Betulin ameliorates 7,12-dimethylbenz(a)anthracene-induced rat mammary cancer by modulating MAPK and AhR/Nrf-2 signaling pathway.

The aim of the present study is to explore the preventive efficacy of betulin (BE) in 7,12-dimethylbenz(a)anthracene (DMBA)-administered mammary cancer by modulating Ahr/Nrf2 signaling in experimental models. The mammary cancer was stimulated by the addition of DMBA (25 mg/kg/b.Wt) mixed in 1 ml of vehicle solution (sunflower oil and saline 1:1) through subcutaneous injection. The DMBA-exposed mammary tumor models showed low bodyweight, elevated quantities of lipid peroxidation molecules (TBARS and LOOH), and low enzymatic (GPx, SOD, and CAT), and nonenzymatic (GSH, vitamin C, and vitamin E) antioxidant activities in plasma and mammary tissues. Moreover, histopathological studies confirmed that invasive ductal carcinoma was observed in DMBA-induced mammary tissue of the experimental model. Dietary oral supplementation of BE prevents the loss of bodyweight, overproduces lipid peroxidation, and restores the antioxidant activities in DMBA-exposed experimental animals. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial antioxidant protein that involves preventing numerous cancers. Therefore, Nrf2-associated signaling concern is a significant target for preventing mammary cancer. This study observed an increased expression of MAPKs, Keap1, ARNT, AhR, and CYP1A1, whereas decreased expression of HO-1 and Nrf2 in DMBA-induced cancer-bearing experimental animals. The oral supplementation of BE effectively modulates the expression of MAPKs, AhR/Nrf2-associated protein expressions in DMBA-exposed experimental animals. This current study concluded that BE is a strong antioxidant, which triggers the MAPKs-mediated oxidative stress and inhibits proliferative markers by restoring the activity of Nrf2 signaling.

Nanocrystalline solid dispersions (NSD) of hesperetin (HRN) for prevention of 7, 12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in Sprague-Dawley (SD) rats.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in females as per the global cancer project (GLOBOCAN 2018) estimates of breast cancer incidence and mortality produced by the International Agency for Research on Cancer (IARC). In 2018, there will be 2,088,849 new cases of breast cancer and 626,679 cases of deaths due to breast cancer in 5 regions (Americas, Africa, Europe, Asia and Oceania) at 20 sites of the world. Epidemiologic studies on diet and cancer have guided the search for some nutraceuticals acting as anti-cancer agents. Hesperetin (HRN), the aglycone of hesperidin, a glycoside found in citrus fruits, has been reported to have anti-cancer effects by apoptosis induction and inhibition of cell proliferation in cancer cells. However, poor solubility of HRN has limited its absorption while deviating from its therapeutic benefit. The present study aimed to develop nanocrystalline solid dispersions (NSD) of HRN and evaluating the oral bioavailability in rats. The study also evaluated the efficacy of NSDs against the carcinogenic activity of DMBA in female rats. NSDs were optimized using design of experiments (DoE) and multivariate analysis (MVA) tools. The optimized NSD formulation showed an average particle size (Zavg) of 558.2 ±â€¯68.1 nm and ~70% release in 30 min. The in vivo pharmacokinetic study also construed remarkable improvement (3.3 and 2.1-fold increase in Cmax and AUC0-∞) in rate and extent of absorption and 4-fold reduction in Tmax by the optimized NSD formulation. In vivo chemoprevention study construed superior efficacy of the NSD formulation by reducing the tumor burden, delaying the onset of tumors and reducing the tumor weight and volume in DMBA-induced breast cancer rats. In conclusion, we present a simple NSD formulation of HRN with enhanced bioavailability and superior chemopreventive efficacy.

Pueraria mirifica Exerts Estrogenic Effects in the Mammary Gland and Uterus and Promotes Mammary Carcinogenesis in Donryu Rats.

Pueraria mirifica (PM), a plant whose dried and powdered tuberous roots are now widely used in rejuvenating preparations to promote youthfulness in both men and women, may have major estrogenic influence. In this study, we investigated modifying effects of PM at various doses on mammary and endometrial carcinogenesis in female Donryu rats. Firstly, PM administered to ovariectomized animals at doses of 0.03%, 0.3%, and 3% in a phytoestrogen-low diet for 2 weeks caused significant increase in uterus weight. Secondly, a 4 week PM application to non-operated rats at a dose of 3% after 7,12-dimethylbenz[a]anthracene (DMBA) initiation resulted in significant elevation of cell proliferation in the mammary glands. In a third experiment, postpubertal administration of 0.3% (200 mg/kg body weight (b.w.)/day) PM to 5-week-old non-operated animals for 36 weeks following initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), respectively, resulted in significant increase of mammary adenocarcinoma incidence. A significant increase of endometrial atypical hyperplasia multiplicity was also observed. Furthermore, PM at doses of 0.3%, and more pronouncedly, at 1% induced dilatation, hemorrhage and inflammation of the uterine wall. In conclusion, postpubertal long-term PM administration to Donryu rats exerts estrogenic effects in the mammary gland and uterus, and at a dose of 200 mg/kg b.w./day was found to promote mammary carcinogenesis initiated by DMBA.

