Unknown,Transcriptomics,Genomics,Proteomics

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Toxicogenomics in the 3T3-L1 cell line, a new approach for screening of obesogenic compounds

ABSTRACT: The obesogen hypothesis states that exposure to environmental compounds early in life or throughout lifetime might have an influence on obesity development. In this paper a new approach for obesogen screening is proposed, namely the use of transcriptomics in the 3T3-L1 pre-adipocyte cell line. Based on the data from a previous study of our group using a lipid accumulation based adipocyte differentiation assay, several humanly relevant obesogenic compounds were selected: reference obesogens [Rosiglitazone (ROSI), Tributyltin (TBT)], test obesogens [Butylbenzyl phthalate (BBP), butylparaben (BP), propylparaben (PP), Bisphenol A (BPA)] and non-obesogens [Ethylene Brassylate (EB), Bis (2-ethylhexyl)phthalate (DEHP)]. In this paper, the high stability and reproducibility of the 3T3-L1 gene transcription patterns over different experiments and cell batches was shown. Obesogens and non-obesogen gene transcription profiles were clearly distinguished using hierarchical clustering. Furthermore, a gradual distinction corresponding to differences in induction of lipid accumulation could be made between test and reference obesogens based on transcription patterns, indicating the potential use of this strategy for classification of obesogens. Marker genes that are able to distinguish between non, test and reference obesogens were identified. Well known genes involved in adipocyte differentiation, as well as genes with unknown functions were selected, implying a potential adipocyte related function of the latter. Cell physiological lipid accumulation was well predicted based on transcription levels of the marker genes, indicating the biological relevance of omics data. In conclusion, this study shows the high relevance and reproducibility of this 3T3-L1 based in vitro toxicogenomics tool for classification of obesogens and biomarker discovery. The hybridization design was a reference design (reference sample was a mixture of equal aliquots from control and exposed samples), recommended for class discovery purposes (Knapen et al. 2009). The reference sample was labeled with Cy5, and the exposed samples with Cy3. Three biological replicates were measured for every condition.

ORGANISM(S): Mus musculus

SUBMITTER: Anna Pereira-Fernandes

PROVIDER: E-GEOD-53004 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Obesogenic compounds are chemicals that might have an influence on obesity development through in utero or chronic lifetime exposure. Tributyltin (TBT) is one of the most studied obesogenic compounds, inducing adipogenesis in vitro and increasing body fat after in utero exposure of mice. This study was designed to unravel the molecular mechanisms of TBT, using microarray analysis, and to evaluate the use of toxicogenomics for obesogen screening. The murine 3T3-L1 cell line was used to study TBT induced adipogenesis. Lipid staining as well as gene expression measurements of an adipocyte specific marker gene were performed to select relevant concentrations (10 nM, 50 nM) and time points (day1, day10) for microarray analysis. Additionally, solvent control and positive control (MDI) treated 3T3-L1 cells were included in the analysis. The microarray results were analysed using GO enrichment and Pathway analysis tools, which revealed enrichment of several GO terms involved in energy and fat metabolism after 10 days of TBT exposure. Pathway analysis unveiled PPAR signalling pathway as the sole pathway significantly enriched after 1 day and the most significantly enriched pathway after 10 days of exposure. By examination of effects on both cell physiological and gene expression level we provide a detailed overview of TBT induced obesogenic mechanisms. To our knowledge, this is the first study delivering an in depth mechanistic outline of the mode of action of TBT as an obesogen. Furthermore, our results show that combining omics with 3T3-L1 cells is promising for screening of potential obesogenic compounds. A separate v+2 A-optimal hybridization design was used for each time-point. Using this design each exposure condition is represented by three biological replicates, with each biological replicate analysed in technical duplicate while applying the dye swap principle to correct for dye bias. Two concentrations of TBT (10 nM, 50 nM), DMSO and MDI (positive control, adipogenic hormone cocktail) were the conditions tested, at two time-points (day 1 and day10).

Project description:Transcriptional profiling of insulin exposed H4IIE cells at different time points (6h-24h) to 100 nM insulin, with untreated solvent control cells (blanks). The central role of hepatic insulin resistance in pandemic metabolic diseases urges investigation of the underlying mechanisms of its development and pathogenesis. Besides genetic susceptibility and lifestyle factors, environmental pollutants are suggested risk factors. In order to appraise the role of the thousands of pollutants we are daily exposed to, stable, robust and high throughput cell-based systems should be developed. In this context, it is of paramount importance to select an in vitro system which mimics in vivo insulin responses as close as possible. Therefore, this study was designed to evaluate if the rat H4IIE hepatoma cell line is a physiologically relevant model to study hepatic insulin responses. DNA microarray analysis, real-time PCR and flow cytometric cell cycle analysis were used to assess the relevance of the insulin response in H4IIE cells. Insulin dose dependently stimulated H4IIE growth. Time dependency of the insulin response was shown with real-time PCR for the known insulin responsive genes: Fasn, Pck1 and Irs2. Based on these results, microarray analysis was performed on H4IIE cells exposed to insulin (100 nM) for 6h and 24h. Genes related to carbohydrate (glycolysis and gluconeogenesis) and lipid metabolism (glycerolipid metabolism, cholesterol synthesis and fatty acid oxidation) were most profoundly afflicted, in accordance with in vivo insulin action in liver. Since changes in carbohydrate and lipid metabolism are pivotal in the pathogenesis of insulin resistance, the presence of a physiological relevant insulin response in H4IIE cells pleads for further testing of its potential use in research on pollutant-driven insulin resistance. Microarray analysis was performed on H4IIE cells exposed to insulin (100 nM) for 6h and 24h. A separate v+2 A-optimal hybridization design was used for each time-point. Using this design each exposure condition is represented by three biological replicates, with each biological replicate analysed in technical duplicate while applying the dye swap principle to correct for dye bias.

Project description:Enhancers are developmentally-controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. Here, we show by genome-wide mapping that the newly discovered DNA modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells as well as during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates like Meis1 in P19 cells and PPARgamma in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5mC hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes Genome-wide 5hmC distribution was determined using hMeDIP-seq. Cells used in this study are P19.6 mouse embryonal carnicoma cells and P19.6 cells treated for 48 hours with 1M-BM-5M all-trans retinoic acid (RA), as well as 3T3-L1 cells and 3T3-L1 derived adipocytes differentiated with dexamethasone, insulin and IBMX (differentiation cocktail - DC). Individual hMeDIP samples from P19.6 ord 3T3-L1 cells were pooled for library preparation. Libraries were prepared and sequenced at the IBL sequencing facility (Lille, France) with an Illumina Genome Analyser II.

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Toxicogenomics in the 3T3-L1 cell line, a new approach for screening of obesogenic compounds

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