinogenesis of the Noble rat (using testosterone and 17beta-estradiol) resembles that of the human counterpart in its growth pattern as well as the histopathology of the tumors induced. This model may provide a paradigm for examination of genetic alterations and changes in gene expression between different histological groups and to make inferences about the role of known and putative oncogenes and tumor suppressor genes. We studied the gene expression profile during sex hormone-induced mammary carcinogenesis using a cDNA array technique; the results were further confirmed by RT-PCR, western blotting and immunohistochemical analyses. From the 10 differentially expressed genes identified, we have studied four highly overexpressed genes, two cell cycle/growth control regulators, the cyclins D1 and D2, a growth factor, IGF-2 and a cytokine TNF-alpha. Cyclins D1 and D2 were highly expressed in the nuclei of carcinoma cells but at low levels in the nuclei of the hyperplastic and normal mammary tissue. IGF-2 was found to expressed in the cytoplasm of the carcinoma cells but not in the stromal cells. Western blot showed expression of big IGF-2 consistent with the tumor derived truncated forms of pro-IGF-2. The matured circulating IGF-2 at 7.5 kDa identified in the serum was not expressed in any of the breast tissue samples. TNF-alpha expression was found not only in the macrophages but also in the mammary carcinoma cells. The result of the present study provides some information on the molecular basis of this sex hormone-induced mammary carcinogenesis and the role of these proteins in tumor progression.

In Vitro Rat Bladder Function After Neonatal Estrogenization

Experiments were done to evaluate the functional effects of neonatal diethylstilbestrol (DES) treatment on bladder function in male and female Noble rats. At 5 months after neonatal DES bladders were removed and weighed. Ventral and dorsal bladder strips were prepared to evaluate the effects of neonatal DES on contractile responses to electrical field stimulation, carbachol, adenosine triphosphate, phenylephrine and KCl. Relaxant responses to the catecholamines arterenol (norepinephrine), epinephrine and isoproterenol were also monitored. Neonatal DES resulted in significant increases in bladder mass in males and females. Contractile and relaxant responses were largely unchanged by neonatal DES treatment and the only change observed was a decreased response of ventral strips from male neonatal DES rats to 4 and 8 Hz. stimulation. Ventral strips from male control and neonatal DES rats responded to field stimulation and carbachol with significantly greater responses than dorsal strips and were more sensitive to the relaxant actions of norepinephrine and epinephrine. The data confirm that neonatal DES causes infravesical obstruction. However, in contrast to published reports of the effects of surgically induced mild outlet obstruction, neonatal DES treatment has little effect on in vitro bladder strip contractile or relaxant function. Thus, the neonatal DES treated rat does not seem to be a useful model in which to study the in vitro effects of partial outlet obstruction on the bladder.

Sex hormone-induced mammary carcinogenesis in the female Noble rats: Expression of Bcl-2 and Bax in hormonal mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal-epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of Bcl-2 and Bax in pre-malignant mammary glands from rats treated with different protocols of sex hormones for 7 weeks as well as sex hormone induced mammary tumours. We observed that Bcl-2 was strongly expressed in most of mammary tumour cells, whereas weak or negative in adjacent normal or hyperplastic ductal structures. On the contrary, Bax immunoreactivity was weak in mammary tumour cells while strongly expressed in adjacent normal or hyperplastic ductal structures. More importantly, the results from comparative study of 'pre-malignant' glands further showed that when animals were treated with 17beta-oestradiol, the mammary epithelial cells expressed high levels of Bcl-2. The results from rats treated with testosterone, either alone or in combination with oestrogen, give rise to high levels of Bax expression in 'pre-malignant' mammary glands. These observations indicate that in 'pre-malignant' mammary glands, treatment with testosterone, either alone or in combination with 17beta-oestradiol, may induce high apoptotic activities. However, in fully developed mammary tumours, the apoptotic activities apparently decrease in tumour cells. TUNEL assay provides further data to support this conclusion. Our study, thus, suggests that androgens may play a promoting role in mammary carcinogenesis by upregulation of Bax expression and induction of high apoptotic activities in 'pre-malignant' stage, which would provide a selective pressure favouring the expansion of the initiated cells.

