Toxicogenomics analysis of placenta samples from mice exposed to different doses of BPA
ABSTRACT: Bisphenol A (BPA) is a widespread Endocrine Disrupter mainly used in food contact plastics. Several evidences support BPA adverse effects, especially on susceptible groups such as pregnant women. The present study considered placental development - relevant for pregnancy outcomes and fetal nutrition/programming - as a potential target of BPA. Pregnant CD-1 mice were administered per os with vehicle, 0.5 (BPA05) or 50 mg/kg (BPA50) body weight (bw)/die of BPA, from gestational day (GD) 1 to GD11. At GD12, BPA50 induced significant degeneration and necrosis of giant cells, increased vacuolization in the junctional zone in absence of glycogen accumulation and reduction of the spongiotrophoblast layer. BPA05 induced a significant nuclear accumulation of β-catenin in trophoblasts of the labyrinthine and spongiotrophoblast layers, supporting an activation of the Wnt/β-catenin pathway. Transcriptomic analysis indicated a BPA05 promotion and a BPA50 inhibition of blood vessels development and branching supported by the morphological observation of decreased and increased total vessels area in the labyrinth layers upon BPA05 and BPA50 treatments, respectively, with BPA50 also inducing a decrease in vessels number. These mechanisms involve, respectively, CREB and Wnt/β-catenin pathways for BPA05 and AhR/ARNT pathway for BPA50, as deducted by the transcription factor binding analysis. These results show that BPA differently affects mouse placental development depending on the dose level. In particular, the 0.5 mg/kw bw, in the range of dose level used to set the Tolerable Daily Intake, may elicit not negligible alterations whose relevance for human health has to be carefully evaluated. Three conidtions experiment: Control, BPA 0.5 mg/kg bw die; BPA 50 mg/kg bw die. Biological replicates: three control replicates; four replicates per treatment. Technical replicates: 2 (dye swap) per biological replica
Project description:Benzo(a)pyrene is a well-established human carcinogen in humans and rodents. In the present study, we sought to determine the dose- and time-dependent changes in gene expression upon oral exposure to benzo(a)pyrene. Adult male MutaTMMouse were exposed to three doses of benzo(a)pyrene or vehicle control (olive oil) for 28 days and sacrificed three days after the final exposure. This experiment examined the forestomach transcriptional response of male mice exposed to BaP for 28 days at three different doses, including D1 (25 mg/kg BW/day), D2 (50 mg/kg BW/day), and D3 (75 mg/kg BW/day) and vehicle control. Each dose group was examined 72 hours following the final exposure. Each dose group and time point had 4-5 biological replicates. There were a total 17 samples (arrays) included in the final analysis using a two-colour reference design.
Project description:Mechanism-based toxicogenomics (tgx) is used as a tool to identify markers reflective of the onset and progression of cholestasis in C57BL/6 mice using Cyclosporin A (CsA) as a model compound. Critical doses for tgx analysis were derived from a dose range finding study in which increase of serum cholesterol, total bile acids, and total bilirubin as well as induction of hepatocyte vacuolization 25 days upon repeated CsA administration through oral gavage were considered as critical effects. For tgx analysis to find early markers, livers of mice repeatedly treated with 3 mg/kg BW, 8.9 mg/kg BW, and 26.7 mg/kg BW for one, four, and eleven days were collected. 60 samples are analyzed; per treatment duration (1, 4, 11 days), time-matched vehicle (olive oil) controls and three dose groups (3, 8.9, 26.7 mg/kg BW) were included; each group consisted of 5 replicates; 3 arrays were excluded, 2 because of quality control restrictions, 1 because of outlier properties. 2 that failed QC are omitted. Final data consists of 58 CEL files.
Project description:To examine whether the BPA-induced morphological alterations of the fetal mouse mammary glands are a) associated with changes in mRNA expression reflecting estrogenic actions and/or b) dependent on the estrogen receptor α (ERα), we compared the transcriptomal effects of BPA and the steroidal estrogen ethinylestradiol (EE2) on fetal mammary tissues of wild type and ERα knock-out mice. Mammary glands from fetuses of dams exposed to vehicle, 250 ng BPA/kg BW/d or 10 ng EE2/kg BW/d from embryonic day (E) 8 were harvested at E19. Total RNA was subjected to transcriptomal profiling using Affymetrix mouse genome 430 2.0 microarray.
Project description:Benzo(a)pyrene is a well-established human carcinogen in humans and rodents. In the present study, we sought to determine the dose- and time-dependent changes in gene expression upon oral exposure to benzo(a)pyrene. Adult male B6C3F1 mice were exposed to four doses of benzo(a)pyrene or vehicle control for three days and sacrificed 4 or 24 hours after the final exposure. This experiment examined the hepatic transcriptional response of male mice exposed to BaP for 3 days at four different doses, including D1 (300 mg/kg BW/day), D2 (150 mg/kg BW/day), D3 (50 mg/kg BW/day) and D4 (5 mg/kg BW/day), and one control. Each dose group was further examined at 2 time points, 4 hours and 24 hours, following the final exposure. Each dose group and time point had 4-5 biological replicates. There were a total 46 samples (arrays) included in the final analysis using a two-colour reference design.
