Project description:There is still a lot of contradiction on whether metal ions are solely responsible for the observed the toxicity of ZnO and CuO nanoparticles to aquatic species. While most tests have studied nanoparticle effects at organismal levels (e.g. mortality, reproduction), effects at suborganismal levels may clarify the role of metal ions, nanoparticles and nanoparticle aggregates. In this study, the effect of ZnO, CuO nanoparticles and zinc, copper salts was tested on the gene expression levels in Daphnia magna. D. magna was exposed during 96 hours to 10% immobilization concentrations of all chemicals, after which daphnids were sampled for a differential gene expression analysis using microarray. When comparing the nanoparticle exposed daphnids (ZnO or CuO) to the metal salt exposed daphnids (zinc or copper salt), the microarray results showed no significantly differentially expressed genes. These results indicate that the toxicity of the tested ZnO and CuO nanoparticles to D. magna caused is solely caused by toxic metal ions. 4 replicate exposures of ZnO nanoparticles, ZnCl2, Blank (for Zn); 4 replicate exposures of CuO nanoparticles, CuCl2.2H2O, Blank (for Cu); Individual reference design with swapped dyes for zinc (e.g. ZnO-REFZn; REFZn-bl) and copper exposure (e.g. CuO-REFCu; REFCu-bl); Zinc reference sample is a mixture of equal aliquots of ZnO nanoparticle, ZnCl2 and blank; Copper reference sample is a mixture of equal aliquots of CuO nanoparticle, CuCl2.2H2O and blank
Project description:There is still a lot of contradiction on whether metal ions are solely responsible for the observed the toxicity of ZnO and CuO nanoparticles to aquatic species. While most tests have studied nanoparticle effects at organismal levels (e.g. mortality, reproduction), effects at suborganismal levels may clarify the role of metal ions, nanoparticles and nanoparticle aggregates. In this study, the effect of ZnO, CuO nanoparticles and zinc, copper salts was tested on the gene expression levels in Daphnia magna. D. magna was exposed during 96 hours to 10% immobilization concentrations of all chemicals, after which daphnids were sampled for a differential gene expression analysis using microarray. When comparing the nanoparticle exposed daphnids (ZnO or CuO) to the metal salt exposed daphnids (zinc or copper salt), the microarray results showed no significantly differentially expressed genes. These results indicate that the toxicity of the tested ZnO and CuO nanoparticles to D. magna caused is solely caused by toxic metal ions. 4 replicate exposures of ZnO nanoparticles, ZnCl2, Blank (for Zn); 4 replicate exposures of CuO nanoparticles, CuCl2.2H2O, Blank (for Cu); Individual reference design with swapped dyes for zinc (e.g. ZnO-REFZn; REFZn-bl) and copper exposure (e.g. CuO-REFCu; REFCu-bl); Zinc reference sample is a mixture of equal aliquots of ZnO nanoparticle, ZnCl2 and blank; Copper reference sample is a mixture of equal aliquots of CuO nanoparticle, CuCl2.2H2O and blank
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:We use the gowth zone of the maize leaf as a model system to study the growth reduction in response to drought stress. The spatial gradient and the relatively large size of the maize leaf allowed us to sample at a subzonal rezolution and to examine different developmental stages at the same time. We compared the response to different levels of drought stress (mild and severe) of proliferating (meristem), expanding (elongation zone) and differentiated (mature zone) tissue. Three separate loop designs were used for the three zones of the maize leaf (meristem, elongation zone, and mature zone). In each loop three treatments were contrasted (control, mild stress, and severe stress). Four biological replicates were used for each zone/condition (4 replicates x 3 zones x 3 conditions = 36 samples).
Project description:Changes in environmental temperature can profoundly change species habitats and result in populations facing suboptimal environments. Many aquatic organisms are restricted in terms of migration by their habitat requirements. Also due to anthropogenic migration barriers (both physical as well as chemical), organisms are often left with no choice but to acclimate (or, in the long run, adapt) to their changing environment. The scope of this study is to investigate thermal acclimation in zebrafish by combining data from several levels of biological organization. Zebrafish were acclimated to a higher temperature (8°C increase compared to controls) or a lower temperature (8°C decrease compared to controls) in an acute as well as a prolonged and a chronic scenario (4, 14 and 28 days). General condition of the fish was assessed by determining organismal (condition factor) and biochemical (energy homeostasis) parameters. Data at the transcriptome level (using printed oligonucleotide microarrays containing 15,208 probes and real time PCR) were applied to clarify the mechanisms underlying the thermal acclimation response in zebrafish. All three biological replicates of liver samples from acute (4 days) and chronic (28 days) controls (26°), warm acclimation (34°) and cold acclimation (18°) were analyzed using microarrays. An A-optimal interwoven loop design was used in which each sample appeared on an array twice in Cy3 and twice in Cy5, resulting in 36 arrays for 18 samples.
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
Project description:To study the mechanisms of Ni resistance in the metal resistant Acidiphilium sp. PM, the transcriptome of Acidiphilium sp. PM was studied 5min and 30 min after the addition of 10mM Ni and compared to the transcriptome in untreated cells. Two-condition hybridization experiments: untreated cells vs cells treated with 10mM Ni (for either 5 or 30 minutes). Eighteen 100ml-cultures: 6 untreated control cultures (not exposed to Ni), 6 cultures exposed to 10mM Ni for 5min, and 6 cultures exposed to 10mM Ni for 30 min. Cultures were independently grown and harvested. Gene expression levels of one untreated replicate were compared separately with one 5min-exposed replicate and with one 30min-exposed replicate (yielding two microarray data sets. One for each hybridization).