Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene Expression Analysis of Human Endometrial Endothelial Cells Exposed to Bisphenol A

ABSTRACT: 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 ÂµM 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Matts Olovsson

PROVIDER: E-GEOD-10802 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene expression analysis of human endometrial endothelial cells exposed to Bisphenol A.

Bredhult Carolina C Sahlin Lena L Olovsson Matts M

Reproductive toxicology (Elmsford, N.Y.) 20090325 1

Bisphenol A (BPA) can affect reproductive tissues in several species. Recently, treatment of human endometrial endothelial cells (HEECs) with 100 microM BPA decreased their proliferation and viability. In the present study, 50 microM BPA decreased HEEC proliferation, and microarray analyses of five HEEC cultures revealed that BPA affected biological processes associated with proliferation. Expression of three of the most differentially expressed genes identified in the gene array analysis, SPBC2 ...[more]

PMID: 19490990

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Project description:The endocrine disrupting chemical o,p'-DDT can affect reproductive organs, tissues and cells in several species. Treatment of human endometrial endothelial cells (HEEC) with 50 µM o,p'-DDT decreased the cell proliferation compared to the control. Microarray analyses revealed that o,p'-DDT affected biological processes such as the cell cycle, cell division, defence response and lipid and steroid metabolism, in cellular components such as the plasma membrane and chromosomes, with molecular functions involved in signalling, receptor and cytokine activity, confirming the results of the proliferation assay. The present study supports our previous findings of decreased proliferation and increased cell death in response to o,p'-DDT, and may offer important clues in the deduction of the mechanisms of action of o,p'-DDT. Keywords: Exposure analysis Two-condition experiments, o,p'-DDT-exposed vs. control HEEC, and individual control vs control pool. Biological replicates: 5 HEEC cultures, independently grown, exposed and harvested. One replicate per array. Dye-swaps for exposed and reference samples between replicates. variable = Exposure: o,p'-DDT-treated: Indiv1 s1 op-DDT Cy3, Indiv2 s19 op-DDT Cy3, Indiv3 s37 op-DDT Cy3, Indiv4 s55 op-DDT Cy5, Indiv5 s73 op-DDT Cy5 variable = Exposure: control: Indiv1 s2 control Cy5, Indiv2 s20 control Cy5, Indiv3 s38 control Cy5, Indiv4 s56 control Cy3, Indiv5 s74 control Cy3 variable = Exposure: individual control: Indiv1 s17 control Cy3, Indiv2 s35 control Cy5, Indiv3 s53 control Cy3, Indiv4 s71 control Cy5, Indiv5 s89 control Cy3 variable = Exposure: control pool: Pooled control s18/36/54/72/90 Cy5, Pooled control s18/36/54/72/90 Cy3, Pooled control s18/36/54/72/90 Cy5, Pooled control s18/36/54/72/90 Cy3, Pooled control s18/36/54/72/90 Cy5 repeat = Biological replicate: Indiv1 s1 op-DDT Cy3, Indiv1 s2 control Cy5, Indiv1 s17 control Cy3, sample 18 in Pooled control repeat = Biological replicate: Indiv2 s19 op-DDT Cy3, Indiv2 s20 control Cy5, Indiv2 s35 control Cy5, sample 36 in Pooled control repeat = Biological replicate: Indiv3 s37 op-DDT Cy3, Indiv3 s38 control Cy5, Indiv3 s53 control Cy3, sample 54 in Pooled control repeat = Biological replicate: Indiv4 s55 op-DDT Cy5, Indiv4 s56 control Cy3, Indiv4 s71 control Cy5, sample 72 in Pooled control repeat = Biological replicate: Indiv5 s73 op-DDT Cy5, Indiv5 s74 control Cy3, Indiv5 s89 control Cy3, sample 90 in Pooled control

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: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).

Dataset Information

Gene Expression Analysis of Human Endometrial Endothelial Cells Exposed to Bisphenol A

Publications

Gene expression analysis of human endometrial endothelial cells exposed to Bisphenol A.

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