Modulatory effect of Pleurotus ostreatus on oxidant/antioxidant status in 7, 12-dimethylbenz (a) anthracene induced mammary carcinoma in experimental rats--A dose-response study.

BACKGROUND: Worldwide, breast cancer is the second most prevalent cancer among women and its incidence is increasing alarmingly. AIM: To determine a dose-response effect of Pleurotus ostreatus on oxidant/antioxidant status in 7,12-dimethylbenz. (a) antheracene induced. (DMBA) mammary carcinoma in experimental rats. MATERIALS AND METHODS: Cancer bearing female Sprague Dawley rats were orally treated with Pleurotus ostreatus ethanolic extract (POEet) (150, 300 and 600 mg/kg body weight) for 16 weeks. By means of high performance liquid chromatography (HPLC) analysis, ergosterol (48.82%) were identified and quantified in POEet. Body weight of experimental rats in each groups and the biochemical parameters of plasma, liver and mammary tissues were carried out. Histopathological analyses were also determined. STATISTICAL ANALYSIS USED: Results were analyzed using SPSS software package, version 16.0. The values were analyzed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT). RESULT: The result showed that depleted activities of enzymatic and non-enzymatic antioxidant level and significant elevated TBARS level were observed in DMBA group of plasma, mammary and liver tissues of experimental rats. The effects were dose.dependent and the above noted parameters were renovated to near normal after supplementation with different dose of POEet (150 mg, 300 mg and 600 mg/kg bwt). The data obtained from the study indicate that POEet at a dose of 600 mg/kg bwt possesses optimum anticancer effects against DMBA induced mammary carcinogenesis. CONCLUSION: Based on the scientific appraisal, we conclude that the POEet is having a potent antioxidant capacity; thereby it offers maximum protection against DMBA-induced mammary carcinogenesis.

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model.

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.

Ontogeny of the major xenobiotic-metabolizing enzymes expression and the dietary lipids modulatory effect in the rat dimethylbenz(a)anthracene-induced breast cancer model.

Breast cancer is the most common malignancy in women worldwide. Environmental factors such as xenobiotic exposure and lifestyle and nutrition play a key role in its etiology. This study was designed to evaluate the age-related changes in the expression of major xenobiotic-metabolizing enzymes (XMEs) in the rat liver and the mammary gland in the dimethylbenz(a)anthracene-induced breast cancer model. The influence of dietary lipids on the ontogeny of XMEs was also evaluated. mRNA and protein levels of phase I (CYP1A1, CYP1A2, and CYP1B1) and phase II (NAD(P)H:quinone acceptor oxidoreductase 1 and GSTP1) enzymes were analyzed, as well as their regulation by AhR and Nrf2, respectively. Results showed differences in the phase I enzymes expression, whereas little changes were obtained in phase II. High corn oil and olive oil diets differentially influenced the expression of age-related changes, suggesting that the different susceptibility to xenobiotic exposure depending upon the age may be modulated by dietary factors.

Effects of Ginkgo biloba on chemically-induced mammary tumors in rats receiving tamoxifen.

BACKGROUND: Ginkgo biloba extract (GbE) is used extensively by breast cancer patients undergoing treatment with Tamoxifen (TAM). Thus, the present study investigated the effects of GbE in female Sprague-Dawley (SD) rats bearing chemically-induced mammary tumors and receiving TAM. METHODS: Animals bearing mammary tumors (≥1 cm in diameter) were divided into four groups: TAM [10 mg/kg, intragastrically (i.g.)], TAM plus GbE [50 and 100 mg/kg, intraperitoneally (i.p.)] or an untreated control group. After 4 weeks, the therapeutic efficacy of the different treatments was evaluated by measuring the tumor volume (cm(3)) and the proportions of each tumor that were alive, necrotic or degenerative (mm(2)). In addition, labeling indexes (LI%) were calculated for cell proliferation (PCNA LI%) and apoptosis (cleaved caspase-3 LI%), expression of estrogen receptor-alpha (ER-α) and p63 biomarkers. RESULTS: Overall, the tumor volume and the PCNA LI% within live tumor areas were reduced by 83% and 99%, respectively, in all TAM-treated groups when compared to the untreated control group. GbE treatment (100 mg/kg) reduced the proportions of live (24.8%) and necrotic areas (2.9%) (p = 0.046 and p = 0.038, respectively) and significantly increased the proportion of degenerative areas (72.9%) (p = 0.004) in mammary tumors when compared to the group treated only with TAM. The expression of ER-α, p63 and cleaved caspase-3 in live tumor tissues was not modified by GbE treatment. CONCLUSIONS: Co-treatment with 100 mg/kg GbE presented a slightly beneficial effect on the therapeutic efficacy of TAM in female SD rats bearing mammary tumors.

