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.

内分泌干扰（endocrine disruption, ED）可对环境种群引发深远影响，因此应拒绝为具有ED特性的化学品核发市场准入许可。为鉴定靶向甲状腺系统的内分泌干扰化学品的分子特征谱，本研究以作为水生脊椎动物模型及动物试验替代方案的斑马鱼（Danio rerio）胚胎为实验对象，开展生态毒理基因组筛选。本研究选用6-丙基-2-硫氧嘧啶（6-Propyl-2-thiouracil, 6PTU, CAS: 51-52-5）作为抗甲状腺活性的模型受试物。在改良版斑马鱼胚胎毒性试验（OECD 236）中，将15枚受精卵在半静态条件下暴露于两种不同的亚致死浓度的6PTU中，持续96小时。每组试验均设置低暴露组（low exposure, LE）、高暴露组（high exposure, HE）与阴性对照组（negative control, NC），并开展三次生物学重复。于受精后96小时，从每个样本组中随机选取10条幼鱼并混合，采用NucleoSpin® RNA/Protein试剂盒（Macherey-Nagel）进行RNA与蛋白质提取。通过2100 Bioanalyzer系统（Agilent）评估RNA质量后，使用TruSeq RNA文库制备试剂盒v2进行polyA选择以纯化编码RNA，再通过Illumina HiSeq 4000测序系统（Illumina）以50 bp单端读取模式完成测序，每个样本可产生约3000万条读段。初始BCL文件通过bcl2fastq软件解复用为fastq文件，使用Trimmomatic去除接头序列，并通过STAR软件将序列比对至斑马鱼参考基因组（GRCz11）。通过featureCounts软件统计比对到基因特征的读段数量，将各文库的比对读段表合并为单个计数矩阵。以该矩阵作为输入数据，使用DESeq2对读段计数进行标准化，以开展差异基因表达分析。