Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Effect of Cu-Nanoparticles versus Cu-salt in Enchytraeus albidus (Oligochaeta): differential gene expression through microarray analysis

ABSTRACT: Despite the increased utilization of nanoparticles, the behavior and effect in the environment is largely unknown and few resources are available for health and environmental effect studies. Enchytraeids are extensively used in studies of soil ecotoxicology and recently, a cDNA microarray for Enchytraeus albidus was developed, allowing also toxicogenomic studies in this species. These organisms are ecologically relevant small worms that indirectly contribute to the regulation and degradation of organic matter. In this study we compared the gene expression profiles of E. albidus when exposed to copper-salt (CuCl2) and copper nanoparticles (Cu-NP) spiked soil. The worms were exposed for 48 hours in soil to a range of concentrations. Microarray hybridizations revealed different response patterns between copper-salt and copper nanoparticles exposed organisms, these differences are mainly related with transcripts involved in the energy metabolism of the organisms. Despite unknown gene function in the data-set there are indications that Cu-salt and Cu-NP exposure induced specific gene level responses. Fluorescently labelled cDNA, from enchytraeids exposed during 2 days to control soil (from Hygum site in Denmark) and to the different exposure conditions (Cy5), was synthesized for microarray analysis and hybridizations were performed. After scanning, spots were identified and ratios quantified using the QuantArray (Packard Biochip Technologies). Statistical analysis of the microarrays was performed using limmaGUI package (1.18.0) (Smyth, 2005) in the R (2.8.0) software environment (http://www.R-project.org/). After being submitted to local background subtraction, microarrays were normalized using global loess method. To statistically evaluate the differential gene expression between the different conditions, a gene-per-gene linear model (limma – linear model for microarray analysis) and empirical Bayes methods were applied. The results were then corrected for multiple testing using the Benjamini-Hochberg’s method (adjusted p<0.05 was considered significant) (Benjamini and Hochberg, 1995).

ORGANISM(S): Enchytraeus albidus

SUBMITTER: Susana Gomes

PROVIDER: E-GEOD-26331 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The soil oligochaete Enchytraeus albidus is a standard test organism used in biological testing for Environmental Risk Assessment (ERA). Although effects are known at acute and chronic level, through survival, reproduction and avoidance behaviour endpoints, very little is known at the sub-cellular and molecular levels. In this study, the effects of soil properties (clay, organic matter and pH) and of the chemicals copper and phenmedipham were studied on gene expression in E. albidus during exposure periods of 2 days, 4 days and 3 weeks, using DNA microarrays based on a normalised cDNA library for this test species (Amorim et al., 2011) The main objectives of this study were: 1) to assess changes in gene expression of E. albidus over time, and 2) to identify molecular markers for natural and chemical exposures. Results showed a clear influence of exposure time in gene expression. Transcriptional responses to phenmedipham were seen at 2 days while the responses to copper and the different soils were more pronounced at 4 days of exposure. Some genes were differentially expressed in a stress specific manner and, in general, the responses were related with effects at energy metabolism and cell growth level. Fluorescently labelled cDNA, from enchytraeids exposed during 4 days and 3 weeks to control soil (LUFA 2.2) and to the different exposure conditions was synthesized for microarray analysis and hybridizations were performed. After scanning, spots were identified and ratios quantified using the QuantArray (Packard Biochip Technologies). Statistical analysis of the microarrays was performed using limmaGUI package (1.18.0) (Smyth, 2005) in the R (2.8.0) software environment (http://www.R-project.org/). After being submitted to local background subtraction, microarrays were normalized using global loess method. To statistically evaluate the differential gene expression between the different conditions, a gene-per-gene linear model (limma – linear model for microarray analysis) and empirical Bayes methods were applied. The results were then corrected for multiple testing using the Benjamini-Hochberg’s method (adjusted p<0.05 was considered significant) (Benjamini and Hochberg, 1995).

Project description:Standard bioassays allow hazard assessment at the population level, but much remains to be learned about the molecular level response of organisms to stressors. The main aim of this study was the development of a DNA microarray for Enchytraeus albidus, a common soil worm species. Further, this microarray was tested using worms exposed to Cu, phenmedipham, and different soil types. Hybridization onto the developed microarray revealed several genes with homology to known sequences. Genes of interest were confirmed through real-time polymerase chain reaction. It was possible to discriminate between natural and chemical stressors and chemical concentrations. Gene responses were detected under conditions known to have effects in the reproduction of individuals. It was confirmed that the integration of different endpoints improves the assessment process and enhances the understanding of the modes of action of stressors. The chemical stressM-bM-^@M-^Sinduced genes were related to factors such as immune response, stress response, metabolic processes, and/or signal transduction. The present study represents the first step of a gene-level study in the ecologically relevant and standard test species E. albidus. It demonstrates the usefulness of cDNA normalization in the production of cDNA libraries of ecotoxicological standard organisms that are not genome models like E. albidus. Fluorescently labelled cDNA, from enchytraeids exposed during 2 days to control LUFA 2.2 (Cy3) soil and to the different exposure conditions (Cy5), was synthesized for microarray analysis and hybridizations were performed. After scanning, spots were identified and ratios quantified using the Genepix software 5.0 (Axon Instruments). Statistical analysis of the microarrays was performed using limmaGUI package (1.18.0) (Smyth, 2005) in the R (2.8.0) software environment (http://www.R-project.org/). After being submitted to local background subtraction, microarrays were normalized using global loess method. To statistically evaluate the differential gene expression between the different conditions, a gene-per-gene linear model (limma M-bM-^@M-^S linear model for microarray analysis) and empirical Bayes methods were applied. The results were then corrected for multiple testing using the Benjamini-HochbergM-bM-^@M-^Ys method (adjusted p<0.05 was considered significant) (Benjamini and Hochberg, 1995).

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Effect of Cu-Nanoparticles versus Cu-salt in Enchytraeus albidus (Oligochaeta): differential gene expression through microarray analysis

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