Diosgenin, a steroidal saponin, exhibits anticancer activity by attenuating lipid peroxidation via enhancing antioxidant defense system during NMU-induced breast carcinoma.

Diosgenin, a natural steroidal saponin, has been reported to be found predominantly in fenugreek and has diverse biological properties. N-Methyl-N-nitrosourea (NMU) is a mammary gland-specific carcinogen that closely mimics human breast cancer in many aspects. The aim of this study was to investigate the anticarcinogenic property of diosgenin with reference to lipid peroxidation, status of antioxidants, and activities of marker enzymes against NMU-induced experimental mammary carcinogenesis. Breast cancer was induced in female Sprague Dawley rats by an intraperitoneal administration of a single dose of NMU (a concentration of 50 mg/kg body weight) diluted in 0.9% saline, and the rats were treated with oral diosgenin, 20 mg/kg body weight, for 45 days. The results were interesting, and the diosgenin treatment remarkably downregulated the peroxidation reaction and marker enzymes and extraordinarily enhanced the indigenous antioxidant defense system. The factor for this remarkable restoration might be due to the effect of the intervention strategy on the downregulation of the peroxidation reaction through the strong antioxidant nature, which ultimately reflected in the downregulation of marker enzyme activities. The histopathological study of breast and liver tissues inevitably confirms the biochemical changes. Thus, it can be concluded that diosgenin exhibits anticarcinogenic activity via reducing peroxidation reaction and marker enzymes through enhancing the intrinsic antioxidant defense system.

In vitro and in vivo antimammary tumor activities and mechanisms of the apple total triterpenoids.

Consumption of apples has been linked to the prevention of various chronic diseases, including tumors and cardiovascular diseases. The apple total triterpenoid content (ATT) was extracted and concentrated from apple peels. The in vitro and in vivo antitumor activities, related antitumor mechanisms, were investigated. In vitro, ATT showed potent antiproliferative activities against human breast cancer (MCF-7, MDA-MB-231), human colon cancer (Caco-2), and human liver cancer (HepG2) cell lines. In vivo antitumor experiments showed that ATT could substantially reduce the occurrence and growth of mammary tumor with a good dose-dependent manner in a rat model. During the apoptosis in MDA-MB-231 cells induced by ATT, the caspase-independent pathway was involved in the process of apoptosis, and the mitochondrial transmembrane potential was markedly reduced. Also the PI3K/Akt/NF-κB pathway was activated. These results indicated that ATT-induced apoptosis of MDA-MB-231 cells may involve a mitochondrial-related pathway.

Morin augments anticarcinogenic and antiproliferative efficacy against 7,12-dimethylbenz(a)-anthracene induced experimental mammary carcinogenesis.

In general, oxidative stress resulting from an imbalance between prooxidant and antioxidant systems plays an important role in the pathogenesis of cancer. Morin (3,5,7,2',4'-pentahydroxyflavone), a member of the flavanol group, has been shown to possess chemopreventive potential against hepatocellular and colon cancer in experimental animals. Given the demonstrated importance of morin, aim of the present study was to evaluate the effect of morin on antiproliferative and anticarcinogenic effect against DMBA-induced experimental mammary carcinogenesis. Oral administration of 7,12-dimethylbenz(a)-anthracene (25 mg/kg body weight) to rats resulted in significant reduction of body weight, enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase), and nonenzymic antioxidants (reduced glutathione, vitamin C, and vitamin E). The levels of lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides) and tumor markers such as CA 15-3, AFP and CEA in serum were increased significantly in cancer-induced animals as compared to control rats. Oral supplementation of morin at a dose of 50 mg/kg body weight significantly improved the body weight, enzymic, and nonenzymic antioxidants and considerably decreased the lipid peroxidation marker and tumor markers levels. Histological observations also correlated with the biochemical parameters. Tumor bearing animals showed marked increase in proliferating cell nuclear antigen-positive cells and also the number of AgNOR/nuclei compared with control rats while this expression levels were significantly reduced upon morin treatment. Thus, this study reveals the possible beneficial effect of morin as chemopreventive agent against the oxidative stress induced during mammary carcinogenesis.

Synergistic effect of lycopene and tocopherol against oxidative stress and mammary tumorigenesis induced by 7,12-dimethyl[a]benzanthracene in female rats.

Breast cancer is one of the most serious problems in oncology. We investigated the antitumor potential of lycopene (Lyco) alone or combined with tocopherol (Lyco + Toco) for 90 days against a single oral dose of (50 mg/kg body weight) 7,12-dimethyl[a]benzanthracene (DMBA)-induced oxidative stress and mammary carcinogenesis in female rats. The treatment protocol started from the day immediately after DMBA administration. Results obtained indicated that there was a significant elevation in the levels of malondialdehyde (MDA) and nitric oxide (NO) in serum and breast tissues of DMBA-injected rats. The combined treatment (Lyco + Toco) group showed a potential reduction of these parameters more than Lyco alone group. The activities of superoxide dismutase, catalase, and glutathione peroxidase were found to be significantly higher when compared to rats treated with Lyco alone. In DMBA group, a positive significant correlation between NO and MDA (r = 0.92) was observed. Histopathological examination revealed the formation of tumor and angiogenesis in DMBA-induced rats and these abnormal changes were ameliorated by combined treatment with Lyco + Toco. In conclusion, these results suggested that supplementation of diet with Lyco and Toco provided antioxidant defense, with strong chemopreventive activity against DMBA-induced mammary tumors.

