MNU-induced Mammary Carcinogenesis Research Articles

Prenatal exposure to a mixture of different phthalates increases the risk of mammary carcinogenesis in F1 female offspring

Phthalates metabolites have been detected in the urine of pregnant and breastfeeding women. Thus, this study evaluated the adverse effects of maternal exposure to a mixture of six phthalates (Pth mix) on the mammary gland development and carcinogenesis in F1 female offspring. Pregnant female Sprague-Dawley rats were exposed daily to vehicle or Pth mix (35.22% diethyl-phthalate, 21.03% di-(2-ethylhexyl)-phthalate, 14.91% dibutyl-phthalate, 15.10% diisononyl-phthalate, 8.61% diisobutyl-phthalate, and 5.13% benzylbutyl-phthalate) by gavage at 20 μg/kg, 200 μg/kg or 200 mg/kg during gestational day 10 (GD 10) to postnatal day 21 (PND 21). After weaning (PND 22), some female offspring were euthanized for mammary gland analyses while other females received a single dose of N-methyl-N-nitrosourea (MNU, 50 mg/kg) or vehicle and then tumor incidence and multiplicity were recorded until PND 180. Maternal Pth mix exposure increased the number of Ki-67 and progesterone receptor-positive epithelial cells in the mammary gland from Pth mix 200 at μg/kg and 200 mg/kg groups. In addition, tumor incidence and mean number were higher only in Pth mix at 200 mg/kg when compared to the vehicle-treated group, and percentage of tumor-free animals was lower in Pth mix at 200 μg/kg and 200 mg/kg groups. The findings indicate that perinatal Pth mixture exposure increased susceptibility to MNU-induced mammary carcinogenesis in adult F1 female offspring.

Chemopreventive Effects of Propolis in the MNU-Induced Rat Mammary Tumor Model.

Currently, one of the central problems in cancer management is the relapse of disease following conventional treatments, yet few therapeutic agents targeting resistance and tolerance exist. Propolis is known as a healing agent since ancient times. Therefore, over time, its curative properties have kept the interest of scientists, thus leading permanently to investigations of its other possible undiscovered effects. In this context, current experiments were performed to establish the chemopreventive potential of propolis extract (PE) (1.05 mg/kg BW/day) in N-methyl-N-nitrosourea- (MNU-) induced rat mammary tumors. MNU-inoculated/PE-treated rats had tumors of different physical attributes compared with control rats MNU-inoculated. The number of developed tumors (mean 49% versus 100%), incidence (mean 49% versus 100%), multiplicity (1.8 versus 3.7 (p < 0.001)), tumor volume (mean 10 cm3 versus 16 cm3 (p < 0.001)), and weight of the tumor mass (mean 7.42 g versus 9.00 g (p < 0.05)) were noted. The numbers of grade I tumors recorded for MNU-inoculated rats were 24 (Group 1) and 7 (Group 2) for MNU-induced/PE-treated rats. In the serum of rats MNU-inoculated/PE-treated were found higher levels of antioxidative enzymes (SOD, CAT, and GPx) than in MNU-induced. Taken together, these data indicate that propolis could be a chemopreventive agent against MNU-induced mammary carcinogenesis.

Anti-Estrogenic and Anti-Cell Proliferative Effect of Allyl Isothiocyanate in Chemoprevention of Chemically Induced Mammary Carcinogenesis in Rats.

The anti-estrogenic and anti-cell proliferative effect of allyl isothiocyanate (AITC) was carried out by analyzing the status of sex hormones and its receptors and cell proliferative markers in chemically induced mammary carcinogenesis in rats. Mammary tumor was induced by a single dose of DMBA (25mg/rat) and MNU (50mg/kg bw) injected subcutaneously near mammary gland. RT-PCR, western blotting and immunohistochemical analysis of mammary tissues show an upregulation of ER-α, PR, aromatase, PCNA, cyclin D1 and AgNORs staining and down regulation of p53 expression as well as plasma estradiol, prolactin and testosterone levels increased in DMBA and MNU-induced tumor bearing rats. Oral administration of AITC at a dose of 20mg/kg bw restored the levels of sex hormones and its receptors, aromatase, cell proliferative markers and AgNORs staining near to normal levels. Molecular docking studies also supported these findings. The results suggest that anti-estrogenic and anti-proliferative effect of AITC prevent the development of DMBA and MNU-induced mammary carcinogenesis in rat.

