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Evaluation of the physiology of medaka for standardized toxicity tests of chemicals

ABSTRACT: The “Fish Acute Toxicity Test” in the “OECD Guideline for Testing of Chemicals” is an essential test for environmental toxicity. Here we have tried to evaluate the physiology of medaka during this test procedure by using genomics. Genomics technology can provide genome-wide expression profiles of mRNA, and these profiles correspond to the physiology of organisms. Thus, a comparison of mRNA expression profiles gives information on the reproducibility of experimental conditions. Expression profiles of mRNA were measured for medakas maintained within the allowable range of the test conditions and also under extreme conditions beyond the guidelines limits. We confirmed the high physiological reproducibility of medaka kept in the recommended conditions of the “Fish Acute Toxicity Test” in the “OECD Guideline for Testing of Chemicals" from the expression profiles obtained under all experimental conditions except for the type of feeding.

ORGANISM(S): Oryzias latipes

PROVIDER: GSE15380 | GEO | 2010/07/22

SECONDARY ACCESSION(S): PRJNA117113

REPOSITORIES: GEO

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Project description:Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the assessment of ED effects on development and reproduction, regulatory decisions mostly rely on apical endpoints from in vivo testing with adult animals. Here, we present a rapid and reproducible mRNA expression profiling experiment for identifying comprehensive molecular fingerprints interfering with the sexual endocrine system in zebrafish (Danio rerio) embryos as an alternative to animal testing. For this, we have analysed tamoxifen as model substances for anti-estrogenic perturbation in a modified zebrafish embryo toxicity test (zFET). These signatures allow for the identification of gene expression changes and the definition of solid biomarkers as tools in screening approaches and for integration in chronic toxicity studies for identifying suspect ED substances, in the fish early life-stage test (OECD TG 210). In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of tamoxifen for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 50 µg/L) and high exposure (HE, 500 µg/L) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Project description:Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the assessment of ED effects on development and reproduction, regulatory decisions mostly rely on apical endpoints from in vivo testing with adult animals. Here, we present a rapid and reproducible mRNA expression profiling experiment for identifying comprehensive molecular fingerprints interfering with the sexual endocrine system in zebrafish (Danio rerio) embryos as an alternative to animal testing. For this, we have analysed methyltestosterone as model substances for androgenic perturbation in a modified zebrafish embryo toxicity test (zFET). These signatures allow for the identification of gene expression changes and the definition of solid biomarkers as tools in screening approaches and for integration in chronic toxicity studies for identifying suspect ED substances, in the fish early life-stage test (OECD TG 210). In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of methyltestosterone for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 1600 µg/L) and high exposure (HE, 3200 µg/L) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

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Evaluation of the physiology of medaka for standardized toxicity tests of chemicals

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