Sex hormone-induced mammary carcinogenesis in female Noble rats: expression of TGF-beta1 and its receptors, TGF-alpha, and EGF-R in mammary carcinogenesis.

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal-epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-beta1) and its receptors (TGF-beta RI, TGF-beta RII), transforming growth factor alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-beta1 was strongly expressed in most mammary tumors, whereas TGF-beta RI and TGF-beta RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17beta-estradiol, the mammary gland epithelial cells expressed high levels of TGF-beta1. This over-expression of TGF-beta1 can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-beta1 in mammary glands. TGF-beta RI and TGF-beta RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17beta-estradiol treated and control mammary epithelial cells. Furthermore, TGF-beta RI and TGF-beta RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-beta1 and its receptors. On the other hand, TGF-alpha was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF-alpha was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17beta-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF-alpha induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF-alpha when the animals were treated with testosterone or 17beta-estradiol alone, respectively. These results suggest that both testosterone and 17beta-estradiol may be required for the over-expression of TGF-alpha in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-beta1, TGF-alpha, and their receptors by testosterone and 17beta-estradiol in mammary carcinogenesis.

Sex hormone-induced mammary carcinogenesis in female noble rats: the role of androgens.

Breast cancer is the most common cancer and the second most frequent cause of cancer death in women. Despite extensive research, the precise mechanisms of breast carcinogenesis remain unclear. We have shown that in female rats, treatment with a combination of oestrogen and testosterone can induce a high incidence of mammary cancer. The dosage of testosterone affects only the latency period of mammary cancer, not the final incidence. Based on these observations, we hypothesize that oestrogen and androgens may act in concert on the mammary gland to induce mammary carcinogenesis, with oestrogen serving as the predominant initiator whereas the androgen acts as a major promoter. In the present study, we report the changes in morphology of the mammary gland with special emphasis on the perialveolar or interlobular stroma after treatment with various sex hormone protocols. Our data showed that after treatment with testosterone, either alone or in combination with 17beta-oestradiol, there was overexpression of the androgen receptor in alveolar or ductal epithelial cells. Concurrent with strong expression of the androgen receptor in epithelium, there was also an increase in the amount of perialveolar and interlobular connective tissue, a decrease in surrounding adipose tissue and an increase in proliferation rate of fibroblast-like cells in the stroma. All these changes were blocked by simultaneous implantation of flutamide, indicating that androgens play a crucial role in the process despite the absence of androgen receptors in stromal cells. We further measured the mammary gland density (MGD), in order to determine the ratio of fatty to non-fatty tissue. The data showed that MGD values were significantly higher in animals treated with testosterone alone or in combination with 17beta-oestradiol than in those treated with 17beta-oestradiol alone or in controls. Furthermore, treatment with different doses of testosterone resulted in an increase in MGD in a dose-dependent manner. These findings highlight the effect of androgens on the stroma, probably through a paracrine action of epithelial cells. The stroma may, in turn, promote mammary carcinogenesis in a reciprocal fashion.

Induction of high incidence of mammary tumour in female Noble rats with a combination of 17beta-oestradiol and testosterone.

