Project description: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.

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:Microarray analyses were used to evaluate patterns of gene transcription following exposure of the waterflea Daphnia magna to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed the water flea Daphnia magna to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Most gene fragments were downregulated under stress (82.4% downregulation compared to 17.6% upregulation) irrespective of the treatment. In approximately 5% of the cases up- or downregulation depended on stressor identity. Based on a PCA, we could identify two different groups within our exposure treatments: small and big gene expression differences compared to the control condition. The treatments parasite 96h exposure, carbaryl 144h of exposure and fish 144h of exposure are combined in the high stress group. 26 samples x 2 biological replicates were analysed in a carriage wheel design (Khanin & Wit, 2005; Knapen et al., 2009) with a reference sample composed of a clutch of non-exposed D. magna that were collected at different time points ranging from 0-24h until three weeks old. The 26 samples included: 4 treatments (control, carbaryl, fish and parasite exposure) x three time points (48h, 96h and 144h) x 2 replicates + 2 start samples (non-exposed individuals younger than 24h old). This led to 52 different microarray hybridizations: 26 hybridizations for the inner part of the wheel, where every sample is hybridized against the reference, and another 26 for the outer part. Color flip experiments were incorporated per sample to fulfill the conditions of technical replicates on the level of labelling; every stressor-time combination was included at least once in red and once in green. One hybridization in the outer part of the wheel had technical problems, and is not included here.

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:Genome-wide transcriptional profiling of Arabidopsis thaliana to a combination of heatwave and drought under ambient and elevated CO2. Goal of this study was elucidate the transcriptional responses to a combination of heat wave and drought, and to see how these responses are modifed under future climate (high) CO2. Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level-analysis (i.e. eco-physiological, biochemical and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under elevated CO2.The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2O2 production and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect. Transcriptome analysis was performed by Agilent Arabidopsis (V4) 4x44K platform which represented all known genes in the Arabidopsisgenome. Experiments were performed using a modified loop design (Knapen et al., 2009). This design consisted of total 8 arrays; sample from each treatment was labelled once and has 4 biological replicates, two of which were labelled in red and two in green

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