Identifying key events from Per- and Polyfluoroalkyl Substances exposure that influence larval zebrafish in light:dark assay using RNA-sequencing analysis of perfluorohexane-1-sulfonate (PFHxS)

Gene expression data from short-term in vivo studies shows efficacy in quantifying concentration-dependent effects that inform toxicity outcomes, particularly for chemicals with limited toxicity data such as Per- and Polyfluoroalkyl Substances (PFAS). Automated behavioral assessments, at non-teratogenic concentrations reveal perfluorohexane-1-sulfonate (PFHxS) exposure causes hyperactivity in larval zebrafish (6 days post fertilization (dpf)) during the light phase of the light:dark assay compared to 0.4% dimethylsulfoxide (DMSO) vehicle control. To identify key events that drive this abnormal behavior, zebrafish embryos were exposed to 7.87-25.1 µM PFHxS or 0.4% DMSO, and RNA was isolated from pooled head tissue collected at 4 and 5 dpf, before the onset of hyperactivity, for RNA-sequencing (NextSeq 500). Differentially expressed genes (DEGs) were identified using Gene Specific Analysis (Partek Flow, St. Louis, MO) and mapped to human orthologs for pathway analyses to inform modes of action. Overall design: At 0 dpf, zebrafish embryos were bleached as previously described (Tal et al. 2017). A single embryo at the dome-to-epiboly stages (Kimmel et al. 1995) was placed into each individual well of a 96-well plate containing a 40-µm nylon mesh filter (Millipore, Catalog No. MANMN4010) with 400µL of 10% Hanks' balanced salt solution (HBSS) per well. Filter inserts containing zebrafish embryos were transferred to 96-well culture trays (Millipore, Catalog No. MAMCS9610) containing 250µL of 10% HBSS (Westerfield 2007) and 1µL of 250× working solutions per well. A final concentration of 0.4% DMSO was used for all exposure groups and as a vehicle control. Daily, from 1–4 or 1-5 dpf, plates underwent 100% media changes to refresh chemical dosing solutions by blotting (Brandel; Catalog No. FPXLR-196) and transferring mesh inserts containing zebrafish to new bottom plates (Millipore; Catalog No. MAMCS9610). To minimize evaporation, plates were sealed (Biorad; Catalog No. MSA5001) and wrapped with parafilm. Plates were maintained on a 14 h:10 h light cycle at 26.0°C and scored daily for death, malformations, hatching, and swim bladder inflation. Zebrafish embryos were exposed to 7.87-25.1 µM PFHxS or 0.4% DMSO with targeting n=6 pooled replicates/treatment. Each pooled replicate consisted of 15 zebrafish heads collected anterior to the swim bladder/treatment.