Breast cancer is the most common cancer and the second most frequent cause of cancer death in women. Despite extensive research, the precise mechanisms of breast carcinogenesis remain unclear. One of the reasons for this is due, at least in part, to a lack of a suitable animal model which can closely mimic the breast carcinogenesis in normal situations without using chemical carcinogens. We have developed an animal model of mammary gland carcinogenesis using a combination of oestradiol and testosterone, and succeeded in inducing a high percentage of female Noble rats to develop mammary cancer in a relatively short time (approximately 6 months). The results showed that androgens might work as a promoter to shorten the latency time of mammary gland carcinogenesis. Histopathological examination revealed that hyperplasia and dysplasia were first observed 2 months after treatment, in situ carcinoma after 3 months, and fully developed carcinoma of various forms including cribriform, papillary and camedo types were observed from 5 to 6 months after hormone implantation. Animals implanted with oestrogen or testosterone alone also developed mammary cancers, though with a lower overall incidence than the two hormones combined. They ranged from well differentiated to poorly differentiated forms with predominantly infiltrating ductal carcinoma. We have also observed a case of secondary cancer in the uterus. In addition to the high incidence of carcinoma, there was also a peculiar unexplained ipsilateral correlation between the site of hormonal implantation and the location of tumours, and the highest incidence of carcinogenesis was found to be in thoracic mammary gland. The study showed that both oestrogens and androgens are important in mammary cancer development. The animal model would prove to be a useful model for analysis of the mechanism(s) of hormonal carcinogenesis.

Profound effects of the weak environmental estrogen-like chemical bisphenol A on the growth of the mammary gland of Noble rats.

In the present study we have examined the effects of the environmental estrogenic chemical bisphenol A on proliferative activity, cell cycle kinetics and differentiation of the mammary gland of female Noble rats. Differentiation measured by the degree of lobular maturation revealed that the conversion of immature structures to mature structures was significantly increased in response to exposure to both low (0.1 mg/kg/day) and high (54 mg/kg/day) doses of bisphenol A compared to controls. The proliferative activity of epithelial cells was increased by 143% over controls by the exposure of animals to the low dose of bisphenol A, whereas a 220% increase over controls was observed for the high dose of bisphenol A. The labelling index and growth fraction were 19% and 27%, respectively, for a low dose of bisphenol A; and 27% and 45%, respectively, for a high dose of bisphenol A, compared to 18% and 31%, respectively, in controls. A significant increase in the conversion of mammary epithelial cells from G0, to G1, and S-phase cells by 1.8 and 4.5-fold, respectively, was observed in animals exposed to the high dose of bisphenol A compared to that of controls. Based on the previously reported estrogenic activity of an equivalent dose of bisphenol A to that of diethylstilbestrol (DES) (0.1 mg/kg/day), a calculated theoretical dose of the order of 10(6)-fold higher of bisphenol A will be required to produce the same biological effects as DES. A comparison of the proliferative activity of DES and that of an equivalent dose of bisphenol A observed in this study, however, revealed that its influence on proliferative activity in the epithelial cells of the mammary gland was profound. The weak estrogenic activity of bisphenol A does not explain its profound effect on cell proliferation observed in this study. Perturbation of the cell cycle is considered a risk factor for the development of cancer. Bisphenol-mediated perturbation of the cell cycle in epithelial cells may produce adverse effects in the mammary glands of Noble rats.

Exposure of environmental estrogenic compound nonlyphenol to noble rats alters cell-cycle kinetics in the mammary gland

