Expression data from in utero exposure to genistein, vinclozolin and the mixture of genistein and vinclozolin on the mammary gland
ABSTRACT: Morphogenesis of the mammary gland relies on the precise developmental control of morphological elements including TEBs, ducts and lobules. In the peripubertal mammary gland, rising levels of ovarian hormones control this development through a tightly controlled genetic program where specific sets of genes are up-regulated. We used microarrays to detail the program of gene expression underlying different classes of up-regulated genes during the peripubertal process after administration of endocrine disruptors during the fetal and neonatal development. Rat mammary glands were selected at two peripubertal periods (days 35 and 50), after administration of genistein and vinclozolin during the fetal and neonatal development, for RNA extraction and hybridization on Affymetrix microarrays.. We sought to obtain homogeneous populations of mammary gland for each treatment, and at each developmental stage, in order to increase the temporal and specific effect resolution of time course and endocrine disruption.
Project description:Background: Post-menopausal obesity is an established risk factor for breast cancer. Consumption of diets high in fat is known to be highly correlated with obesity. In this, we sought to evaluate the interaction(s) between high fat diet, weight gain and mammary carcinogenesis using an obese-resistant and obese-prone rat model with direct correlates to human disease. Methods: Female obese-prone (OP) and obese-resistant (OR) weanling rats were placed on either a low fat (10% kcal) or a high fat (39% kcal) n-6 polyunsaturated (PUFA) safflower diet for 30 days. At post natal day (PND) 50, global gene expression profiling was performed on microdissected mammary epithlelium from one cohort of rats and another cohort of rats were given a single oral gavage of either 7,12-dimethylbenz[a]anthracene (DMBA at 14 mg/kg) or vehicle. Rats were then maintained on the diets and body weights, food consumption and development of mammary lesions were monitored weekly. Results: The DMBA-treated OR rats on the 39% safflower diet had significantly greater incidence of ductal carcinoma-in-situ (DCIS) lesions and significantly greater DCIS multiplicity than DMBA-treated OR rats on the 10% safflower diet. These differences were not seen in the OP strain. Gene expression analysis of mammary ductal epithelium from OR rats on the high fat diet showed significant upregulation of proliferation-related genes compared to those consuming the low fat safflower diet. Again, these differences were not seen in the OP strain. Conclusion: Our findings indicate that consumption of high fat safflower diet enhances mammary carcinogenesis in an OR rat strain through increased proliferation of mammary epithelium at the time of exposure, but not in the OP rat strain. Thus, the diet-induced increase in sensitivity was strain-specific and independent of weight gain or obesity level. Female obese-prone (OP) and obese-resistant (OR) weanling rats were placed on either a low fat (10% kcal) or a high fat (39% kcal) n-6 polyunsaturated (PUFA) safflower diet for 30 days. At post natal day (PND) 50, global gene expression profiling was performed on microdissected mammary epithlelium from one cohort of rats and another cohort of rats were given a single oral gavage of either 7,12-dimethylbenz[a]anthracene (DMBA at 14 mg/kg) or vehicle. Rats were then maintained on the diets and body weights, food consumption and development of mammary lesions were monitored weekly.
Project description:Soy foods have been suggested to have both positive health benefits and potentially adverse effects largely as a result of their content of isoflavone phytoestrogens. Since soy protein isolate (SPI) contains isoflavones, in addition to purported health benefits, safety concerns have been raised regarding the use of SPI and soy formulas, because of potential estrogenic actions during the neonatal period, including the potential for reproductive toxicity, infertility, and the possibility of increased risk for development and recurrence of estrogen sensitive cancers such as breast cancer. In the current study, we used a rat model to compare the effects of SPI with those of 17b-estradiol (E2), on global gene expression profiles and morphology in the female rat mammary gland. Rats were either fed AIN-93G diets containing casein (CAS) or SPI beginning on postnatal day (PND) 30. Rats were ovariectomized (OVX) on PND 50 and treated with E2 or vehicle for 14 days. Microarray analysis was carried out to compare the effects of SPI and E2 alone or in combination on the mammary gene expression. The data suggest a non-estrogenic effect of SPI on the rat mammary even in the absence of endogenous estrogens.
