Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Whole transcriptomic analysis of mouse embryonic fibroblasts BALB/c 3T3 clone A31-1-1 after treatment with bis (2-ethylhexyl) phthalate for 24 hrs

ABSTRACT: Di(2-ethylhexyl) phthalate (DEHP; CAS No. 117-81-7) belongs to the phthalate class of chemicals, and is commonly added to plastics for flexibility. DEHP has been identified as an index compound for group-TDI calculations owing to its extensive toxicological dataset. Humans are exposed to this ubiquitous environmental contaminant through multiple routes. DEHP has been designated as probably and possibly carcinogenic to humans based on its ability to induce rodent carcinogenicity, although the relevance of its mode of action (MoA) in humans remains unclear. The aim of this study was to investigate the carcinogenic potential of DEHP using an alternative method and explore the possible mode and mechanisms of action at the molecular level. Special attention has been paid to DEHP dissolution in cell media, leading to the use of a final concentration of 0.5% DMSO. Transcriptomics were conducted on cells treated with a cytotoxic concentration of DEHP (19.7 µg/mL) for 24 h. The aim of the microarray experiment was to analyze the molecular effects of the substance under the specific conditions of the Cell Transformation Assay protocol (BALB c/ 3T3 clone A31-1-1 CTA, according to the method validated by ECVAM, Sasaki et al., 2012), in order to provide mechanistic explanations of the test results derived from the CTA.

INSTRUMENT(S): Agilent SureScan Microarray Scanner G2600D

ORGANISM(S): Mus musculus

SUBMITTER: Gelsomina Pillo

PROVIDER: E-MTAB-13716 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This analysis utilised the \"Transformics Assay\" and evaluated the effect of cell exposure to 0.02 μg/ml of Benzo(a)pyrene, the lowest transforming concentration, using the BALB / c 3T3 A31-1-1 cell line. The Transformic assay is an integrated approach combining the Cell Transformation Assay (CTA), with the high throughput technology of transcriptomics. The CTA is a widely used in vitro assay that has been designed to evaluate the effects of chemicals on the growth of specific cell types and their potential progression through a transformation process from being normal cells to fully malignant cells. It has the potential to detect both genotoxic and some non-genotoxic carcinogens and shows a good correlation with rodent bioassay data. However lack of understanding of the mechanistic basis of the assays, and particularly of the mechanisms leading to cell malignancy, has limited its acceptance for regulatory purposes. The Transformics Assay introduces the RNA extraction and microarray experiment performed at specific time points along the process of in vitro transformation, helping to identify mechanisms of action leading to chemical-induced transformation. The experiment was conducted testing the carcinogen Benzo(a)pyrene (CAS No.50-32-8), which is a polycyclic aromatic hydrocarbon (PHA) and classified as carcinogenic to humans (Group 1) by IARC. Particularly Benzo(a)pyrene is a prototypical member of this class of chemicals and its toxic effects has been extensively studied, both in laboratory animals and human populations. Benzo(a)pyrene and others PAHs-induced carcinogenesis is a multi-step process, which since the last decade, was considered to be initiated and substantially sustained by the reaction and binding to DNA (oncogenic targets). Indeed, as other PHAs, Benzo(a)pyrene is metabolically activated to a series of reactive intermediates by CYP450 and related enzymes under control of the aryl-hydrocarbon receptor (AhR). These events support the genotoxic mode of carcinogenic action. Nevertheless it is now widely recognized that Benzo(a)pyrene can act via non-genotoxic modes of action too, including epigenetic mechanisms involving the AhR signaling pathway, oxidative stress, disruption of mitogenic and other cellular signaling. This study aimed to explore the molecular and cellular events necessary to sustain the transforming activity of Benzo(a)pyrene, at the lowest in vitro concentration capable of sustaining the process of oncotransformation. In order to find the lowest transforming concentration, a preliminary CTA has been conducted according to the EURL-ECVAM (EU Reference Laboratory European Centre for the Validation of Alternative Methods) validated protocol. The exposure time recommended by EURL-ECVAM, 72 hrs, represents both the exposure time of cells and the RNA extraction time point. According to previous studies 72 hrs exposure could be a critical timing to study the cell transformation process, during which differences in adverse and adaptive cell responses began to become visible at the transcriptional level.