Maternal western-style diet enhances the effects of chemically-induced mammary tumors in female rat offspring through transcriptome changes

Previous studies have shown that early life intake of high-fat diet or western-style diet (WD) enhances the development of mammary tumors in adult female rats. Thus, we hypothesized that maternal WD throughout pregnancy and the lactation period could speed up the development of MNU-induced mammary tumors and alter their gene expression. For this, the present study investigated the gene expression profile of chemically-induced mammary tumors in female rat offspring from dams fed a WD or a control diet. Pregnant female Sprague-Dawley rats received a WD (high-fat, low-fiber and oligoelements) or a control diet from gestational day 12 until post-natal day (PND) 21. At PND 21, female offspring received a single dose of N-Methyl-N-Nitrosourea (MNU, 50 mg/kg body weight) and were fed a control diet for 13 weeks. Tumor incidence, multiplicity, and latency were recorded and mammary gland samples were collected for histopathology and gene expression analysis. Tumor multiplicity and histological grade were significantly higher and tumor latency was lower in WD offspring compared to control offspring. Transcriptome profiling identified 57 differentially expressed genes in tumors from WD offspring as compared to control offspring. There was also an increase in mRNA expression of genes such as Emp3, Ccl7, Ets1, Abcc5, and Cyr61, indicative of more aggressive disease detected in tumors from WD offspring. Thus, maternal WD diet increased MNU-induced mammary carcinogenesis in adult female offspring through transcriptome changes that resulted in a more aggressive disease.

Effect of allyl isothiocyanate on NF-κB signaling in 7,12-dimethylbenz(a)anthracene and N-methyl-N-nitrosourea-induced mammary carcinogenesis.

Inflammation plays a pivotal role in the process of carcinogenesis and phytochemicals have anti-inflammatory properties gaining more importance in cancer chemoprevention. The present study aimed to investigate the anti-inflammatory effect of allyl isothiocyanate (AITC) on 7,12-dimethylbenz(a)anthracene (DMBA)- and N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis in female Sprague-Dawley rats. RT-PCR and western blot analysis showed that inflammatory markers such as NF-κB p65, TNF-α, and IL-6 were overexpressed in mammary tumor tissues. Histological analysis of tumor tissues shows abnormality in hematoxylin and eosin (H&E) staining and toluidine blue (TB) staining of mast cell content, and lipid accumulation in oil red O staining. Administration of AITC (20mg/kg bw) to carcinogen-injected rats significantly decreased the expression of NF-κB p65, TNF-α, and IL-6 in mammary tissues. Further, molecular docking study demonstrates the binding of AITC to NF-κB p65. Remarkably, AITC treatments control the growth of cancer cells as clearly evidenced by histopathological analysis. Staining of mammary tissues for mast cells and lipids indicates that AITC treatment to carcinogen-administrated rats significantly reduced mammary tumorigenesis. The result suggests that AITC has anti-inflammatory potential to prevent DMBA- and MNU-induced mammary carcinogenesis in rats.

Electron Microscopy Findings in N-Methyl-N-Nitrosourea-Induced Mammary Tumors.

Although the rat model of mammary tumors chemically induced by N-methyl-N-nitrosourea (MNU) has been frequently used by several research teams, there is a lack of ultrastructural studies in this field. The main aim of this work was to perform an ultrastructural characterization of MNU-induced mammary tumors in female rats. Some alterations previously reported in human mammary tumors, such as nucleus size and shape, accumulation of heterochromatin in the perinuclear region, and interdigitating cytoplasmic processes between cancer cells were also observed in MNU-induced mammary tumors. Although a low number of samples were analyzed by transmission electron microscopy in the present study, we consider that it may contribute to a better understanding of MNU-induced mammary carcinogenesis in a rat model. The ultrastructural characteristics of the two most frequently diagnosed mammary carcinomas described in the present work can be useful to differentiate them from other histological patterns. In addition, the loss of cytoplasm in neoplastic cells and formation of vacuoles were described.

