Single-cell RNA sequencing to reveal tissue-specific transcriptomic changes induced by perfluorooctanesulfonic acid (PFOS) in larval zebrafish (Danio rerio)

Per- and poly-fluoroalkyl substances (PFAS), a class of ubiquitous, persistent, and toxic anthropogenic chemicals that exert harmful effects on environmental and human health, but the mechanisms that drive these effects are not well understood. Perfluorooctanesulfonic acid (PFOS) is a legacy PFAS that elicits numerous adverse effects on the endocrine, immune, nervous, reproductive, and developmental processes. The aim of this study was to identify transcriptomic changes in zebrafish (Danio rerio) exposed to PFOS that provide insight into sensitive target tissues and molecular mechanisms of developmental toxicity. Zebrafish embryos were exposed to DMSO (0.01%) or PFOS (16 µM) from 3–72 hours post fertilization (hpf). Larvae were collected, cells isolated and assessed for viability, and single-cell gene expression profiling was performed. Data analysis was conducted in three different ways: pseudo-bulk analysis, untargeted cluster analysis, and a targeted cluster analysis where unique clusters were defined for liver (fabp10a, and apoa2). Results from the pseudo-bulk analysis showed that 8.63% (2390/ 27698) genes were significantly differentially expressed between DMSO and PFOS groups. The top five pathways impacted by these significant DEGs were neuron development, visual perception, central nervous system development, eye development, and synaptic signaling. Results from untargeted cluster analysis revealed 22 distinct clusters that were annotated to specific tissues. The top five clusters with the highest number of significant DEGs were digestive organs, muscle, otolith, cardiac muscle, and optic lens. Additionally, we looked at the distribution of pathway-specific genes for epigenetic modifications, Nfe2l2 pathway, and PPAR pathway within clusters to identify sensitive tissues—blood-related tissue, retinal rod, and cardiac muscle were more likely be impacted by epigenetic modifications by DNA methylation; while blood-related tissue and periderm are sensitive to be impacted by both Nfe2l2 and PPAR pathway. Another potential mechanism induced by PFOS is beta-oxidation of fatty acids—which was significantly downregulated in blood-related tissue and upregulated in digestive organs. Results of targeted or curated liver cluster showed 220 DEGs in the liver were significant with PFOS exposure with Notch signaling pathway more likely to be impacted with PFOS exposures. The sensitivity pathways for PFOS-induced toxicity in larval zebrafish are PPAR signaling (overall, and digestive organs) and Notch signaling pathway (liver). Overall, scRNA-seq is a powerful tool to identify tissue-specific changes and novel mechanisms by which toxicants like PFOS disrupt embryonic development. Single cells were obtained from whole body zebrafish larvae exposed to DMSO and PFOS.

全氟和多氟烷基物质（Per- and poly-fluoroalkyl substances, PFAS）是一类广泛存在、性质稳定且具有毒性的人工合成化学物，可对环境与人类健康造成损害，但其介导这些损害的分子机制尚未得到充分阐明。全氟辛烷磺酸（Perfluorooctanesulfonic acid, PFOS）作为一种遗留型全氟和多氟烷基物质，可对内分泌、免疫、神经、生殖及发育过程引发诸多不良影响。本研究旨在探究全氟辛烷磺酸（PFOS）暴露下斑马鱼（Danio rerio）的转录组变化，以解析其发育毒性的敏感靶组织与分子机制。
将斑马鱼胚胎于受精后3~72小时（hours post fertilization, hpf）期间暴露于0.01%二甲基亚砜（Dimethyl sulfoxide, DMSO）或16 μM全氟辛烷磺酸（PFOS）环境中。收集幼虫并分离细胞，检测细胞活力，随后开展单细胞基因表达谱分析。数据分析采用三种不同方法：伪批量分析、无监督聚类分析，以及针对肝脏的靶向聚类分析——该分析中以fabp10a与apoa2作为肝脏特异性聚类标记基因。
伪批量分析结果显示，二甲基亚砜组与全氟辛烷磺酸组间共有8.63%（2390/27698）的基因呈现显著差异表达。上述显著差异表达基因（differentially expressed genes, DEGs）所影响的前五大通路依次为神经元发育、视觉感知、中枢神经系统发育、眼部发育及突触信号传导。
无监督聚类分析结果显示，共得到22个可注释到特定组织的独立聚类簇。差异表达基因数量最多的前五大聚类簇分别为消化器官、肌肉、耳石、心肌及晶状体。此外，本研究还分析了表观遗传修饰、核因子红细胞2相关因子2通路（Nfe2l2 pathway）及过氧化物酶体增殖物激活受体通路（PPAR pathway）的通路特异性基因在各聚类簇中的分布，以识别敏感组织：血液相关组织、视杆细胞与心肌更易受DNA甲基化介导的表观遗传修饰影响；而血液相关组织与表皮层则对Nfe2l2及PPAR通路的调控作用更为敏感。
全氟辛烷磺酸诱导的另一潜在机制为脂肪酸β-氧化：该过程在血液相关组织中显著下调，而在消化器官中则显著上调。经人工注释的肝脏靶向聚类分析结果显示，全氟辛烷磺酸暴露后肝脏内共有220个差异表达基因呈现显著变化，其中Notch信号通路（Notch signaling pathway）更易受全氟辛烷磺酸暴露的影响。
总体而言，单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）是识别组织特异性变化及全氟辛烷磺酸等有毒物质破坏胚胎发育的新型机制的有力工具。本研究的单细胞样本来源于经二甲基亚砜与全氟辛烷磺酸暴露的斑马鱼幼虫全身组织。