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.

Project description:Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the Aryl Hydrocarbon Receptor (AHR) in a structurally-dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at a concentration that induces developmental malformations by 120 hours post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burden was analyzed at these time points using GC-MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the smallest number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure and may provide a path for unraveling the toxicity of complex PAH mixtures. Gene expression was measured in zebrafish embryos after exposure to PAHs. Embryos were batch-exposed in groups of 40 to 25 μM BAA, 25 μM DBT, 25 μM PYR or 1% DMSO vehicle control starting at 6 hpf and collected at 24 or 48 hpf. Four independent biological replicates were prepared for each treatment. The reference was a pool of zebrafish embryos exposed to DMSO control until 24 and 48 hpf.

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Comparison of zebrafish toxicity among different developmental windows of exposure to three environmentally relevant PFAS compounds

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