Effect of Thyroid Function on MNU-Induced Mammary Carcinogenesis

Mammary cancer is a disease that affects many women. Extensive research has been conducted to elucidate which variables are involved in the development of this cancer. Studies have highlighted thyroid function as a modulator of tumor growth and development. Thyroxine and 3,3',5-triiodothyronine are responsible for regulating the development, differentiation, homeostasis, and metabolism of cells in the body including mammary tissue. Thyroid hormones also have estrogen-like effects on mammary cancer cell growth by regulating the estrogen receptor. The present study was designed to determine whether medically induced hyperthyroidism increases the multiplicity, prevalence, and mammary tumor burden in rats; and to elucidate whether surgically induced hypothyroidism conversely attenuates the rate of mammary cancer cell growth. Female Sprague-Dawley rats were randomly divided into three groups (euthyroid-control, hyperthyroid, and hypothyroid). Hyperthyroidism was induced via oral administration of levothyroxine; whereas, hypothyroidism was induced by thyroidectomy. Mammary carcinogenesis was induced with a single intraperitoneal injection of N-methyl-N-nitrosurea (MNU). Rats were sacrificed at 38 weeks, and the mammary tumors were excised, fixed for histology and analyzed. Analysis included evaluation of malignancy and immunohistochemistry for ER. MNU-induced mammary carcinogenesis among the groups resulted in a significant difference in tumor burden. The hyperthyroid group had a statistically higher tumor burden than did the euthyroid group, and the hypothyroid group had no tumors of mammary tissue origin at 38 weeks. All excised mammary tumors were ER alpha negative. These data support the hypothesis that thyroid function is one of potentially many factors that contribute to modulation of MNU-induced mammary tumor growth.

The effect of physical training on the N-methyl-N-nitrosourea-induced mammary carcinogenesis of Sprague-Dawley rats.

The impact of physical activity on carcinogenesis has been demonstrated in many studies. Taking into account the discrepant results of physical exercise on the cell proliferation and apoptosis of breast cancer, we aimed to examine the impact of physical training on N-methyl-N-nitrosourea-(MNU)-induced mammary carcinogenesis. Fifty female rats were divided into four groups according to the intensity of physical activity they undertook. The number of developed tumors, tumor volume, and histopathological diagnoses were noted. Apoptosis and cell proliferation were studied by the number of TUNEL-positive and Ki-67-expressing cells. We demonstrated a statistically significant decrease in the tumor number between all trained groups and the control group. The results were most pronounced in the group with a moderate intensity of training. Moreover, we showed a decrease in tumor volume as training intensity increased, though the differences were not statistically significant. The mean number of TUNEL-positive cancer cells was significantly higher in the training groups than in the control group. These data suggest that physical training, especially of moderate intensity, may alleviate MNU-induced mammary carcinogenesis. The results could suggest that physical exercise-induced apoptosis may be a protective mechanism.

Abstract 2486: Plasma proteomic changes induced by effective chemopreventive ratios of N-3:N-6 fatty acids against MNU-induced mammary carcinogenesis of the rat.