Project description:Environmental factors during perinatal development influence developmental plasticity and disease susceptibility via alterations to the epigenome. Developmental exposure to the endocrine active compound, bisphenol A (BPA), has previously been associated with altered methylation at candidate gene loci. Here, we undertake the first genome-wide characterization of DNA methylation profiles in the liver of murine offspring perinatally exposed to multiple doses of BPA through the maternal diet. Using a tiered focusing approach, our strategy proceeds from unbiased broad DNA methylation analysis using methylation-based next generation sequencing technology to in-depth quantitative site-specific CpG methylation determination using the Sequenom EpiTYPER MassARRAY platform to profile liver DNA methylation patterns in offspring maternally exposed to BPA during gestation and lactation to doses ranging from 0 BPA/kg (Ctr), 50 µg BPA/kg (UG), or 50 mg BPA/kg (MG) diet (N=4 per group). Genome-wide analyses indicate non-monotonic effects of DNA methylation patterns following perinatal exposure to BPA, corroborating previous studies using multiple doses of BPA with non-monotonic outcomes. We observed enrichment of regions of altered methylation (RAMs) within CpG island (CGI) shores, but little evidence of RAM enrichment in CGIs. An analysis of promoter regions identified several hundred novel BPA-associated methylation events, and methylation alterations in the Myh7b and Slc22a12 gene promoters were validated. Using the Comparative Toxicogenomics Database, a number of candidate genes that have previously been associated with BPA-related gene expression changes were identified, and gene set enrichment testing identified epigenetically dysregulated pathways involved in metabolism and stimulus response. In this study, non-monotonic dose dependent alterations in DNA methylation among BPA-exposed mouse liver samples and their relevant pathways were identified and validated. The comprehensive methylome map presented here provides candidate loci underlying the role of early BPA exposure and later in life health and disease status. For this study, liver DNA from a subset of a/a wild-type animals was analyzed for full methylome characteristics: 1) standard diet (Ctr, n = 4 offspring; 2 male and 2 female); 2) 50 µg BPA/kg diet (UG, n = 4 offspring; 2 male and 2 female); 3) 50 mg BPA/kg diet (MG, n = 4 offspring; 1 male and 3 female).
Project description:Rodent studies have indicated that gestational and perinatal bisphenol A (BPA) exposure increase the risk of developing breast cancer during adulthood. In contrast, some dietary compounds such as genistein (GEN) and indole 3-carbinol (I3C) present potential protective effects against inducing hormone-dependent cancers, including that of the mammary gland. Thus, we aimed to evaluate the role of these dietary compounds on early mammary gland development and carcinogenesis in female Sprague-Dawley offspring. Pregnant Sprague-Dawley (SD) rats were treated with BPA at 25 or 250µg/kg b.w./day by gavage from gestational day (GD) 10 to 21 with or without dietary GEN (250 mg/kg chow, ~5.5 mg/kg b.w./day) or I3C (2000 mg/kg chow, ~45.0 mg/kg b.w./day). At post-natal day (PND) 21, some female offspring from different litters were euthanized for mammary gland development and gene expression analyses while other female offspring received a single dose of N-methyl-N-nitrosourea (MNU) for mammary carcinogenesis initiation. The findings this study indicated the prenatal exposure to BPA, GEN and I3C did not significantly alter ductal elongation, number of terminal end buds (TEB) or cell proliferation, and estrogen receptor alpha (ER-α) immunostaining expression in epithelial mammary cells at PND 21. BPA treatment modulated the expression of several genes, but these changes were not associated with a dose dependent response. Dietary GEN and I3C treatment causally and consistent with the mammary gland structures outcomes. Besides, maternal BPA exposure associated with dietary GEN and I3C did not alter the susceptibility to the mammary cancer development in adulthood when the carcinogen was administered in a window of immature mammary gland development. Overall design: Pregnant Sprague-Dawley (SD) rats were treated with BPA at 25 or 250µg/kg b.w./day by gavage from gestational day (GD) 10 to 21 with or without dietary GEN (250 mg/kg chow, ~5.5 mg/kg b.w./day) or I3C (2000 mg/kg chow, ~45.0 mg/kg b.w./day). At post-natal day (PND) 21, some female offspring from different litters were euthanized for mammary gland development and gene expression analyses while other female offspring received a single dose of N-methyl-N-nitrosourea (MNU) for mammary carcinogenesis initiation.