p-Nonylphenol is an environmental estrogen-like chemical. Nonylphenol has previously been shown to mimic the actions of estrogen both in vivo and in vitro. In the present study, we have examined the effects of an environmental estrogenic chemical, nonlylphenol, on the proliferative activity, cell-cycle kinetics, and differentiation of the mammary gland of female Noble rats. The exposure of animals to two different doses of nonlylphenol (0.01 and 7.1 mg/24 h) significantly increased proliferation of the mammary epithelial cells. Both labeling index and growth fraction were increased by nonlylphenol treatment. Labeling index and growth fraction were 24 and 38%, respectively, for a low dose of nonylphenol; and 32 and 67%, respectively, for a high dose of nonlyphenol, compared to 18 and 35%, respectively, of controls. In addition, nonlylphenol exposure altered cell-cycle kinetics. Both low and high doses of nonylphenol significantly increased the conversion of mammary epithelial cells from G0 to G1 and S-phase cells by 2.2-and 2.6-fold, and by 11- and 4-fold, respectively, compared to that of controls. Differentiation measured by the degree of lobular maturation revealed that the conversion of immature structures to mature structures was significantly increased in response to nonylphenol exposure compared to that of control. Based on the previously reported estrogenic activity of an equivalent dose of nonylphenol to that of DES (0.01 mg/d), a calculated theoretical dose of the order of 10(5)- to 10(6)-fold higher of nonlylphenol will be required to produce the same biological effects as DES. However, the data of this study showed that exposure of 0.01 mg/d of nonlylphenol produced profound effect on cell proliferation in the mammary gland. The weak estrogenic activity of nonlylphenol does not explain its profound effect on cell proliferation observed in this study. Perturbation of cell cycle is considered as a risk factor for the development of cancer. Changes in proliferation and cell cycle have been shown to lead to genetic instability, ultimately resulting in cell transformation. Our results indicated an increase in labeling index and growth fraction, and a perturbation in cell-cycle kinetics from nonlylphenol exposure. Perturbation of cell cycle in response to nonlylphenol exposure may produce adverse effects in the mammary glands of the Noble rats.

Perturbation of cell cycle kinetics in the mammary gland by stilbene estrogen, diethylstilbestrol (DES).

In the present study we have examined the effect of stilbene estrogen on the proliferative activity, cell cycle kinetics and the differentiation of the mammary gland of female Noble rats. The exposure of animals to stilbene estrogen (diethylstilbestrol) or natural estrogen (estrone) (10 micrograms/24 h) significantly increased proliferation in mammary epithelial cells. Both labeling index and growth fraction were increased by DES or estrone treatment. In addition, both stilbene and natural estrogen exposure altered cell cycle kinetics. Stilbene estrogen exposure to animals increased the conversion of mammary epithelial cells from G0 to G1 and G1 to S-phase cells by 8.3- and 8.5-fold, respectively, compared to that of control. Differentiation measured by the degree of lobular maturation revealed that the conversion of immature structures to mature structures was significantly increased by 2.4-fold in response to DES or estrone exposure compared to that of control. Perturbation of cell cycle is considered as a risk factor for the development of cancer. Changes in proliferation and cell cycle have been shown to lead to genetic instability, ultimately resulting in cell transformation. Our results indicated an increase in labeling index and growth fraction and a perturbation in cell cycle kinetics from DES or estrone exposure. Perturbation of cell cycle in response to stilbene estrogen exposure may produce adverse effects in the mammary glands of the Noble rats.

Estrone-induced cell proliferation and differentiation in the mammary gland of the female Noble rat.

In this study we examined the influence of estrone on proliferation and differentiation in the mammary gland of female Noble rats. Estrone treatment increased proliferation in mammary epithelial cells at days 3 and 7, and peaked by day 11 of estrone exposure. In addition, estrone exposure altered cell cycle kinetics. Animals exposed to estrone for 11 days demonstrated a 9-fold increase in the cells in G1 phase and a 48-fold increase in the cells in S phase, compared to those of controls. Differentiation, measured by the degree of lobular maturation, was significantly increased in the treatment group by day 7 and continued to mature through day 21 post estrone implant. The appearance of the mammary glands in these animals approached the morphology normally found during pregnancy and lactation. In a subset of animals we examined the effects of a concurrent treatment with luteolin, a bioflavonoid which has been shown to inhibit estrone binding to type II estrogen receptors. Luteolin significantly reversed the effects of estrone treatment on both proliferation and differentiation in these animals. Changes in proliferation and cell cycle kinetics have been shown to lead to genetic instability, ultimately resulting in cell transformation. Our results indicated an increase in proliferative cells by > 2-fold and a perturbation in cell cycle kinetics from estrone exposure. These changes may play a role in the induction of estrone-induced mammary cancer in the Noble rat model. In addition, the anti-proliferative action of luteolin suggests that it may play a protective role in estrone-induced mammary carcinogenesis.