4-Vinylcyclohexene diepoxide (VCD) inhibits mammary epithelial differentiation and induces fibroadenoma formation in female Sprague Dawley rats.

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that targets ovarian follicles and accelerates ovarian failure in rodents, was used to test the effect of early-onset reproductive senescence on mammary fibroadenoma formation. One-month female Sprague Dawley rats were dosed with VCD (80 mg/kg or 160 mg/kg) and monitored for 22 months for persistent estrus and tumor development. Only high-dose VCD treatment accelerated the onset of persistent estrus relative to controls. However, both doses of VCD accelerated mammary tumor onset by 5 months, increasing incidence to 84% (vs. 38% in controls). Tumor development was independent of time in persistent estrus, 17 ß-estradiol, androstenedione and prolactin. Delay in VCD administration until after completion of mammary epithelial differentiation (3 months) did not alter tumor formation despite acceleration of ovarian senescence. VCD administration to 1-month rats acutely decreased mammary alveolar bud number and expression of ß-casein, suggesting that VCD's tumorigenic effect requires exposure during mammary epithelial differentiation.

Anti-/pro-apoptotic effects of hesperetin against 7,12-dimetylbenz(a)anthracene-induced alteration in animals.

This study investigated the anticancer effects of hesperetin on 7,12-dimethylbenz(a)anthracene (DMBA)-treated animals and explored its anticancer mechanism. The experiment consisted of two parts. First, Sprague-Dawley rats were given hesperetin daily at a dose of 50 mg/kg for 8 weeks after a single dose of DMBA (100 mg/kg). As controls, rats were divided into vehicle alone and DMBA alone groups. Secondly, ICR mice were given hesperetin daily at a dose of 10 and 50 mg/kg BW/day for 7 weeks before a single dose of DMBA (34 mg/kg/week). In rats with DMBA-induced mammary gland tumors, hesperetin pretreatment significantly reduced the tumor burden and PCNA overexpression. The administration of hesperetin significantly inhibited mammary gland carcinoma from developing by restoring the decreased Bcl-2 and increased Bax expression. By contrast, in the livers of mice treated with DMBA, obvious DNA fragmentation was observed. Moreover, apoptosis-related gene expression in the livers of the mice differed from that in mammary gland carcinomas in rats. These changes were restored in mice treated with hesperetin, indicating the inhibition of apoptosis. Based on these results, hesperetin may act not only as a proapoptotic agent, but also as an antiapoptotic agent, depending on the circumstance.

Fresh apples suppress mammary carcinogenesis and proliferative activity and induce apoptosis in mammary tumors of the Sprague-Dawley rat.

Whole apple extracts possess potent antioxidant activity and antiproliferative activity against cancer cells in vitro. The objectives of this study were to determine the anticancer activity of apple extracts in a rat mammary cancer model induced by 7,12-dimethylbenz(a)anthracene (DMBA) in vivo and to determine if apple extracts inhibited cell proliferation and affected apoptosis in mammary cancer tissues in vivo. Rats were given the whole apple extracts (0, 3.3, 10.0, or 20.0 g/kg of body weight) by gavage starting 2 weeks prior to DMBA administration and continuing for 24 weeks. Rats treated with DMBA (positive control) developed mammary tumors with 71.4% tumor incidence during the 24-week study. No tumors were detected in the negative control group untreated with DMBA. A dose-dependent inhibition of mammary carcinogenesis by apple extracts was observed (P < 0.01). Tumor multiplicity decreased with increasing apple extracts. Histopathological evaluations of tumors were performed. The proportions of adenocarcinoma masses decreased with increasing apple extracts. The expression of proliferating cell nuclear antigen (PCNA), cyclin D1, and Bcl-2 decreased, and Bax expression and apoptosis increased with increasing apple extracts. These results demonstrate the potent capacity of fresh apples to suppress DMBA-initiated mammary cancers in rats.

Multigenerational reproductive study of genistein (Cas No. 446-72-0) in Sprague-Dawley rats (feed study).