Project description:Dibenzo[a,h]anthracene DB[a,h]A is a polycyclic aromatic hydrocarbon potent carcinogen. Few studies have investigated the role of DB[a,h]A on mRNA and miRNA expression. In this study a 10-week old male MutaTM Mouse were exposed to 6.25, 12.5, and 25 mg/kg/day DB[a,h]A by oral gavage for 28 consecutive days. DNA adducts were detected in the livers at each DB[a,h]A dose tested, and a dose-dependent increase in lacZ mutants was observed in the same samples. MAANOVA analysis revealed minor changes in the mRNA expression for the two lowest doses. Differential expression of 19 up-regulated and 22 down-regulated transcripts with fold-change > 1.5 (FDR-adjusted P < 0.05) were identified in the 6.25 mg/kg/day DB[a,h]A treatment group. For the 12.5 mg/kg/day treatment group 13 transcripts were up-regulated and 32 down-regulated (FDR-adjusted P < 0.05 and fold-change > 1.5). Major effect on mRNA expression resulted from exposure to the highest dose (25 mg/kg/day) of DB[a,h]A with 135 up-regulated and 104 down-regulated genes with fold-change > 1.5 (FDR-adjusted P < 0.05). The significantly regulated genes are involved in circadian rhythm, drug metabolism, glucose metabolism, cholestrol and lipid metabolism, immune response, cell cycle, and apoptosis. We also investigated miRNA response to the three doses of DB[a,h]A. MiRNA expression was relatively unaffected. Only miR-34a showed significant (FDR-adjusted P < 0.05) up-regulation with a fold change above 1.3-fold. RNA samples from 5 control and 4-5 mice per treatment group (6.25mg/kg, 12.50mg/kg, and 25mg/kg) containing 100 ng were labelled using Agilent’s miRNA complete labelling and Hyb Kit (Agilent Tech, Mississauga, ON, Canada).
Project description:We report here hippocampal RNA-seq data from adult (20 weeks) male offspring exposed to 0 mg (control) or 50 mg BPA/kg diet during gestation and lactation. Overall design: RNA-seq in adult hippocampus from control and BPA-exposed males, four independent biological replicates per treatment group, sequenced on Illumina NextSeq 500 platform.
Project description:Furan is a widely used industrial chemical and a common contaminant in heated foods. Chronic furan exposure has been shown to cause cholangiocarcinoma and hepatocellular tumors at doses of 2 mg/kg bw/day with gender differences in frequency and severity. The hepatic transcriptional alterations induced by low doses of furan (doses below those inducing liver tumors) and the potential mechanisms underlying gender differences are largely unexplored. We used DNA microarrays to examine the global hepatic mRNA and microRNA transcriptional profiles of male and female rats exposed to 0, 0.03, 0.12, 0.5 or 2 mg/kg bw/day furan over 90 days. Marked gender differences in gene expression responses to furan were observed, with many more altered genes in exposed males than females, confirming the increased sensitivity of males even at the low doses. Pathway analysis supported that key events in furan-induced hepatotoxicity in males included gene expression changes related to oxidative stress, apoptosis and inflammatory response, while pathway changes in females were consistent with primarily adaptive responses (regeneration). Pathway benchmark doses (BMDs) were estimated and compared to relevant apical endpoints. Transcriptional BMDs could be examined in males, they ranged from 0.08 – 1.43 mg/kg bw/day and approximated those derived from traditional histopathology. MiR-34a (a target of P53 signalling) was the only microRNA significantly increased at the 2 mg/kg bw/day, providing evidence in support of the importance of apoptosis and cell proliferation in furan hepatotoxicity. Overall, this study demonstrates the use of transcriptional profiling to discern mode of action and mechanisms involved in gender differences. Total RNAs from 16 liver samples (4 males and 4 females from control and 2 mg/kg bw/day dose groups) were processed using the miRNA Complete Labelling and Hybridization kit (Agilent Technologies Inc.). Labelled RNA was hybridized on 8 X 15K Agilent rat miRNA microarray slides. Arrays were scanned using an Agilent G2505B scanner (5 μM resolution). Feature extraction (version 10.7.3.1, Agilent Tech. Inc.) was used to acquire the fluorescence intensity of each probe.
Project description:We treated gestating female mice with vinclozolin (VZ), bisphenol A (BPA), di-(2-ethylhexyl) phthalate (DEHP), or control oil, during the time when the prospermatogonia of the exposed fetus undergo global de novo DNA methylation. Using genome-wide assays we detected changes in transcription and DNA methylation, respectively, in fetal prospermatogonia. Our results suggest that EDs exert direct epigenetic effects in the exposed fetal germ cells, but the germline corrects against deleterious effects in the subsequent generation. Pregnant mice were administered endocrine distruptors (VZ at 100 mg/kg/day, DEHP at 750 mg/kg/day, and BPA at 0.2 mg/kg/day) startin g at 12.5 days post coitum (dpc) and tissue samples were collected at 17.5 dpc.