Project description:BACKGROUND: Phthalates are manmade industrial additives used mostly as plasticizers. In addition to their deleterious effects on male genital development, population studies have recently documented correlations between phthalates exposure and subtle impacts on reproductive tract development and on the metabolic syndrome in male adults. In mature rodents liver di-(2-ethylhexyl)-phthalate (DEHP) activates the peroxisome proliferators-activated receptor (PPARalpha), a member of the nuclear receptor (NR) superfamily. OBJECTIVES: Using a systems biology approach, we aimed at defining potential mechanisms underlying the impacts of DEHP on adult mouse liver and testis. METHODS: Thus, we performed a parallel analysis of transcript and metabolic profiles in the liver from adult mice exposed to varying DEHP doses. Moreover, we obtained pangenomic mRNA profiles of laser-captured Leydig and Sertoli cells from mature animals exposed to DEHP. RESULTS: Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of some prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways. Integration of hepatic transcriptomic and metabonomic profiles further revealed a correlation between the impacts of DEHP on a cluster of genes and metabolites linked to heme synthesis and on a cluster of Rev-erbalpha target genes related to metabolic and circadian clock pathways. Cell-specific effects of DEHP were investigated in the adult testis and a noticeable impact of DEHP was observed on Leydig cells transcriptome. CONCLUSIONS: We report a detailed analysis of DEHP interference with hepatic Constitutive Androstane Receptor (CAR) and Rev-erbalpha pathways and a novel transcriptional impact of DEHP on adult endocrine cells of the testis. Experiment Overall Design: two condition experiment, Sertoli cells-rich areas from DEHP-treated mice vs. Sertoli cells-rich areas from vehicle-treated mice. Biological replicates: 4 DEHP-treated samples and 4 vehicle-treated samples. Dye-swap design. N=4 DEHP-treated vs vehicle-treated x 2 microarrays (dye-swap)=8 microarrays

Project description:BACKGROUND: Phthalates are manmade industrial additives used mostly as plasticizers. In addition to their deleterious effects on male genital development, population studies have recently documented correlations between phthalates exposure and subtle impacts on reproductive tract development and on the metabolic syndrome in male adults. In mature rodents liver di-(2-ethylhexyl)-phthalate (DEHP) activates the peroxisome proliferators-activated receptor (PPARalpha), a member of the nuclear receptor (NR) superfamily. OBJECTIVES: Using a systems biology approach, we aimed at defining potential mechanisms underlying the impacts of DEHP on adult mouse liver and testis. METHODS: Thus, we performed a parallel analysis of transcript and metabolic profiles in the liver from adult mice exposed to varying DEHP doses. Moreover, we obtained pangenomic mRNA profiles of laser-captured Leydig and Sertoli cells from mature animals exposed to DEHP. RESULTS: Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of some prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways. Integration of hepatic transcriptomic and metabonomic profiles further revealed a correlation between the impacts of DEHP on a cluster of genes and metabolites linked to heme synthesis and on a cluster of Rev-erbalpha target genes related to metabolic and circadian clock pathways. Cell-specific effects of DEHP were investigated in the adult testis and a noticeable impact of DEHP was observed on Leydig cells transcriptome. CONCLUSIONS: We report a detailed analysis of DEHP interference with hepatic Constitutive Androstane Receptor (CAR) and Rev-erbalpha pathways and a novel transcriptional impact of DEHP on adult endocrine cells of the testis. Experiment Overall Design: two condition experiment, Leydig cells from DEHP-treated mice vs. Leydig cells from vehicle-treated mice. Biological replicates: 2 DEHP-treated samples and 3 vehicle-treated samples. Each treated sample has been hybridized against each vehicle-treated sample in a dye-swap design. N=2 DEHP-treated x 3 vehicle-treated x 2 microarrays=12 microarrays

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Whole transcriptomic analysis of mouse embryonic fibroblasts BALB/c 3T3 clone A31-1-1 after treatment with bis (2-ethylhexyl) phthalate for 24 hrs

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