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. Consumption of soy and genistein has been associated with a variety of beneficial effects in animals and humans, but concerns have also been raised concerning potential adverse effects of genistein, particularly with regard to reproductive toxicity and the induction or potentiation of carcinogenesis, due primarily to its weak estrogenic activity. Because of these concerns, genistein was selected as one of the compounds to be examined in a protocol utilizing Sprague-Dawley rats to evaluate the effects of multigenerational and long-term exposures to doses of estrogenic agents that produce subtle reproductive tract lesions in developmentally exposed Sprague-Dawley rat pups. Results from the multigenerational reproductive toxicology feed study are reported in this report, and results of the 2-year feed study are reported separately (NTP, 2008a). Data from a preliminary reproductive dose range-finding feed study (NTP, 2007) that utilized exposure concentrations of up to 1,250 ppm genistein were used to select dietary exposure concentrations of 0, 5, 100, and 500 ppm for the current study. These dietary doses resulted in ingested genistein doses of approximately 0, 0.3, 7, or 35 mg genistein/kg body weight per day for males and 0, 0.5, 10, or 51 mg/kg per day for females during the time that the rats were directly consuming dosed feed. The current study was a multigenerational study (F(0) through F(4), with F(5) litters terminated at weaning) focused on reproductive endpoints. Animals were continuously exposed to genistein from the time that the F(0) generation was 6 weeks old through weaning of the F(3) generation, and animals of the F(0) through F(4) generations were sacrificed and necropsied on postnatal day 140 (PND 140). Dosed feed was removed from the F(3) pups at the time of weaning, and this generation and subsequent generations were maintained on control feed for the remainder of the study. For this study, 140 animals of each sex were obtained from the NCTR CD (Sprague-Dawley) rat colony at weaning and placed on a soy- and alfalfa-free diet that was used throughout the study in an attempt to maintain consistently low background exposure to phytoestrogens. Thirty-five animals per sex were assigned to exposure groups by a weight-ranked randomization procedure prior to the start of dietary exposure of the parental (F(0)) generation at 6 weeks of age. At the time of mating, males were paired with females from the same exposure group, and they were housed together until evidence of successful mating was detected or for a maximum of 14 days. Litters were randomly standardized to four males and four females on PND 2, and 25 litters per exposure group and their associated sires and dams were randomly selected to continue on study to produce the next generation and then necropsied at termination at 20 weeks of age (PND 140). Similar procedures were used to produce each generation. Results of the current study are summarized below. In the postweaning period, exposure to 500 ppm genistein reduced body weights predominantly in females of generations in which rats were ingesting the compound throughout adulthood (F(0) through F(2)). In the unexposed F(4) generation, female body weight was also depressed, although to a lesser extent than in the earlier generations. In the F(1) generation, postweaning body weights were reduced in all 100 and 500 ppm groups, with a more pronounced effect in the females. While pup birth weights were not significantly affected by genistein in the F(1) through F(4) generations (with the exception of 100 ppm males in the F(1) generation), both sexes showed depressed body weight gains during the preweaning period in the 500 ppm groups in all of these generations. Male pup preweaning body weight gains were also depressed in the 5 and 100 ppm groups in the F(1) generation. In the unexposed F(5) generation, pup birth weights in all exposed groups of both sexes were significantly lower than those in the controls, although it seems likely that this is a chance observation rather than a carryover effect from exposures in earlier generations. Measures of fertility were not adversely affected by genistein except for litter size. Litter size of the 500 ppm group in the F(2) generation was significantly smaller than that in the corresponding control group. The litter sizes in the F(1), F(2), and F(3) generations showed negative exposure concentration trends. Male and female 500 ppm pups in the F(1) generation had slightly reduced anogenital distances (AGDs) relative to controls when covaried by body weight. Female pups also had reduced AGDs in the F(2) (500 ppm) and F(3) (100 ppm) generations, although