Abstract The development of sensitive, specific, and reliable biomarkers for early detection of breast cancer and the discovery of novel chemopreventive agents are two strategies with promising clinical applications. It has been postulated that the risk of breast cancer can be influenced by certain dietary compounds such as the type and amount of dietary fats ingested. We have shown that a diet high in an n-3:n-6 ratio (10:1 and 25:1), that provided 30% calories from fat (10% monounsaturated, 10% polyunsaturated, and 10% saturated fatty acids), significantly reduced rat mammary cancer induced by 1-methyl-1-nitrosourea(MNU). In the present study we used a proteomic approach to gain insights into the mechanisms of protection by a high n-3:n-6 ratio with the goal of identifying biomarkers that can be employed to monitor efficacy of chemopreventive agents. The plasma from six rats from each of three groups, a control group(n-3:n-6=1:1) and two groups fed a high n-3 diet(n-3:n-6=10:1 and 25:1) was pooled and submitted to the isobaric Tags for Relative and Absolute Quantitation(iTRAQ) method, which includes matrix-assisted laser desorption/ionization tandem mass spectroscopy(MALDITOF/TOF) to identify and quantify proteins that were regulated as a result of n-3 fatty acid supplementation. There were 148 proteins identified in our study with 95% confidence(unused score 1.3 or greater) by ProteinPilot 4.0. In plasma of rats fed 10:1 n-3:n-6, and 25:1 n-3:n-6 the number of proteins that met our criteria(p&amp;lt;0.05, error factor≤2) were 10 and 14 proteins, respectively. Our results illustrated that many proteins were modified in a manner consistent with chemoprevention. Validation of the iTRAQ by Western Blotting showed up-regulation of vitamin D binding protein and gelsolin postulated to have a tumor suppressive effect through modulation of inflammation and actin depolarization, respectively. We also observed that 14-3-3 sigma, a well known tumor suppressor in breast tissue, was increased in a ratio dependent fashion. In contrast, we observed down-regulation of alpha-1b-glycoprotein which has been reported to be elevated in the serum of breast cancer patients. These proteins may serve in future studies as candidate biomarkers in early detection and clinical chemoprevention trials with well-defined ratios of n-3:n-6 fatty acids. Support: Susan G. Komen Foundation(KG081632) Citation Format: Christine G. Skibinski, Henry J. Thompson, Arunangshu Das, Andrea Manni, Karam El-Bayoumy. Plasma proteomic changes induced by effective chemopreventive ratios of N-3:N-6 fatty acids against MNU-induced mammary carcinogenesis of the rat. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2486. doi:10.1158/1538-7445.AM2013-2486

Fish Oil Alters Tamoxifen-Modulated Expression of mRNAs That Encode Genes Related to Differentiation, Proliferation, Metastasis, and Immune Response in Rat Mammary Tumors

We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publisher's online edition of Nutrition and Cancer to view the supplemental file.

Mammary cancer promotion by ovarian hormones involves IGFR/AKT/mTOR signaling

In a previous study, we observed that N-methyl-N-nitrosourea (MNU)-induced mammary lesions are promoted to overt mammary cancers by exogenous administration of estradiol (E) and progesterone (P). The purpose of the present study was to identify the early molecular events occurring during the hormonal promotion of mammary carcinogenesis and persistent activation of molecular pathways responsible for tumor growth. Seven-week-old female Copenhagen (COP) rats, which are resistant to MNU-induced mammary carcinogenesis, were intraperitoneally administered a single dose of MNU (50mg/kg body weight). Six weeks after carcinogen administration, the rats were treated with E+P, killed at 15th week and 43rd week to obtain mammary lesions and tumor tissues and the molecular analysis were performed. Quantitative RT–PCR experiments showed increased mRNA expression of Igfr, Grb2, Sos1, and Shc1 in mammary lesions and tumors. Immunoblot data also showed increased protein levels of IGFR, GRB2 and SHC1 in mammary lesions and tumors, which is in correlation with their respective RT–PCR data. Activation of AKT and ERK1/2 were up regulated in E+P treated mammary lesions and tumors. Molecular analysis of mTOR pathway proteins revealed increased phosphorylation of p70S6K and 4EBP1 in the hormone treated tumors indicating the activation of mTOR signaling. E+P treatment reduced the protein expression of BAX and increased BCL2 expression along with down regulation of active caspase 3 and 8. Together, these data demonstrate that ovarian hormones promote the lesions to mammary tumors by enhancing IGFR and Akt/mTOR signaling along with inhibition of apoptotic stimuli.

