Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Combined transcriptomics classifier analysis reveals a set of adverse effect genes for use as potential endpoint in effect based screening

ABSTRACT: Environmental risk assessment relies heavily on the use of bioassays to assess the environmental impact of chemicals. Gene expression is gaining acceptance as a valuable mechanistic endpoint in bioassays and effect-based screening. Data analysis and its results however, are often complex and not directly applicable in risk assessment. Classifier analysis is a promising method to turn complex gene expression analysis results into answers suitable for risk assessment. We have assembled a large gene expression dataset assembled from multiple studies and experiments in the springtail Folsomia candida, with the aim of selecting a set of genes that can be trained to classify general toxic stress. By performing differential expression analysis prior to classification we were able to select a set of 135 genes which was enriched in stress related processes. This set was then used to classify two test sets comprised of chemical spiked soils, polluted soils and clean soils and compared to another, more traditional feature selection for classification. The gene set presented here outperformed the more traditionally selected gene set. This gene set has the potential to be used as a biomarker to test for adverse effects caused by chemicals in springtails to provide endpoints in environmental risk assessment. The data presented in our manuscript is part of a larger experiment which was performed in single, large loop design. Only the samples used in the study presented here are named while the other samples will remain unnamed. All the data can still be used for normalization after which the analysis presented in the manuscript can be replicated. A single channel, interwoven loop design was used to test animals exposed to a control and to 5 concentrations of cadmium and phenanthrene (cadmium concentrations are: cad_1 till cad_5: 5.8, 14.5, 28.9, 57.9 and 115.8 mg/Kg respectively and phenanthrene concentrations are: phe_1 till Phe_5: 0, 4.6, 11.4, 22.9, 45.8 and 91.6 mg/Kg soil respectively). Exposures lasted for 2 days and used 4 biological replicates per condition each containing 30 grams soil and 30 individuals.

ORGANISM(S): Folsomia candida

SUBMITTER: Tjalf de Boer

PROVIDER: E-GEOD-70877 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Combined Transcriptomics Analysis for Classification of Adverse Effects As a Potential End Point in Effect Based Screening.

de Boer Tjalf E TE Janssens Thierry K S TK Legler Juliette J van Straalen Nico M NM Roelofs Dick D

Environmental science & technology 20151113 24

Environmental risk assessment relies on the use of bioassays to assess the environmental impact of chemicals. Gene expression is gaining acceptance as a valuable mechanistic end point in bioassays and effect-based screening. Data analysis and its results, however, are complex and often not directly applicable in risk assessment. Classifier analysis is a promising method to turn complex gene expression analysis results into answers suitable for risk assessment. We have assembled a large gene expr ...[more]

PMID: 26523736

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Project description:Polycyclic Aromatic Hydrocarbons (PAHs) continue to cause environmental challenges due to their release in the environment by a great variety of anthropogenic activities and their accumulation in soil ecosystems. Here we studied the toxicological effect of the model PAH phenanthrene (Phe) on the soil invertebrate model Enchytraeus crypticus at the individual, tissue and molecular level. Organisms were exposed to Phe for 2 and 21 days to the (previously estimated) EC10 and EC50 (population reproduction over 3 weeks). Gene expression profiling did not reveal a typical Phe-induced biotransfor-mation signature, as it usually does in arthropods and vertebrates. Instead, we observed only general metabolic processes to be affected after 2 days of exposure, such as translation and ATP synthesis-coupled electron transport. Histological sections of tissues of 2-day exposed animals did not show any deviations from the control situation. In contrast, prolonged exposure up to 21 days showed histopathological effects: chloragogenous cells were highly vacuolated and hypertrophic. This was corroborated by differential expression of genes related to immune response and oxidative stress at the transcriptomic level. The data exemplify the complexity and species-specific features of PAH toxicity among soil invertebrate communities, which restricts read-across and extrapolation in the context of soil ecological risk assessment. The data presented in our manuscript is an exposure experiment where E. cryticus is exposed to phenanthrene EC10 and EC50 on reproduction for 2 and 21 days. A single channel, interwoven loop design was used to test animals. 4 biological replicates per condition were used containing 25 grams of soil and 5 - 7, adult old animals per replicate. The platform is a 4*180k Agilent platform containing some 86k E. crypticus probes in duplicate. However, only a subset of the probes (23k) was used for the analysis. To see which probes were used in the analysis see the raw data files control type column, only probes which are denoted with a 0 were used.

