Expression Analysis of Bisphenol A and 17β-estradiol Exposure in Embryonic Zebrafish
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ABSTRACT: Investigation of whole genome expression pattern of 24 hours post fertilization Danio rerio embryos exposed to bisphenol A, 17β-estradiol, or 0.1% DMSO vehicle control This data represents 2 experiments using 2 separate microarrays. Microarray 1 [Low]: Embryos were exposed to 0.1 µM BPA, 0.1 µM E2, or control from 8 hpf to 24 hpf. Three biological replicates were collected for each treatment. 40 embryos were pooled to comprise a replicate. Microarray 2 [High]: Embryos were exposed to 80 µM BPA, 15 µM E2, or control from 8 hpf to 24 hpf. Two biological replicates were collected for each treatment. 40 embryos were pooled to comprise a replicate. These 2 microarray experiments are cross-comparable, as described in the associated publication.
Project description:Bisphenol A (BPA) is a xenobiotic endocrine disrupting chemical. In vitro and in vivo studies indicated that BPA alters endocrine-metabolic pathways in adipose tissue increasing the risk of developing metabolic disorders. BPA effects on human adipocytes, specifically in children, are poorly investigated. To investigate in childhood the effect of exposure to BPA on metabolic disorders we analyzed in vitro the effects of environmentally relevant doses of BPA on gene expression of mature human adipocytes from pre-pubertal lean patients and on related physiological outcomes. Adipocytes from children were treated in vitro with BPA and gene expression was evaluated by qRT-PCR. Genome wide analyses were performed using GeneChip Human Gene 1.0 ST array. Lipid content in adipocytes was estimated by ORO staining and Triglyceride Quantification Kit. Secreted IL-1beta, in adipocytes culture medium, and insulin, in PANC-1 culture medium, were performed using ELISA assays. BPA was found to promote up-regulation of ER? and ERR?, and down-regulation of GPR30 expression modulating estrogen signaling and following a non-linear dose-response. Microarray data analysis demonstrated that BPA increases the gene expression of pro-inflammatory cytokines and lipid metabolism-related FABP4 and CD36 in adipocytes. PCSK1 resulted the most interesting gene being down-regulated by BPA thus impairing insulin production in pancreas. BPA promotes inflammation and lipid metabolism dysregulation in adipocytes from lean children. Moreover, PCSK1 can be a key gene in BPA action modulating insulin production. Exposure to BPA in childhood may be an important risk factor in developing obesity and metabolic disorders. Three prototypic situations were analyzed (5 replication each): untreated cells (C), 10 nM BPA treated cells (BPA), 1 nM 17-beta Estradiol treated cells (ES).
Project description:Bisphenol A (BPA) is an environmental endocrine disruptor which has been detected in almost all human bodies. Many studies have implied that BPA exposure is harmful to human health. Previous studies mainly focused on BPA effects on ER-positive cells. Genome-wide impact of BPA on gene regulation in ER-negative cells is unclear. In the present study, we performed RNA-seq to characterize BPA-induced gene regulation on ER-negative HEK 293 cells.
Project description:The endocrine disrupting chemical bisphenol A (BPA) can affect reproductive organs, tissues and cells in several species. Treatment of human endometrial endothelial cells (HEECs) with 50 µM BPA decreased their proliferation compared with the control. Microarray analyses revealed that BPA affected biological processes such as the cell cycle, cell division, and cytoskeleton organization, confirming the results of the proliferation assay. Expression of three of the most differentially expressed genes identified in the microarray analysis was verified by real-time quantitative reverse transcription polymerase chain reaction in five HEEC cultures obtained from women in the proliferative phase and in five cultures obtained from women in the secretory phase of the menstrual cycle after treatment with BPA. The present study supports our previous findings of decreased proliferation and increased cell death in response to BPA, and may offer important clues to the mechanisms of action of BPA. Keywords: Exposure analysis Two-condition experiments, BPA-exposed vs. control HEEC. Biological replicates: 5 HEEC cultures, independently grown, exposed and harvested. One replicate per array. Dye-swaps for exposed and reference samples between replicates.
Project description:Embryonic period is sensitive window of bisphenol A (BPA) exposure. However, embryonic development is a highly dynamic process with changing cell populations and gene expression profiles. Heterogeneity effects of BPA on fish embryonic development remain not clear. This study applied single-cell RNA sequencing to analyze the impact of BPA exposure on transcriptome heterogeneity of 64683 cells from zebrafish embryos at 8, 12 and 30 hpf. A total of 38 cell populations were identified, and gene expression profiles of 16 cell populations were significantly altered by BPA exposure. The strongest toxic effects of BPA were found at 12 hpf of segmentation stage, which is an active stage of cell differentiation. At 8 hpf, BPA mainly influenced the outer layer cell populations of embryos, such as neural plate border and enveloping layer cells. At 12 and 30 hpf, nervous system formation and heart morphogenesis were disturbed. Differential process of neural plate border, neural crest, and neuron cells was altered, leading to increased neurogenesis. For the forebrain, midbrain, neurons, and optic cells, the altered cell division and DNA replication and repair were identified. Our study for the first time provides the comprehensive understanding of BPA toxicity on fish embryo development at single-cell levels.
