Dataset Information

Quantitative proteome fin(ger)prints of propylthiouracil interference with the thyroid system in zebrafish embryos as 3R alternative in environmental hazard assessment

ABSTRACT: 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 the thyroid system, regulatory decisions mostly rely on amphibian studies. Here we present a rapid and reproducible data dependent proteomics approach for identifying comprehensive molecular signatures of interference with the thyroid system in zebrafish (Danio rerio) embryos as an alternative to animal testing. For this, we have analysed the thyroid peroxidase inhibitor 6-propyl-2-thiouracil (6-PTU) as model substances for anti-thyroidal activity in a modified zebrafish embryo toxicity test (zFET). These fingerprints allow for a definition of solid biomarkers as tools in screening approaches and for integration in chronic toxicity studies for suspect substances, such as the fish early life-stage test (OECD TG 210).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

TISSUE(S): Embryo

SUBMITTER: Steve Ayobahan

LAB HEAD: Dr. Sebastian Eilebrecht

PROVIDER: PXD019079 | Pride | 2021-01-15

REPOSITORIES: Pride

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Toxicogenomic fin(ger)prints for thyroid disruption AOP refinement and biomarker identification in zebrafish embryos.

Reinwald Hannes H König Azora A Ayobahan Steve U SU Alvincz Julia J Sipos Levente L Göckener Bernd B Böhle Gisela G Shomroni Orr O Hollert Henner H Salinas Gabriela G Schäfers Christoph C Eilebrecht Elke E Eilebrecht Sebastian S

The Science of the total environment 20201210

Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the hazard assessment of ED effects on the thyroid system, regulatory decisions mostly rely on amphibian studies. Here, we used transcriptomics and proteomics for identifying molecular signatures of interference with thyroid hormone signaling preceding physiological effects in zebrafish embryos. For this, we analyzed the thyroid ho ...[more]

PMID: 33333401

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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.

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 flutamide as model substances for anti-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 flutamide for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 200 µg/L) and high exposure (HE, 2000 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.

			Action	DRS
	ZE_6-PTU_Rep1_001.raw	Raw
	ZE_6-PTU_Rep1_002.raw	Raw
	ZE_6-PTU_Rep1_003.raw	Raw
	ZE_6-PTU_Rep2_001.raw	Raw
	ZE_6-PTU_Rep2_002.raw	Raw