Project description:Background: This study examines the impact of dietary fatty acids on regulation of gene expression in the mammary epithelial cells before and during puberty. Methods: The diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Dietary-induced changes in gene expression were examined in the LCM (Laser Capture Microdissected) captured mammary ductal epithelial cells at day of weaning (21 days) and at the end of puberty (50 days after birth). PCNA immunohistochemistry analysis was used to compare proliferation rates between diets. Results: Genes differentially expressed between each of the test diets and the reference diet in both age groups were significantly enriched by cell cycle genes. Some of these genes were involved in the activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. Conclusion: Fatty acid-enriched diets significantly up-regulated proliferation above the normal physiological level at day 50. The higher cellular proliferation during puberty caused by enriched fatty acid diets pose a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle cluster of rat genes are included in a human breast cancer cluster of 45 cell cycle related genes further emphasizing the importance of our findings in the rat model. Female, virgin Spraque-Dawley rats were obtained from Taconic Farms (Germantown, NY) at approximately 7 weeks of age and placed on one of 9 pelleted purified diets for one month. After one month of diet exposure, female rats were bred with male Sprague-Dawley rats of approximately 3 months of age. Litters were weighed and monitored throughout gestational period. Pups had access to dam’s milk as well as tap water and food throughout gestation. Pups were weaned at DOL 21 and individually housed with tap water and diets ad libitum. Rats were killed by CO2 asphyxiation and decapitated at the following two ages, DOW (DOL21) and DOL 50 with no other treatments.
Project description:Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improving dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate and supplemented), and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P<0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding RARß, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log26 in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naïve rats. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling RBP4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change. All rats were housed in a room maintained at 22°C with a 12-h dark:light cycle, and food and water were freely available. For the Steady-State Vitamin A Study (experiment 1) and the Retinoic Acid 16-hour Kinetic Study (experiment 2), lactating female Sprague-Dawley rats with 12 female pups (purchased from Charles River Laboratories, Willmington, MA) were fed a vitamin A-deficient purified diet [AIN-93G diet, prepared by Research Diets, New Brunswick, NJ] to reduce the transfer of vitamin A in milk from mother to pups prior to the start of the study. For experiment 1, from weaning, the offspring were fed the same diet modified to contain vitamin A at one of four levels: 0 (vitamin A deficient), 0.4 mg retinol/kg diet (vitamin A marginal), 4 mg retinol/kg diet (vitamin A adequate control), or 100 mg retinol/kg diet (vitamin A supplemented). All rats were studied at 8 weeks of age. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. In experiment 2, at 8 weeks of age female vitamin A-deficient rats were treated with ~100 ug of All-trans-Retinoic acid (at-RA) for 0 (vehicle only), 3, 6, 10 or 16 h (n=3-4/group). Tissues were collected and RNA was prepared in the same manner as in experiment 1. In the 90-minute "first pass" kinetic study (experiment 3), female rats were purchased at 6 weeks of age and fed a stock rodent diet. When the rats were 8 weeks old they were assigned to a control (naïve) group. Rats in the naive group received an equal amount of vehicle only (vegetable oil/5% ethanol). Food was removed immediately. Sixteen hours after priming, each rat was lightly anesthetized by isoflurane-oxygen inhalation and treated with ~25 ug all-trans-RA bound to albumin [10 ug RA per 100 g bo dy weight], injected into the exposed left common iliac vein. The incision was closed with a surgical staple. The rats were allowed to recover from the anesthesia. Rats were killed at 0 minute (vehicle injection), and 30, 60, and 90 min (n = 3/group) after injection of the RA test dose. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. For experiment 1, five biological repeats were Vitamin A deficient, seven biological repeats were Vitamin A marginal, six biological repeats were Vitamin A adequate, and two biological repeats were Vitamin A supplemented. In experiment 2, three biological repeats were performed for each of the following treatments: untreated, at-RA for 3 hours, at-RA for 6 hours and at-RA for 16 hours. Four biological repeats were treated with at-RA for 10 hours. For experiment 3, two biological repeats were untreated and three biological repeats were performed for each of the following at-RA treatment times: 30, 60 and 90 minutes. A total of 47 samples were analyzed.