Comparative aspects regarding MNU-induced mammary carcinogenesis in immature Sprague-Dowley and Whistar rats

The research carried out on an experimental mammary cancer model by using two breeds of rat, such as Sprague-Dawley (n=10) and Wistar (n=9) female rats. There have been used immature rats that received intraperitonealy a single dose of methyl-nitroso-urea (55 mg/kg MNU). The rats were kept in optimal conditions of temperature, food and hygiene for 8 months. The Wistar rats were sacrificed by deep narcosis with Halothane, and were harvested samples for histopathology exam. The Sprague-Dawley rats were not slaughtered being utilized still to test the efficacy of some antioxidative agents in mammary cancer prevention. In Wistar rats, mammary tumor incidence was reduced representing 33,3% of all cases. In 22,2% of all cases precancerous mammary lesions occurred. On the other hand, MNU generated a great variability of tumor types in various tissues and organs, such as: liposarcoma, nephroblastoma, bronchial gland carcinoma, hemangiosarcoma, squamous carcinoma, sebaceous gland carcinoma, small cell lung carcinoma and lipoma. Comparing to this, in Sprague-Dawley rats mammary tumor incidence following to MNU-intake was 80% of all cases. Concluding, the most appropriate breed of rat that could be utilized as experimental model to study canine and human mammary cancer is Sprague-Dawley.

Effects of short-term estrogen treatment on the progression of N-methyl-N-nitrosourea-induced premalignant mammary lesions in female Lewis rats

We studied the effects of short-term estrogen treatment (STET) on the progression of mammary lesions from ductal hyperplasia (DH) through ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) in the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis model. Three-week-old female Lewis rats (n = 40) received an intraperitoneal injection of MNU (50 mg/kg). Three weeks later, a 3-week-release, 0.25-mg, 17β-estradiol pellet was subcutaneously implanted for 2 weeks in 20 rats (STET); the remaining 20 rats did not receive the estradiol pellets (age-matched control). All rats were killed at 12 weeks of age, and their abdominal-inguinal mammary glands were histologically examined. The incidence and multiplicity of DHs were similar between groups (STET, 90% and 3.9 ± 0.6 vs. age-matched controls, 80% and 3.0 ± 0.5). However, DCIS and IDC did not develop in STET rats, whereas DCIS (25% and 1.4 ± 0.2) and IDC (35% and 1.4 ± 0.3) developed in the age-matched controls. Immunoscores of estrogen and progesterone receptors and positive rate of proliferative cell nuclear antigen (PCNA) in DH were similar in both groups, while the positive rate of cyclin D1 was significantly reduced in the STET group (P < 0.05). Thus, STET blocked the progression from DH to DCIS in MNU-induced mammary carcinogenesis, and decreased expression of cyclin D1 may play an important role in the blockade of cell transition from DH to DCIS.

MNU-induced mammary gland carcinogenesis: Chemopreventive and therapeutic effects of vitamin D and Seocalcitol on selected regulatory vitamin D receptor pathways

The effects of administration of vitamin D 3 and Seocalcitol on MNU-induced carcinogenesis of mammary gland in Sprague-Dawley rats have been investigated. Administration of both substances in a weekly dose of 7 μg/kg caused prolonged latency of mammary gland tumors. The latency of tumors was markedly prolonged for 30–40 days by Seocalcitol. Using PET analysis, reduction in [ 18F]2-fluoro-2-deoxy- d-glucose (FDG) uptake or tumor volume in tumors chemopreventively treated with vitamin D 3 were detected in MNU-induced tumors, vitamin D 3 reduced expression of 25-hydroxylase (25OHase) ( p < 0.01) and 24-hydroxylase (24OHase) ( p < 0.01) and Seocalcitol 24OHase. Positive regulation of 25OHase mRNA level after the treatment with vitamin D 3 was observed in liver, while in kidney, vitamin D 3 and Seocalcitol induced expression of 24OHase was significant. Our observations indicate a cross talk between respective pathways of VDR, RARs/RXRs, TRs and ERs in carcinogenesis process.