the statistical significance was dependent on the analysis method applied. Females exposed to 500 ppm showed an accelerated time of vaginal opening (approximately 3 days) in the F(1) and F(2) generations, while the 5 ppm group showed an earlier time of vaginal opening (1.3 days) in the F(3) generation. Body weight at vaginal opening was lower in 500 ppm females of the F(1) through F(3) generations and in the 5 ppm females of the F(1) generation. When examined shortly after vaginal opening, estrous cycles of 500 ppm females in the F(1) and F(2) generations were significantly longer (approximately 3 days and 1 day, respectively) than those of their respective control groups. Other estrous cycle disturbances (with the exception of decreased time in diestrus for 100 ppm females in the F(4) generation) were confined to the 500 ppm group of the F(1) generation and included reduced time in proestrus and an increase in the number and percentage of aberrant cycles. When the estrous cycles of older animals were examined prior to termination, the sole significant effects were a decreased time in estrus and increased time in diestrus in 5 ppm females of the F(2) generation and an increased number of abnormal cycles in 500 ppm females of the F(3) generation. No effects of genistein on male sexual development were noted with the exception of an increased time to testicular descent in 500 ppm males of the F(3) generation. Significant organ weight effects in both sexes were largely confined to single exposed groups in single generations; no clear patterns indicating toxicity to reproductive or nonreproductive organs were observed. Exposure-related microscopic lesions were confined to males, with the mammary gland and kidney affected. Incidences of mammary gland alveolar/ductal hyperplasia were significantly increased in 500 ppm males in the F(0) through F(2) generations and in 100 ppm males in the F(1) and F(2) generations. In the F(3) generation, a significant positive linear exposure concentration trend in the incidences of mammary gland hyperplasia occurred, but no exposed group differed significantly from the controls in pairwise comparisons. The more pronounced effect of genistein on the incidences of male mammary gland hyperplasia in the continuously exposed F(1) and F(2) generations as compared to the late adolescent and adult exposures of the F(0) generation and the preweaning-only exposure of the F(3) generation indicates that both developmental and adult exposures contribute to the maintenance of this effect into adulthood. Statistically significant effects of genistein on the incidences of generally minimal to mild kidney lesions in males were confined to the continuously exposed F(1) and F(2) generations. Incidences of renal tubule mineralization were significantly increased in 100 and 500 ppm males in the F(1) and F(2) generations, and incidences of inflammation and renal tubule regeneration were significantly increased in 500 ppm males in the F(1) generation. In addition to the results reported above for animals from the main study, ancillary studies were conducted with pups derived from the current study or from animals treated under similar conditions. These results have been reported elsewhere (Appendix P) and are not presented in detail in this report. Of particular importance are the data on blood and tissue genistein concentrations obtained from adult animals in the F(1) generation (Chang et al., 2000), from dams and fetuses (Doerge et al., 2001), and from dams and nursing pups (Doerge et al., 2006). These data provide measures of the internal dose resulting from the dietary exposure concentrations used in the current study and indicate that while fetal and adult exposures to genistein were at concentrations relevant to the full range of human exposures, only very low exposures were achieved during the early neonatal period when the pups were receiving exposures exclusively from the milk. The minimal exposure to genistein during this critical developmental period must be considered in the interpretation of the data derived from the current study. In summary, although genistein did show adverse effects with dietary exposures of 100 or 500 ppm, there were no clear adverse effects on the reproductive or developmental parameters measured at genistein concentrations ranging from less than 1 ppm (control diet) to 100 ppm, a range of doses producing serum concentrations achievable from the phytoestrogen content of human diets. There were few clear, overtly toxic effects that carried over across directly exposed generations or appeared to be imprinted to carry over into unexposed descendents under the conditions of exposure in this study. (ABSTRACT TRUNCATED).

