ABSTRACT: 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.
