Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Exploring gene expression dynamics in zebrafish embryos exposed to trenbolone: A 96-Hour RNA-seq Study

ABSTRACT: Endocrine active substances present significant risks to both human health and the environment, particularly by disrupting essential endocrine-regulated functions like organism development and reproductive capacity. Regulatory frameworks mandate animal testing for chemical risk assessment, placing specific emphasis on suspected endocrine disruptors. For example, aquatic vertebrate chronic toxicity testing is mandatory at a chemical tonnage of 100 t/y using the Fish Early Life Stage (FELS, OECD TG 210) test. However, suspected endocrine disruptors are subjected to testing from a lower tonnage threshold of 10 t/y. Currently, the assessment of endocrine effects adheres to an OECD Conceptual Framework (CF) that employs a tiered approach. This entails evaluating existing data for potential endocrine effects (Level 1), conducting mechanism of action-specific in vitro studies (Level 2), and undertaking in vivo mechanistic screening studies (Level 3). If screening studies indicate potential endocrine disruption, further investigation is conducted using a Fish Sexual Development Test (FSDT, OECD TG 234) to clarify (anti)estrogenic, (anti)androgenic, and steroidal effects (EAS effects) in fish. Nevertheless, this testing process is resource-intensive, time-consuming, and involves extensive animal use. Therefore, this study aims to identify the androgenic activity of trenbolone using the zebrafish embryo model. Trenbolone, a synthetic anabolic steroid, primarily exerts its effects by binding to androgen receptors. The data collected will be compared with those obtained from androstenedione exposure to zebrafish embryos.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Danio rerio

SUBMITTER: Dr. Steve Ayobahan

PROVIDER: E-MTAB-14032 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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 ethinylestradiol as model substances for 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 ethinylestradiol for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 20 µg/L) and high exposure (HE, 200 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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Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in zebrafish (Danio rerio) embryos as aquatic vertebrate non-target model exposed to sub lethal concentrations of synthetic organochloride insecticide Methoxychlor (CAS 72-43-5). It is structurally highly similar to its precursor molecule DDT (Dichlorodiphenyltrichloroethane). Like its precursor, today Methoxychlor is banned from the use as pesticide in the United States and the European Union due to its acute toxicity, high bioaccumulation potential and endocrine disruption activity. The Insecticide Resistance Action Committee (IRAC) classified Methoxychlor after its mode of action (MoA) in the target organism as a sodium channel modulator (Group 3B). Our goal is to identify toxicogenomic profiles with predictive character and potential molecular key events (KE) explaining upstream adverse effects in aquatic non-target organisms for this substance of particularly high environmental concern. This will provide useful information to refine and improve existing adverse outcome pathways (AOP). Furthermore, integrating the obtained profiles for this and other tested chemicals in a collective database will enable us in the future to derive predictions about the ecotoxicological hazard for chemcials with unknown apical effects, based on similarly altered transcriptomic and proteomic profiles. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of Methoxychlor for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 20 µg/L), mid exposure (ME, 60 µg/L), high exposure (HE, 180 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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Exploring gene expression dynamics in zebrafish embryos exposed to trenbolone: A 96-Hour RNA-seq Study

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