Abstract P4-02-07: A Progesterone Receptor Modulator, CDB-4124, Inhibits Mammary Carcinogenesis by Suppressing Cell Proliferation In Vitro and In Vivo

Abstract CDB-4124 (Proellex or Telapristone acetate) is a modulator of progesterone receptor (PR) signaling which has been employed in preclinical studies for the treatment of uterine fibroids and endometriosis. Here we provide evidence for its action in breast cancer from both in vitro and in vivo studies. When CDB-4124 is given to rats at 200 mg/kg for 24 months it prevents the development of spontaneous mammary hyperplastic and premalignant lesions. In a second set of experiments, we found that CDB-4124, given as subcutaneous pellets at two different dose suppressed MNU-induced mammary carcinogenesis in rats in a dose dependent manner (see table below). The high dose (30 mg over 84 days) increased tumor latency from 66 to 87 days (P&amp;lt;0.02), decreased incidence from 85% to 35% (P&amp;lt;0.01), and multiplicity, from 3.0 to 1.1 tumors/animal (P&amp;lt;0.01). Tumor burden (total of all tumor weights in an animal) decreased from 2.6 g to 0.26 g/animal (P&amp;lt;0.01). Moreover in suport of our observations, CDB-4124 significantly suppressed Ki-67 positive cells from 30.5±7.1% in placebo-treated to 25.4±14.4% in low-dose and further to 10.3±4.5% (P&amp;lt;0.001) in the high-dose treated animals. CDB-4124 increased latency and inhibited the incidence, multiplicity and burden of mammary tumors CDB-4124 inhibited cell proliferation and induced apoptosis in these mammary tumors and this correlated with decreased proportion of PR+ tumor cells. Serum progesterone was lowered but there were no effects on estradiol. This progesterone receptor modulator appears to exert its effects in the presence of estrogen. In a mechanistic study employing human T47D breast cancer cells we found that CDB-4124 suppressed G1/0-S transition by inhibiting cdk-2 and cdk-4 expression and this correlated with inhibition of ER expression. Taken together, these data indicates that CDB-4124 can suppress the development of spontaneous and carcinogen-induced ER+ mammary tumors in rats and thus newer progesterone receptor modulators may have implications for prevention and treatment of human breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-07.

Abstract B60: Exploitation of a common food/traditional medicine of South America, yacon, in breast cancer prevention in the U.S. using preclinical rat model

