RNA-Seq of zebrafish embryos (96hpf) treated with different concentrations of 6-Propyl-2-thiouracil (6-PTU) below acute toxicity levels against untreated control groups. RNA-Seq of zebrafish embryos (96hpf) treated with different concentrations of 6-Propyl-2-thiouracil (6-PTU) below acute toxicity levels against untreated control groups

Endocrine disruption can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. Ecotoxicogenomic screening was performed to identify molecular fingerprints for endocrine disrupting chemicals affecting the thyroid system in zebrafish (Danio rerio) embryos as aquatic vertebrate model and alternative to animal testing. 6-Propyl-2-thiouracil (6PTU, CAS: 51-52-5) was tested as a model substance for anti-thyroidal activity. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of 6PTU for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE), high exposure (HE) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization 10 larvae were randomly picked from each sample group and pooled for RNA and protein extraction with NucleoSpin© RNA/Protein kit (Macherey-Nagel). RNA quality was assessed via 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 ~ 30 million reads per sample. Initial BCL files were demultiplexed to fastq files via bcl2fastq. 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 mapped read tables were then merged to a single count matrix. Using this matrix as input, read counts were normalized with DESeq2 for differential gene expression analysis.