Project description:Liver gene transcripts patterns were used to characterize toxicity from exposure to polybrominated diphenyl ethers (PBDEs), flame retardant components. In this study, Wistar Han dams were exposed by gavage to the PBDE mixture (DE71) starting at gestation day 6 (GD 6) and continuing to weaning on postnatal day 21 (PND 21). Offspring from the dams began PBDE direct dosing on PND 12 and were dosed daily through PND 21. After weaning, they were dosed 5 days per week for another 13 weeks. Liver samples were collected at PND 22 and week 13 for liver gene expression analysis and interrogated with the Affymetrix Rat Genome 230 2.0 Array. PBDE treatment induced 1,066 liver gene transcript changes in females and 1,200 transcriptional changes in males at PND 22 (false discovery rate (FDR) < 0.01), but only 263 liver transcriptional changes at 13 weeks in male rats (FDR <0.05). No significant differences in dose response were found between male and female pups. There were a total of 6 groups and 5x replication for each group, for 30 total samples that were analyzed. The groups were (1) pup-male-CTL, (2) pup-female-CTL, (3) pup-male-PBDE, (4) pup-female-PBDE, (5) rat-male-CTL, (6) rat-male-PBDE. We generated the following pairwise comparisons using R/maanova: malePups(PBDE vs CTL), femalePups(PBDE vs CTL), maleRats(PBDE vs CTL), CTLpups(male vs female), PBDEpups(male vs female). We also performed ANOVA test for SEX-by-DOSE (pups) and AGE-by-DOSE (males). For pups, genes with an FDR≤1% were selected; for rats, genes with FDR < 5% were selected.
Project description:Transcriptional profiling of rat liver comparing male rats with congenital hypothyrodism (CH) vs intact at adulhood. Here we studied how CH influences liver gene expression program in adulthood. Thyroid hormones are required for normal growth and development in mammals. Congenital-neonatal hypothyroidism (CH) has a profound impact on physiology but its specific influence in liver is less understood. Here we studied how CH influences liver gene expression program in adulthood. Pregnant rats were given anti-thyroid drug methimazole (MMI) from GD12 until PND30 to induce CH in male offspring. Growth defects due to CH were evident as a reduction in body weight and tail length from the second week of life. Once the MMI treatment was discontinued, feed efficiency increased in CH and this was accompanied by significant catch-up growth. On PND80, significant reduction in body mass, tail length, and circulating IGF-I remained in CH rats. On the other hand, mRNA levels of known GH targeted genes were significantly up-regulated. Serum levels of thyroid hormones, cholesterol, and triglycerides showed no significant differences. In contrast, CH rats showed significant changes in expression for hepatic genes involved in lipid metabolism with an increased transcription of PPAR and reduced expression of genes involved in fatty acids and cholesterol uptake, cellular sterol efflux, triglycerides assembly, bile acid synthesis, and lipogenesis. These changes were associated with a decrease of intrahepatic lipids. Finally, CH rats responded to hypothyroidism onset in adulthood with a reduction of serum fatty acids and hepatic cholesteryl esters, and to T3 replacement with enhanced activation of lipogenic transcriptional program. In summary, we provided in vivo evidence that neonatal hypothyroidism causes long-lasting effects on hepatic transcriptional program and tissue sensitivity to hormone treatment. This highlights the critical role that a euthyroid state during development plays on normal liver physiology in adulthood. Two conditions CH vs INTACT male rats. Biological replicates: Four independent hybridizations: 4 controls (age-matched intact rats) vs 4 CH (male rats with congenital hypothyroidism) on postnatal day 80 for a total of four arrays. One replicate per array.
