Project description:Perfluoroalkyl substances (PFAS) are a class of synthetic chemicals that are persistent in the environment. Due to concerns linked with longer chain PFAS, shorter chain chemicals were used as replacements, but developmental toxicity assessments of the shorter chain chemicals are limited. The aim of this study was to compare the developmental toxicity of three perfluoroalkyl acids (PFAAs): perfluorooctanoic acid (PFOA), composed of 8 carbon (C8), perfluorohexanoic acid (PFHxA, C6), and perfluorobutanoic acid (PFBA, C4) using the zebrafish (Danio rerio). LC50s at 120 hour post fertilization (hpf) were determined to assess the potency of each PFAA by exposing developing zebrafish (1-120 hpf) to a range of concentrations. In sublethal assessments, zebrafish were exposed to 0, 4, 40, or 400 parts per billion (ppb; µg/L) of the PFAAs throughout embryonic development (1-72 hpf). Effects of the embryonic exposure on locomotor activities was completed with the visual motor response test at 120 hpf. At 72 hpf, morphological changes including total body length, head length, and head width were assessed. Additionally, transcriptomic profiles of PFOA, PFHxA, and PFBA were determined at 72 hpf to compare altered molecular and disease pathways. The LC50 ranking was PFOA > PFHxA > PFBA, which followed trend as expected based on chain length. PFOA caused hyperactivity and PFBA hypoactivity, while PFHxA did not change behavior. PFOA, PFHxA, and PFBA caused morphological and transcriptomic alterations that were unique for each chemical and were concentration dependent. Cancer was a top enriched disease for PFOA, while FXR/RXR activation was a top canonical pathway in all PFBA exposures. Overall, an embryonic exposure to PFOA, PFHxA, or PFBA resulted in morphological alterations in zebrafish eleuthero-embryos; however, only PFOA and PFBA induced neurobehavioral alterations. The transcriptomic profile of each chemical was unique indicating different toxicity mechanisms and were aligned with human adverse health outcomes supporting predictive value.

Project description:Purpose: The goals of this study are to investigate developmental toxicity and hazard potential of a short-chain PFAS homologous series in zebrafish, specifically interrogating the impact of different functional head groups. Developmental toxicity findings were supplemented with investigations into transcriptom-wide effects of body burdens following PFAS exposures. Methods: For developmental toxicity assessments, dechorionated embryonic zebrafish were statically exposed at 8 hours post fertilization (hpf) to either vehicle control (0.5% methanol) or to FBSA, PFBS, PFPeA, or 4:2 FTS across a range of concentrations (1-100 µM, normalized to 0.5% methanol) in 96-well plate format. Morphological and Behavior endpoints were assessed at 24 and 120 hpf. For investigations into transcriptomic effects and body burdens, embryos were exposed to vehicle control or 47 µM of each PFAS and then sampled at 48 hpf, before morphological effects were observable. Transcriptomic effects of FBSA exposure were phenotypically anchored to abnormal morphology at 120 hpf as 47 µM was the concentration at which morphological effects were observed in 80% of the exposure group. At 48 hpf, 10 embryos were pooled per replicate, euthanized via ice bath, and total RNA extracted using a Zymo Research Direct-zol RNA MiniPrep kit and then sequenced on the BGISEQ-500 platform (Beijing Genomics Institute; BGI) for the transcriptomic investigation. Data analysis and visualization was conducted by BGI and their Dr. Tom platform. Briefly, sequencing data was filtered using SOAPnukes to remove adaptor sequences, N content greater than 5%, and low-quality reads. Clean reads were mapped to the reference genome (Danio rerio GRCz11) using HISAT2. Gene fusions and differential splicing were detected using Ericscript and rMATS, clean read alignments using Bowtie2 and expression level calculations using RSEM. Differential expression analysis was performed using DESEq2 (q-value ≤ 0.05), followed by gene ontology enrichment analysis using Phyper and transcription factor enrichment analysis using MetaCore. For body burden measurments, 4 replicates of 40 embryos were pooled per exposure group, euthanized on ice, homogenized, and analyzed by high pressure liquid chromatography and triple quadrupole mass spectrometry. Results: Across the 4-fluorinated carbon short-chain PFAS series, all chemicals induced larval behavior effects, but only FBSA caused morphological, behavioral and large-scale transcriptomic disruptions (specifically at 47 µM). Notably, exposures at concentrations shown to induce only behavior effects resulted in a small number of differentially expressed genes. Biological processes enriched following FBSA exposure at 47 µM were related to lipid metabolism and biosynthesis, cellular homeostasis, brain and nervous system development, and immune processes. The bioaccumulative potential of the series at 47 µM was FBSA > PFBS > 4:2 FTS > PFPeA.

Dataset Information

Assessment of unique behavioral, morphological, and molecular alterations in the comparative developmental toxicity profiles of PFOA using the zebrafish model system

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