Unravelling the mechanisms of PFOS toxicity by combining morphological and transcriptomic analyses in zebrafish embryos

Purpose: the goal of this project is to study the effects of the PFOS (perfluorooctanesulfonate) in the transcriptome profiling (RNA-seq) of exposed zebrafish larvae. Methods: Total RNA was isolated from the samples using AllPrep DNA/RNA Mini Kit (Qiagen, CA, USA) as described by the manufacturer. Three high quality sample per condition were chosen to the mRNA enrichment using KAPA Stranded mRNA-Seq Kit Illumina® Platforms (Kapa Biosystems). Transcriptomic profiles were generated by deep sequencing using Illumina TruSeq SBS Kit v3-HS (pair-ended; 2x76bp) on a HiSeq2000 sequencing system. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA. Statistical analysis: RNA-seq reads were aligned to the D. rerio reference genome (GRCz10) using STAR version 2.5.1b . Genes annotated in GRCz10.84 were quantified using RSEM version 1.2.28 with default parameters. Differential expression analysis between all PFOS conditions was performed with the DESeq2 (v.1.10.1) R package with the Likelihood ratio test option. ANOVA-PLS was performed on the normalized data using the lmdme package in R (v. 1.0.136, R Core Team). Results: We generated on average 39 million paired-end reads for each sample and identified aproximatelly 24500 transcripts. 1434 differentially expressed genes (DEGs) were detected which could be divided in 2 clusters including 767 and 667 genes, respectively. Affected metabolic pathways were analyzed from the DEGs: lipid transport and metabolism, protein ubiquination, antigen processing, immune system, apoptosis, trans-membrane, cell matrix, Zn-ion binding, cytokines and JAK-STAT signaling pathways', among others, were down or upregulated. Conclusions: Our results suggest a complex, multiple endocrine disruption-like toxic effects at a concentrations well bellow the 1 mg/L, considered as the LOAEC/NOAEC for many of the macroscopic effects traditionally linked to PFOS toxicity in zebrafish embryos. While our results confirm the known effect of PFOS in lipid metabolism, we found a clear decrease on expression of many genes related to natural immunity and defense against infections. We propose that this transcriptional pattern may be a marker for the immunotoxic effects of PFOS and other related substances in fish and other vertebrates, including humans. We concluded that our analysis allowed us the identification of underlying molecular mechanisms occurring simultaneously at the exposed animals. While this approach is very useful to analyze the effects of compounds, like PFOS, able to interact with different cellular targets, we believe that it can be also applied to the characterization of the different toxic components present in complex natural mixtures. Whole embryo (5 dpf; wild type zebrafish) mRNA profiles of 4 groups (control, 0.03, 0.3 and 1 ppm of PFOS) were generated by deep sequencing, in triplicate, using Illumina TruSeq SBS Kit v3-HS (pair-ended) on a HiSeq2000 sequencing system.

研究目的：本项目旨在探究全氟辛烷磺酸（PFOS, perfluorooctanesulfonate）对暴露于该物质的斑马鱼幼体转录组测序（RNA-seq）的影响。
实验方法：按照厂商说明书，采用AllPrep DNA/RNA Mini Kit（Qiagen, CA, USA）从样本中提取总RNA。每个实验条件选取3份高质量样本，采用KAPA Stranded mRNA-Seq Kit Illumina® Platforms（Kapa Biosystems）进行mRNA富集。通过Illumina TruSeq SBS Kit v3-HS（双端测序；2×76 bp）在HiSeq2000测序系统上完成深度测序，以生成转录组图谱。测序运行的图像分析、碱基识别与质量评分采用厂商提供的Real Time Analysis（RTA 1.13.48）软件处理，随后通过CASAVA生成FASTQ序列文件。
统计分析：采用STAR version 2.5.1b软件将RNA-seq读段比对至斑马鱼（Danio rerio）参考基因组GRCz10；以默认参数通过RSEM version 1.2.28对GRCz10.84注释的基因进行表达量定量。采用搭载似然比检验选项的DESeq2（v.1.10.1）R包完成所有PFOS暴露组间的差异表达分析；基于标准化后的数据，使用R语言lmdme包（v.1.0.136，R核心团队）完成ANOVA-PLS分析。
研究结果：本研究为每份样本平均生成3900万条双端读段，共鉴定得到约24500条转录本。共检测到1434个差异表达基因（DEGs），可分为2个基因簇，分别包含767和667个基因。基于差异表达基因分析受影响的代谢通路发现：脂质转运与代谢、蛋白质泛素化、抗原加工、免疫系统、细胞凋亡、跨膜过程、细胞基质、锌离子结合、细胞因子及JAK-STAT信号通路等均出现显著上调或下调。
结论：本研究结果表明，在远低于传统上认为与斑马鱼胚胎PFOS毒性相关的多数宏观效应的LOAEC/NOAEC（1 mg/L）的浓度下，PFOS即可引发复杂的、类似多内分泌干扰物的毒性效应。本研究不仅验证了PFOS对脂质代谢的已知影响，还发现大量与天然免疫及抗感染防御相关的基因表达水平显著下调。我们提出，该转录特征可作为PFOS及其他相关物质在鱼类、包括人类在内的其他脊椎动物中产生免疫毒性效应的标志物。本研究的分析方法成功鉴定了暴露动物体内同时发生的潜在分子机制。该方法在分析如PFOS这类可与多种细胞靶点结合的化合物的毒性效应时极具价值，我们认为其同样可用于表征复杂天然混合物中不同毒性组分的特性。本研究对4组（对照组、0.03、0.3及1 ppm PFOS暴露组）5日龄（5 dpf）野生型斑马鱼全胚胎进行了转录组测序，每组设置3次生物学重复，采用Illumina TruSeq SBS Kit v3-HS（双端测序）在HiSeq2000测序系统上完成深度测序。