Project description:Background: The differential expression pattern of microRNAs (miRNAs) during mammary gland development might provide insights into their role in regulating the homeostasis of the breast epithelium. Our aim was to analyse these regulatory functions by deriving a comprehensive tissue-specific combined miRNA and mRNA expression profile of post-natal mouse mammary gland development. We measured the expression of 318 individual murine miRNAs by bead-based flow-cytometric profiling of whole mouse mammary glands throughout a 16-point developmental time course, including juvenile, puberty, mature virgin, gestation, lactation, and involution stages. In parallel whole-genome mRNA expression data were obtained. Results: One third (n = 102) of all murine miRNAs analysed were present during mammary gland development. MicroRNAs were represented in seven temporally co-expressed clusters, which were enriched for both miRNAs belonging to the same family and breast cancer-associated miRNAs. Global miRNA and mRNA expression was significantly reduced during lactation and the early stages of involution after weaning. For most detected miRNA families we did not observe systematic changes in the expression of predicted targets. For miRNA families whose targets did show significant changes, we observed inverse patterns of miRNA and target expression. The datasets are made publicly available and the combined expression profiles represent an important community resource for mammary gland biology research. Conclusions: MicroRNAs were expressed in co-regulated clusters during mammary gland development. Breast cancer-associated miRNAs were significantly enriched in these clusters. The mechanism and functional consequences of this miRNA co-regulation and its correlation with mRNA expression provide new avenues for research into mammary gland biology and generates candidates for functional validation. Developmental time course over 16 time points with 1-3 independent biological replicates per time point and three pairs of technical replicates, 40 samples in total
Project description:Prenatal iron deficiency (pID) has been described to increase the risk for neurodevelopmental disorders such as autism and schizophrenia; however, the precise molecular mechanisms are still unknown. Here, we utilized high throughput mass spectrometry to examine the proteomic effects of pID in adulthood on the rat frontal cortex area (FCA). In addition, the FCA proteome was examined in adulthood following risperidone treatment in adolescence to see if these effects could be prevented. We identified 1501 proteins of which 100 were significantly differentially expressed in the FCA at post-natal day 90. Pathway Analysis of proteins affected by pID revealed changes in metabolic processes, including the tricyclic acid cycle, mitochondrial dysfunction, and P13K/Akt signaling. Interestingly, most of these protein changes were not present in the adult pID offspring who received risperidone in adolescence. Behavioral testing of pID rats demonstrated social impairment and poor performance during novelty-induced exploration in pID animals in line with a abnormal neurodevelopmental phenotype. Considering the link between prenatal iron deficiency and several neurodevelopmental disorders such as autism and schizophrenia these presented results bring new perspectives to understand the role of iron in metabolic pathways and provide novel biomarkers for future studies of prenatal iron deficiency.
Project description:Sexual selection involves mate preference behavior and is a critical determinant for natural selection and evolutionary biology. Previously an environmental compound (fungicide vinclozolin) was found to promote epigenetic transgenerational inheritance of modified mate selection characteristics in all progeny for three generations after exposure of a gestating female. The current study investigated gene networks involved in various regions of the brain that correlated with the mate preference behavior altered in F3-Vinclozolin lineage animals. Statistically significant correlations of differentially expressed gene clusters and modules were identified to associate with specific mate preference behaviors. This novel systems biology approach identified critical gene networks involved in mate preference behavior and demonstrated the ability of environmental factors to promote epigenetic transgenerational inheritance of this altered evolutionary biology determinant. Combined observations elucidate the potential molecular control of mate preference behavior and suggests environmental epigenetics can have a role in evolutionary biology. We used Affymetrix Rat Gene 1.0 ST microarrays to determine genes expressed differentially in F3 Vinclozolin lineage male or female rats' 6 brain areas - amygdala (Amy), hippocampus (Hipp), olfactory bulb (OlfB), cingulate cortex (CngCtx), entorhinal cortex (EnCtx), and preoptic area-anterior hypothalamus (POAH) - due to Vinclozolin treatments of their grand-grandmothers (F0). For each of 6 brain areas of male or female rats (female: amygdala (F-Amy), cingulate cortex (F-CngCTX), enterorhinal cortex (F-EnCTX), hippocampus (F-Hipp), olfactory bulbs (F-OlfB), and preoptic area-anterior hypothalamus (F-POAH); male: amygdala (M-Amy), cingulate cortex (M-CngCTX), enterorhinal cortex (M-EnCTX), hippocampus (M-Hipp), olfactory bulbs (M-OlfB), and preoptic area-anterior hypothalamus (M-POAH)), RNA samples from 2 treatment groups - F3 Control lineage (Con) or F3 Vinclozolin lineage (Vin) - were compared to each other. Each of treatment groups contained 4-6 biological replicas for each brain region. RNA for each replica was isolated from an individual animal in order to compare to individual animal mate preference behavior studied with the same rats before sacrifice. Totally, 132 RNA samples from 24 animals (6 male F3 Control, 6 male F3 Vinclozolin,6 female F3 Control, and 6 female F3 Vinclozolin) were isolated and studied.