Abstract Background: Yacon has recently been introduced into farmer's markets and natural food stores in the U.S., but its preventive activity for breast cancer has rarely been evaluated. Yacon contains a large amount of non-digestible oligosaccharide called inulin that belongs to a class of carbohydrates known as fructans. Inulin-type fructans (ITF) decreases the rate of aberrant crypt foci (ACF), a pre-neoplastic lesion found in colon. The mechanism by which ITF inhibits ACF is associated with butyrate produced by the anaerobic bacterial fermentation of ITF in the colon. Butyrate can also be absorbed through the colonic epithelial cells into the portal blood and exert its effects within the body. Butyrate modulates gene transcription by inhibiting HDAC. Cancer cells appear to be more sensitive than non-transformed cells to HDAC inhibitory compounds. In addition, the yacon ITF promotes satiety and retard the absorption of food-derived energy via reducing a gastrointestinal peptide-ghrelin that is a growth hormone secretagogue. Reduction of serum ghrelin results in decreases of IGF-1. Subsequently, the PI3K/Akt-mTOR signaling pathway will be inactivated via increasing AMPK activity and its downstream events, inhibition of cell proliferation and induction of cell apoptosis. However, few studies have been shown the effect of the ITF on breast cancer. Objective: 1) To determine mammary carcinogenic responses to yacon in experimental animals; 2) To evaluate the association of the circulating growth factors and cytokines with the carcinogenic responses; 3) To determine the HDAC and identify AMPK/PI3K/Akt-mTOR cell signaling pathway. Methods: Mammary carcinogenesis was initiated by injection of female Sprague Dawley rats with 50 mg of 1-methyl-1-nitrosourea/kg body weight (i.p.) at 21 days of age. One week later, the rats were fed diets containing yacon powder at 0%, 15%, 30% or 60% (30 rats/group) for 8 weeks, respectively. Effects of yacon on mammary carcinomas, circulating growth factors and cytokines and molecular biological assessments in carcinomas, including HDAC and regulators that are associated with AMPK/PI3K/Akt-mTOR pathway, were evaluated. Difference between among different dietary groups was statistically analyzed by ANOVA and Chi-square test. Results/Relevance: Dietary yacon reduced the promotion and progression of MNU-induced mammary carcinogenesis in rat, which is associated with downregulation of IGF-1/HDAC/Akt/mTOR signaling pathway and anti-inflammation, i.e. reduction of plasma IL-6, TNFα, and C-reactive protein. Future work will focus on the effect of yacon on obesity and antiinflammation. The study provides crucial biological information to complement the knowledge of natural functional foods that is relevant to a number of foods rich in non-digestible, fermentable oligosaccharides. This could lead the way to develop a new type of functional whole food with a low calorie density and the ability to promote the intra-intestinal production of cancer inhibitory factors. Consequently, the new type of functional food will significantly contribute to the goal of preventing initiation of breast cancer and reduce survivor recurring because it is natural, tolerant and acceptable for the clinic patients. Supported by W81XWH-09-1-0428 from the Department of Defense-Army. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B60.

Coexistence of different tissue tumourigenesis in an N-methyl-N-nitrosourea-induced mammary carcinoma model: a histopathological report in Sprague-Dawley rats

N-methyl-N-nitrosourea (MNU), a highly potent carginogen, is widely used to generate mammary tumours in murine species. In a model of MNU-induced mammary carcinogenesis using immature female Sprague-Dawley rats, large mammary tumours (largest dimension > or =0.5 cm) were obtained within a very short period of time. In addition, in the rats bearing MNU-induced mammary carcinomas, there were a number of tumours whose origins were not from mammary tissue but from several different tissues and from mammary non-epithelial tissue. The tumours were of mesenchymal or epithelial origin and they were located in the inguinal region. These tumours were diagnosed as fibroadenoma, combined tubular adenoma and fibroadenoma, hyperkeratotic papilloma, keratinous cyst and malignant peripheral nerve sheath tumour (MPNST) with smooth muscle differentiation. The occurrence of these other tumours in addition to the development of the mammary carcinomas may be attributed to a direct local effect of the intraperitoneal administration of MNU during the sexual development of the immature rats. In the MNU-induced mammary tumour model, coexistence of tumourigenesis in various non-mammary tissues should be considered an important factor that may interfere with experimental procedures and results and also the quality of life of the tumour-bearing animals.

MNU-induced carcinogenesis of rat mammary gland: Effect of thyroid hormone on expression of retinoic acid receptors in tumours of mammary gland

The rat model of N-methyl- N-nitrosourea (MNU)-induced mammary carcinomas is well-established animal model for breast cancer. This study was carried out to investigate whether hypothyroid (thyroidectomy or PTU treatment) or hyperthyroid status of female rats would affect MNU-induced mammary carcinogenesis with specific focus on both retinoid and rexinoid receptor expression in mammary tumours. Application of PTU before and during MNU-induced mammary gland carcinogenesis yielded in a marked decrease of the number and volume of tumours per animal, however, there was no effect of hypothyroid state in thyroidectomized rats as well as hyperthyroid state concerning the number and volume of tumours. Mammary tumours of in euthyroid group of MNU animals showed that there was no tumour, in which all of subtypes of retinoid and rexinoid receptors were expressed. A different pattern of expression of retinoid or rexinoid receptors was found either in MNU-induced mammary carcinomas in both hypothyroid and hyperthyroid rats.