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 M-BM-5g 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 M-BM-5g 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:Purpose: To identify differential expressed genes for loss of function of Mycn in zebrafish Methods: wild-type and mycn mutant zebrafish embryos were collected for RNA sequencing at 48 hpf (hours post fertilisation) and 72 hpf Results: We found 85 (overlapped) down-regulated genes in mycn mutant embryos at 48 hpf and 72 hpf (padj < 0.1, log2 fold change < 0) Conclusions: 85 down-regulated genes were identified
Project description:Xenoestrogens are part of a group of agents termed endocrine disruptors because of their capacity to perturb normal hormonal actions. It has been suggested that xenoestrogens may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. Bisphenol A (BPA) is polymerized to manufacture polycarbonate plastic and epoxy resins. Human exposure occurs when BPA leaches from plastic-lined food and beverage cans. In the present work we are aiming to determine if BPA has carcinogenic properties by using an in vitro system. For this purpose the human breast epithelial cells MCF-10F were treated with 10-3M to 10-9M BPA continuously for two weeks. The MCF-10F cells treated with 10-3M and 10-4M BPA died, indicating that these concentrations were toxic for the human breast epithelial cells. The cells treated with 10-5M to 10-9M BPA were evaluated for formation of solid masses in collagen and invasion capacity, both phenotypes that are indicators of cell transformation. MCF-10F treated with 10-5M, 10-6M, 10-7M, 10-8M and 10-9M BPA formed a high percentage of solid masses (34.6%, 20%, 42.4%, 31.8% and 32.2%, respectively). Ten passages after BPA treatments, the invasive capacities of the cells were evaluated using Boyden chambers. The invasion capacity was lower in the cells treated at high concentrations of BPA (10-5M and 10-6M), and there was an increased invasion for the cells treated at low BPA concentration (10-9M) although, in all the cases the differences were not significant to the controls. Expression and DNA methylation analyses were performed with the cells treated with 10-5M and 10-6M BPA. We found that these cells showed an increased expression of BRCA1, BARD1, CtIP, RAD51 and BRCC3, all genes involved in DNA repair, and downregulation of PDCD5 and BCL2L11 (also known as BIM), both involved in apoptosis. The upregulation of CtIP was related to hypomethylation of the promoter/exon1 of this gene. Furthermore, BPA induced silencing of BCL2L11 by hypermethylation. This is the first demonstration that BPA induces neoplastic transformation of breast epithelial cells and that aberrant DNA methylation of genes involved in DNA repair and apoptosis could be involved in the initiation of the neoplastic process. MCF-10F, a normal-like human breast epithelial cell (HBEC) line that is ERalpha-negative PR-negative, was maintained in high-calcium medium at 37°C and 5% CO2 until confluent. These cells were treated with different concentrations of BPA continuously for two weeks, adding fresh media every day. As controls, MCF-10F cells were not treated and maintained in the regular media or treated with 0.284% DMSO (vehicle). The cells treated with 10-5M or 10-6M BPA after the invasion assay were collected and expanded during 6 passages. RNA was isolated from the cells using an RNA extraction kit (Qiagen).
Project description:Xenoestrogens are part of a group of agents termed endocrine disruptors because of their capacity to perturb normal hormonal actions.It has been suggested that xenoestrogens may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. Bisphenol A (BPA) is polymerized to manufacture polycarbonate plastic and epoxy resins. Human exposure occurs when BPA leaches from plastic-lined food and beverage cans. In the present work we are aiming to determine if BPA has carcinogenic properties by using an in vitro system. For this purpose the human breast epithelial cells MCF-10F were treated with 10-3M to 10-9M BPA continuously for two weeks. The MCF-10F cells treated with 10-3M and 10-4M BPA died indicating that these concentrations were toxic for the human breast epithelial cells. The cells treated with 10-5M to 10-9M BPA were evaluated for formation of solid masses in collagen and invasion capacity, both phenotypes that are indicators of cell transformation. MCF-10F treated with 10-5M, 10-6M, 10-7M, 10-8M and 10-9M BPA formed a high percentage of solid masses (34.6%, 20%, 42.4%, 31.8% and 32.2%, respectively). Ten passages after BPA treatments, the invasive capacities of the cells were evaluated using Boyden chambers. The invasion capacity was lower in the cells treated at high concentrations of BPA (10-5M and 10-6M) and, there was an increased invasion for the cells treated at low BPA concentration (10-9M) although, in all the cases the differences were not significant to the controls. Expression and DNA methylation analysis were performed with the cells treated with 10-5M and 10-6M BPA. We found that these cells showed an increased expression of BRCA1, BARD1, CtIP, RAD51 and BRCC3, all genes involved in DNA repair, and downregulation of PDCD5 and BCL2L11 (also known as BIM), both involved in apoptosis. The upregulation of CtIP was related to hypomethylation of the promoter/exon1 of this gene. Furthermore, BPA induced silencing of BCL2L11 by hypermethylation. This is the first demonstration that BPA induces neoplastic transformation of breast epithelial cells and that aberrant DNA methylation of genes involved in DNA repair and apoptosis could be involved in the initiation of the neoplastic process. MCF-10F, a normal-like Homo sapiens breast epithelial cell (HBEC) line that is ERa-negative PR-negative was maintained in high calcium medium at 37°C and 5% CO2 until confluent. These cells were treated with different concentrations of BPA continuously for two weeks, adding fresh media every day. As controls, MCF-10F cells were not treated and maintained in the regular media or treated with 0.284% DMSO (vehicle). The cells treated with 10-5M or 10-6M BPA after the invasion assay were collected and expanded during 6 passages. RNA was isolated from the cells using RNA extraction kit (Quiagen).