Toxicology and carcinogenesis studies of genistein (Cas No. 446-72-0) in Sprague-Dawley rats (feed study).

UNLABELLED: Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. Consumption of soy and genistein has been associated with a variety of beneficial effects in animals and humans, but concerns have also been raised regarding potential adverse effects of genistein, particularly with regard to reproductive toxicity and the induction or potentiation of carcinogenesis, due primarily to its weak estrogenic activity. Because of these concerns, genistein was selected as one of the compounds to be examined using a protocol designed to evaluate the effects of multigenerational and long-term exposures to doses of estrogenic agents that produce subtle reproductive tract lesions in developmentally exposed Sprague-Dawley rat pups. Results from the 2-year study are reported here, and results from the multigenerational reproductive toxicology feed study are reported separately (NTP, 2008a). Data from a preliminary dose range-finding feed study (NTP, 2007) that utilized exposure concentrations up to 1,250 ppm genistein were used to select dietary exposure concentrations of 0, 5, 100, and 500 ppm for the current study. The multigenerational reproductive toxicology study examined F(0) through F(4) generations with F(5) litters terminated at weaning and focused on reproductive endpoints (NTP, 2008a). Animals were exposed from the time that the F(0) generation was 6 weeks old through weaning of the F(3) generation, and animals of the F(0) through F(4) generations were necropsied at 20 weeks of age. The current study was a 2-year dietary study utilizing three exposure arms: continuous exposure from conception through 2 years (designated F(1) continuous, or F(1)C), exposure from conception through 20 weeks followed by control diet to 2 years [designated F(1) truncated at postnatal day (PND) 140, or F(1)T140], and exposure from conception through weaning followed by control diet to 2 years (designated F(3) truncated at PND 21, or F(3)T21). The "F(3)" designation for the F(3)T21 arm indicates that these animals were siblings of the F(3) animals from the multigenerational reproductive toxicology study (NTP, 2008a). The F(1)C and F(1)T140 animals were also siblings but were derived from a separate breeding that was identical to the procedure used to produce the F(1) generation of the multigenerational reproductive toxicology study. The animals in this study were exposed to genistein during various phases of their lives from conception until termination at 2 years, and the ingested doses varied over the course of the study. During pregnancy, the ingested doses of the dams were approximately 0, 0.5, 9, or 45 mg/kg body weight per day. During lactation, the dams' ingested doses were 0, 0.7, 15, or 75 mg/kg per day. Supplementary studies, which are described in the multigenerational reproductive toxicology study, indicated minimal transfer of genistein to pups via the dams' milk. The mean directly ingested genistein doses during the period prior to PND 140 were approximately 0.4, 8, or 44 mg/kg per day for females and 0.4, 7, or 37 mg/kg per day for males. For the period between PND 140 and the end of the study, mean ingested doses were approximately 0.3, 5, or 29 mg/kg per day for females and 0.2, 4, or 20 mg/kg per day for males. For the current study, 50 animals per sex were initially assigned to each exposure group in each arm of the study. In control groups, histopathology data from one to four additional animals that had been assigned as sentinels but that became moribund or died early were also included in the analysis and presentation. Survival was similar in all control and exposed groups and ranged from 62% to 86% for males and 43% to 64% for females. Mean body weights of 500 ppm F(1)C females were less than those of the controls throughout the study. Mean body weights of 500 ppm F(1)T140 rats were less than those of the controls throughout the study. In females of all study arms (F(1)C, F(1)T140, and F(3)T21) an early onset of aberrant estrous cycles, suggesting early reproductive senescence, was observed in the 500 ppm groups. In the F(3)T21 arm, there were also significant effects on the onset of aberrant estrous cycles in the 5 and 100 ppm groups. Pituitary gland weights were significantly increased in females in the 500 ppm groups of the F(1)C and F(1)T140 study arms and in the 100 ppm group of the F(3)T21 arm. In F(1)C females, there was a significant positive trend in the incidences of mammary gland adenoma or adenocarcinoma (combined) regardless of whether an unmodified or natural log-transformed dose scale was used in the analysis, and the incidence in the 500 ppm group was significantly greater than that in the control group. A significant negative trend occurred in the incidences of benign mammary gland fibroadenoma in F(1)C females, and the incidence in the 500 ppm group was significantly less than that in the control group. In 5 and 100 ppm F(1)T140 females, the combined incidences of adenoma and adenocarcinoma were less than those in the control or 500 ppm groups, although these were not statistically significant differences. When the natural log-transformed dose scale was used, a marginally significant positive trend occurred in the incidences of adenoma or adenocarcinoma (combined) in F(3)T21 females. There were positive trends in the incidences of adenoma or carcinoma (combined) in the pars distalis of the pituitary gland of females in the F(1)C and F(1)T140 arms, and the incidence in the 500 ppm group was significantly greater than that in the controls in the F(1)C study arm. In F(1)C males, a significant positive trend (unmodified dose scale only) occurred in the incidences of combined adenoma or carcinoma of the pancreatic islets. While the incidence in the 500 ppm group was elevated relative to that in the control group (6/49 versus 1/49), this was not statistically significant. The fact that transitional lesions (i.e., hyperplasia) were not observed combined with variable control rates in males of this substrain of rats led to the conclusion that this lesion was not likely to be related to genistein treatment. CONCLUSIONS: Under the conditions of this 2-year feed study with continuous exposure to the test compound from conception through termination (F(1)C), there was no evidence of carcinogenic activity of genistein in male Sprague-Dawley rats exposed to 5, 100, or 500 ppm. There was some evidence of carcinogenic activity of genistein in female Sprague-Dawley rats based on increased incidences of mammary gland adenoma or adenocarcinoma (combined) and pituitary gland neoplasms. The incidence of benign mammary gland fibroadenoma in female rats was significantly decreased in the 500 ppm group. Under the conditions of this 2-year feed study with exposure to the test compound from conception through 20 weeks followed by control feed until termination (F(1)T140), there was no evidence of carcinogenic activity of genistein in male Sprague-Dawley rats exposed to 5, 100, or 500 ppm. There was equivocal evidence of carcinogenic activity of genistein in female Sprague-Dawley rats based on marginally increased incidences of pituitary gland neoplasms. Under the conditions of this 2-year feed study where offspring of three prior generations of animals exposed to the test compound were exposed from conception through weaning (PND 21) followed by control feed until termination (F(3)T21), there was no evidence of carcinogenic activity of genistein in male Sprague-Dawley rats exposed to 5, 100, or 500 ppm. There was equivocal evidence of carcinogenic activity of genistein in female Sprague-Dawley rats based on increased incidences of mammary gland adenoma or adenocarcinoma (combined). Exposure to genistein was also shown to accelerate the onset of aberrant estrous cycles in female Sprague-Dawley rats whether exposures were continuous or truncated at PND 140 or at weaning. The effects of genistein on estrous cycling and the incidences of common hormonally related spontaneous neoplasms of female Sprague-Dawley rats are consistent with an estrogenic mechanism of toxicity.

Chemopreventive effect of lycopene alone or with melatonin against the genesis of oxidative stress and mammary tumors induced by 7,12 dimethyl(a)benzanthracene in sprague dawely female rats.