Chemomodulatory influence of Ferula asafoetida on mammary epithelial differentiation, hepatic drug metabolizing enzymes, antioxidant profiles and N-methyl-N-nitrosourea-induced mammary carcinogenesis in rats.

The present study was conducted to ascertain the modulatory influences of Ferula asafoetida L. (asafoetida, flavoring agent) on the mammary epithelial tissue differentiation, hepatic drug metabolizing enzymes, antioxidant profiles and N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis in Sprague-Dawley rats. Feeding with two doses of asafoetida (1.25 and 2.5% w/w in diet) showed a remarkable increase in the development and differentiation of ducts/ductules (p < 0.01-0.005), lobules (p < 0.005) and a decrease in terminal end buds (p < 0.05-0.005) as compared to both normal and MNU-treated control animals. To assess the biochemical parameters, effect of asafoetida on drug-metabolizing enzymes was evaluated in the liver of rats. Asafoetida treatment significantly reduced (p < 0.05) the levels of cytochrome P450 and b5. There was an enhancement in the activities of glutathione S-transferase (p < 0.05-0.005), DT-diaphorase (p < 0.05-0.01), superoxide dismutase (p < 0.01-0.005) and catalase (p < 0.05-0.005) and in the level of reduced glutathione (p < 0.05-0.005), followed by asafoetida treatment. Also, asafoetida significantly restored the level of antioxidant system, depleted by MNU-treatment. The strengthening of antioxidant system by the lower and higher doses of asafoetida in the presence and absence of MNU was further substantiated by a significant inhibition (p < 0.005) in lipid peroxidation as measured by thiobarbituric acid-reactive substances (TBARS) in the liver of rat. Further, in long-term animal studies, where MNU was used to induce mammary carcinogenesis, asafoetida treatment resulted in a significant reduction in the multiplicity (p < 0.001) and size of palpable mammary tumors (p < 0.005-0.001) and a delay in mean latency period of tumor appearance (p < 0.005). Together, these findings indicate the chemopreventive potential of asafoetida against MNU-induced mammary carcinogenesis. Thus, asafoetida needs further investigation with regard to identification and characterization of its active principle(s) and mechanism of action, for this compound to be developed as a potential chemopreventive agent for human cancers.

Dietary Docosahexaenoic Acid Suppresses N-Methyl-N-Nitrosourea-Induced Mammary Carcinogenesis in Rats More Effectively Than Eicosapentaenoic Acid

We comparedthe effects of identical amounts but different proportions of dietary n-3 polyunsaturated fatty acids (PUFAs) on N-methyl-N-nitrosourea (MNU)-induced mammary cancer in a rat model. The ability of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to suppress mammary cancer was evaluated. Female Sprague-Dawley rats were randomly assigned to three groups and maintained on diets containing 10% fatty acid consisting of EPA, a 1:1 mixture of EPA-plus-DHA, or DHA. The experimental diet was started after administration of MNU at 49 days of age, and the rats were maintained on the respective diets until the largest mammary tumor reached ≷ 1 cmin diameter or until the end of the study period (20 wk after MNU). All histologically detected mammarycarcinomas were evaluated, irrespective of size. The DHAdiet was associated with significant suppression of the carcinogenic effect of MNU compared with the EPA and EPA-plus-DHA diets: tumor incidence decreased to 23% (3/13) compared with 73% (11/15) and 65% (12/17) (P<0.01 andP<0.05, respectively); tumor multiplicity decreased to 0.23 compared with 1.67 and 1.59 (P < 0.01 and P < 0.05, respectively). There was no significant difference in tumor latency among the DHA, EPA, and EPA-plus-DHA groups (119, 105, and 117 days, respectively). Over 20 wk, the fatty acid composition of serum and mammary fat tissue reflected differences in the dietary n-3 PUFAs. Although DHA suppressed MNU-induced mammary carcinogenesis more effectively than EPA, generalized steatosis including mammary fat tissue appeared in all three groups.