Project description:Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to identify estrogen regulated genes during the first 4 days of development. Zebrafish embryos were exposed to 1 M-BM-5M 17M-NM-2-estradiol from 3 hours post fertilization to 4 days post fertilization, harvested daily and subjected to RNA extraction for transcriptome analysis using microarrays. Estrogen responsive genes were analyzed with hierarchical clustering followed by gene function and tissue expression analysis. Markedly distinct sets of genes were up and down-regulated by estrogen treatment at different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by estrogen were similar throughout zebrafish development. Estrogen responsive genes were enriched mainly in the liver, pancreas and brain. In conclusion, our data shows that in zebrafish distinct cohorts of E2 responsive genes are expressed in a tissue specific manner at different developmental stages. However, the biological pathways that are affected are conserved. 30 embryos were pooled as one sample and exposed to 1 M-NM-<M E2 or vehicle (0.1% DMSO) at approximately 3 hours post fertilization (hpf). At different time points, 1 dpf (24 hpf), 2 dpf (48 hpf), 3 dpf (72 hpf) and 4 dpf (104 hpf), embryos were collected for total RNA extractions. Time points 1 and 2 dpf were performed in biological triplicates of independent pools of RNA while time points 3 and 4 dpf were performed in quadruplicates.
Project description:Xenoestrogens are part of a group of agents termed endocrine disruptors because of their capacity to perturb normal hormonal actions. It has been suggested that xenoestrogens may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. Bisphenol A (BPA) is polymerized to manufacture polycarbonate plastic and epoxy resins. Human exposure occurs when BPA leaches from plastic-lined food and beverage cans. In the present work we are aiming to determine if BPA has carcinogenic properties by using an in vitro system. For this purpose the human breast epithelial cells MCF-10F were treated with 10-3M to 10-9M BPA continuously for two weeks. The MCF-10F cells treated with 10-3M and 10-4M BPA died, indicating that these concentrations were toxic for the human breast epithelial cells. The cells treated with 10-5M to 10-9M BPA were evaluated for formation of solid masses in collagen and invasion capacity, both phenotypes that are indicators of cell transformation. MCF-10F treated with 10-5M, 10-6M, 10-7M, 10-8M and 10-9M BPA formed a high percentage of solid masses (34.6%, 20%, 42.4%, 31.8% and 32.2%, respectively). Ten passages after BPA treatments, the invasive capacities of the cells were evaluated using Boyden chambers. The invasion capacity was lower in the cells treated at high concentrations of BPA (10-5M and 10-6M), and there was an increased invasion for the cells treated at low BPA concentration (10-9M) although, in all the cases the differences were not significant to the controls. Expression and DNA methylation analyses were performed with the cells treated with 10-5M and 10-6M BPA. We found that these cells showed an increased expression of BRCA1, BARD1, CtIP, RAD51 and BRCC3, all genes involved in DNA repair, and downregulation of PDCD5 and BCL2L11 (also known as BIM), both involved in apoptosis. The upregulation of CtIP was related to hypomethylation of the promoter/exon1 of this gene. Furthermore, BPA induced silencing of BCL2L11 by hypermethylation. This is the first demonstration that BPA induces neoplastic transformation of breast epithelial cells and that aberrant DNA methylation of genes involved in DNA repair and apoptosis could be involved in the initiation of the neoplastic process. MCF-10F, a normal-like Homo sapiens breast epithelial cell (HBEC) line that is ERa-negative PR-negative, was maintained in high calcium medium at 37°C and 5% CO2 until confluent. These cells were treated with different concentrations of BPA continuously for two weeks, adding fresh media every day. As controls, MCF-10F cells were not treated and maintained in the regular media or treated with 0.284% DMSO (vehicle). The cells treated with 10-5M or 10-6M BPA after the invasion assay were collected and expanded during 6 passages. DNA was isolated from the cells using a DNA extraction kit (Qiagen).