Breast cancer is the principle cause of death among women worldwide. In this study, we investigated the anti-tumor potential of lycopene (Lyco) alone or combined with melatonin (Lyco + Mel) for 120 days against a single oral dose of (50 mg/kg B.W.) 7,12-dimethylbenz(a)anthracene (DMBA)-induced oxidative stress and mammary carcinogenesis in female rats. The treatment protocol started from the day immediately after DMBA administration. Results obtained indicated that there was an elevation in the levels of malondialdhyde and nitric oxide in serum and breast tissues of DMBA injected rats. The combined treatment (Lyco + Mel) group showed a potential reduction of these parameters more than lyco individually. The activities of SOD, CAT, and GPx were found to be significantly high than lyco alone treated rats. In DMBA group a negative significant correlation between weight and serum nitric oxide (r = -0.59), and a positive significant correlation between NO and MDA (r = 0.81) was observed. Histopathological examination revealed the formation of tumor and angiogenesis in DMBA-induced rats and these abnormal changes were ameliorated by combined treatment with Lyco + Mel. In conclusion, these results suggested that supplementation of diet with lycopene with melatonin provided antioxidant defense with strong chemo preventive activity against DMBA-induced mammary tumors.

High corn oil and high extra virgin olive oil diets have different effects on the expression of differentiation-related genes in experimental mammary tumors.

Dietary lipids can modify the clinical behavior and morphological features of experimental breast tumors. We previously demonstrated that a high corn oil diet has a tumor-enhancing effect in 7,12-dimethylbenz(alpha)anthracene (DMBA)-induced rat mammary adenocarcinomas, whereas a high olive oil diet acts as a negative modulator of carcinogenesis. In this study, we investigated whether these high fat diets modulate the expression of genes related to differentiation. Rats were induced with DMBA and fed a low fat diet, a high corn oil diet, a high olive oil diet, or both high fat diets. The expression levels of the mammary differentiation biomarkers alpha-casein, beta-casein and transferrin and of beta-actin and its transporter zipcode binding protein 1 (ZBP1) were analyzed by Northern and/or Western blot in the mammary adenocarcinomas. The high fat diets did not induce changes in the expression of caseins, while transferrin expression was increased as a result of the high olive oil diet. beta-actin mRNA levels were higher in the high fat diet groups, though no changes in the protein levels were observed. The expression of ZBP1, a protein reported as having a role in carcinogenesis, was significantly increased by the high corn oil diet. These results suggest that in this model caseins are not good biomarkers of the changes in tumor morphological differentiation conferred by the high fat diets. The modulation of transferrin and ZBP1 expression by the high olive oil and the high corn oil diets could be one of the mechanisms by which such diets have a different influence on mammary carcinogenesis.

Glutamine affects glutathione recycling enzymes in a DMBA-induced breast cancer model.

Malignancy depletes host glutathione (GSH) levels to increase treatment-related toxicity and increases itself to resist the treatments. Our previous studies have shown that dietary glutamine (GLN) prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors through enhancing gut GSH release and reducing tumor GSH level. In addition, GSH synthesis, metabolism, and recycling are accomplished in gamma-glutamyl cycle. We hypothesized that the GLN prevention might be through a differential regulation of the gamma-glutamyl cycle enzymes. Female Sprague-Dawley rats were randomized into DMBA-tumor bearing, DMBA-treated, and control groups subdivided into GLN and water groups. GLN supplementation was given at 1 g/kg/day by gastric gavage. The activities and messenger RNA levels of gamma-glutamyl transpeptidase (GTP), gamma-glutamylcysteine synthetase (GCS), 5-oxo-L-prolinase (OPase), gamma-glutamyl transferase (GTF), and glutaminase (GLNase) were determined in gut mucosa and breast tumor using specific enzyme assays and semiquantitative reverse transcription polymerase chain reaction. GLN upregulated gut GTP, GCS, OPase, and GLNase in DMBA-tumor bearing, DMBA-treated, and/or control rats; however, it downregulated these enzymes in the tumor. The paradoxical effect of GLN on key GSH recycling enzymes in the gut versus tumor suggests that dietary supplemental GLN could be used in the clinical practice to increase the therapeutic index of cancer treatments by protecting normal tissues from, and sensitizing tumor cells to, chemotherapy and radiation-related injury.

Diphenyl diselenide supplementation delays the development of N-nitroso-N-methylurea-induced mammary tumors.

The effect of dietary diphenyl diselenide (1 ppm) on N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis was examined in female Wistar rats. Beginning at 5 weeks of age, the animals were fed with either control or diphenyl-diselenide-supplied diets until the end of the study (210 days). At 50 days of age, mammary tumor was induced by the administration of three doses of NMU (50 mg/kg body wt, intraperitoneally) once a week for 3 weeks. In experimental trials, latency to tumor onset was extended in rats fed with diet supplemented with diphenyl diselenide (P < 0.05). The incidence and frequency of tumors were significantly small in animals supplemented with diphenyl diselenide. However, the multiplicity of tumors was not altered by dietary diphenyl diselenide. Diphenyl diselenide supplementation also restored superoxide dismutase (SOD) activity and vitamin C levels altered in the NMU group (P < 0.05). Our results suggest that diphenyl diselenide can be considered a chemopreventive agent, even when supplemented at a relatively low concentration.