Project description:The soil worm Enchytraeus crypticus (oligochaete) is an ecotoxicology model species although without genome or transcriptome sequence information. The present research aimed at studying, via high-throughput pyrosequencing, the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies. A pyrosequencing run retrieved approximately 1.5 million reads representing 645 million bases. After assembly, 27,296 contigs and 87,686 singletons were obtained. from which 44% and 25% were annotated as protein-coding genes. We show that the high amount of orphan genes is not due to poor sequence or assemble quality: 84% of the contig sequences contains an open reading frame with a start codon and E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65 and 77% of the unknown singletons and contigs, respectively, showed transcriptional activity. An Agilent 180K microarray platform was designed and validated by hybridizing cDNA from 3 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction (EC50). Overall, 70% of all probes exerted a hybridization signal above background level. More specifically, the probes derived from contigs showed a wider range of average intensities when compared to probes derived from singletons. In total, 522 significantly regulated transcripts were identifying upon zinc exposure. Several significantly regulated genes exerted predicted functions (e.g. zinc efflux, zinc transport) associated with zinc stress. Unexpectedly, the microarray data suggest that zinc exposure alters retrotransposon activity in the E. crypticus genome. In conclusion, characterization of the presented E. crypticus transcriptome and associated microarray platform is a valuable and high quality resource that permits further functional genomics experiments examining gene expression patterns underlying distinct environmental stress conditions. We show that unknown sequences are not the result of technical errors but mostly represent functional genes that are actively transcribed. The data presented in our manuscript is part of a larger experiment which was performed in single, large loop design. The analysis presented here can be replicated only by including all raw data from the larger experiment (all raw files are included in the archive linked to this submission). A single channel, interwoven loop design was used to test animals exposed to zinc EC50 on reproduction as compared to untreated controls for 4 days. 4 biological replicates per condition were used containing 25 grams of soil and 5 - 7, adult old animals per replicate. T4_con stands for untreated control soil while T4_50 are the samples exposed to EC50 of zinc on reproduction.

Project description:It has long been recognized that species occupy a specific ecological niche within their ecosystem. The ecological niche is defined as the number of conditions and resources that limit species distribution. Within their ecological niche, species do not exist in a single physiological state but in a number of states we call the Natural Operating Range. In this paper we link ecological niche theory to physiological ecology by measuring gene expression levels of collembolans exposed to various natural conditions. The soil-dwelling collembolan Folsomia candida was exposed to 26 natural soils with different soil characteristics (soil type, land use, practice, etc). The animals were exposed for two days and gene expression levels were measured. The main factor found to regulate gene expression was the soil type (sand or clay), in which 18.5% of the measured genes were differentially expressed. Gene Ontology analysis showed animals exposed to sandy soils experience general stress, affecting cell homeostasis and replication. Multivariate analysis linking soil chemical data to gene expression data revealed that soil fertility influences gene expression. Land-use and practice had less influence on gene expression; only forest soils showed a different expression pattern. A variation in gene expression variation analysis showed overall low variance in gene expression. The large difference in response to soil type was caused by the soil physicochemical properties where F. candida experiences clay soils and sandy soils as very different from each other. This collembolan prefers fertile soils with high organic matter content, as soil fertility was found to correlate with gene expression and animals exposed to sandy soils (which, in general, have lower organic matter content) experience more general stress. Finally, we conclude that there is no such thing as a fixed physiological state for animals in their ecological niche and the boundary between the ecological niche and a stressed state depends on the genes/pathways investigated. Test animals were exposed to 26 natural soils + 2 control soils. 4 biological replicates per soil containing 25 grams of soil and 30 23-day-old animals per replicate, RNA was isolated after two days of exposure. for the micro-array hybridization design we made use of an interwoven loop design. from the four replicates per soil two were labeled with Cy3 and 2 with Cy5. It was made sure that now two replicates of the same soil were ever hybridized against the same soil.

Project description:Polycyclic aromatic hydrocarbons are common pollutants in soil, have negative effects on soil ecosystems, and are potentially carcinogenic. The Springtail (Collembola) Folsomia candida is often used as an indicator species for soil toxicity. Here we report a toxicogenomic study that translates the ecological effects of the polycyclic aromatic hydrocarbon phenanthrene in soil to the early transcriptomic responses in Folsomia candida. Microarrays were used to examine two different exposure concentrations of phenanthrene, namely the EC10 (24.95 mg kg-1 soil) and EC50 (45.80 mg kg-1 soil) on reproduction of this springtail, which evoked 405 and 251 differentially expressed transcripts, respectively. Fifty transcripts were differential in response to either concentration. Many transcripts encoding xenobiotic detoxification and biotransformation enzymes (phases I, II, and III) were upregulated in response to either concentration. Furthermore, indications of general and oxidative stress were found in response to phenanthrene. Chitin metabolism appeared to be disrupted particularly at the low concentration, and protein translation appeared suppressed at the high concentration of phenanthrene; most likely in order to reallocate energy budgets for the detoxification process. Finally, an immune response was evoked especially in response to the high effect concentration, which was also described in a previous transcriptomic study using the same effect concentration (EC50) of cadmium. Our study provides new insights in the molecular mode of action of the important polluting class of polycyclic aromatic hydrocarbons in soil animals. Furthermore, we present a fast, sensitive, and specific soil toxicity test which enhances traditional tests and may help to improve current environmental risk assessments and monitoring of potentially polluted sites. Folsomia candida was exposed to phenanthrene spiked soil or untreated (reference/control) soil for 2 days. Two different concentrations of phenanthrene were used, 24.95 and 45.80 mg/kg soil which represent the EC10 and EC50 on reproduction, respectively. For each concentration treatment 4 biological replicates were used, replicate samples consisted of total RNA extracted from ~30 animals exposed in the same jar to either reference or phenanthrene spiked soil. Phenanthrene treated samples were always hybridized to reference samples in an evenly distributed dye-swap manner, which resulted in total in 8 hybridizations of 16 samples.

Dataset Information

Combined transcriptomics classifier analysis reveals a set of adverse effect genes for use as potential endpoint in effect based screening

Publications

Combined Transcriptomics Analysis for Classification of Adverse Effects As a Potential End Point in Effect Based Screening.

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