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Per- and polyfluoroalkyl substances (PFAS) are a widespread and persistent class of contaminants posing significant environmental and human health concerns. Comprehensive understanding of the modes of action underlying toxicity among structurally diverse PFAS is mostly lacking. To address this need, we recently reported on our application of developing zebrafish to evaluate a large library of PFAS for developmental toxicity. In the present study, we prioritized 15 bioactive PFAS that induced significant morphological effects and performed RNA-sequencing to characterize early transcriptional responses at a single timepoint (48 h post fertilization) after early developmental exposures (8 h post fertilization). Internal concentrations of 5 of the 15 PFAS were measured from pooled whole fish samples across multiple timepoints between 24-120 h post fertilization, and additional temporal transcriptomics at several timepoints (48-96 h post fertilization) were conducted for Nafion byproduct 2. A broad range of differentially expressed gene counts were identified across the PFAS exposures. Most PFAS that elicited robust transcriptomic changes affected biological processes of the brain and nervous system development. While PFAS disrupted unique processes, we also found that similarities in some functional head groups of PFAS were associated with the disruption in expression of similar gene sets. Body burdens after early developmental exposures to select sulfonic acid PFAS, including Nafion byproduct 2, increased from the 24-96 h post fertilization sampling timepoints and were greater than those of sulfonamide PFAS of similar chain lengths. In parallel, the Nafion byproduct 2-induced transcriptional responses increased between 48 and 96 h post fertilization. PFAS characteristics based on toxicity, transcriptomic effects, and modes of action will contribute to further prioritization of PFAS structures for testing and informed hazard assessment.
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Early-life exposure to environmental toxicants like Benzo[a]pyrene (BaP) is associated with several health consequences in vertebrates (i.e., impaired or altered neurophysiological and behavioral development). Although toxicant impacts were initially studied relative to host physiology, recent studies suggest that the gut microbiome is a possible target and/or mediator of behavioral responses to chemical exposure in organisms, via the gut-brain axis. However, the connection between BaP exposure, gut microbiota, and developmental neurotoxicity remains understudied. Using a zebrafish model, we determined whether the gut microbiome influences BaP impacts on behavior development. Embryonic zebrafish were treated with increasing concentrations of BaP and allowed to grow to the larval life stage, during which they underwent behavioral testing and intestinal dissection for gut microbiome profiling via high-throughput sequencing. We found that exposure affected larval zebrafish microbiome diversity and composition in a manner tied to behavioral development: increasing concentrations of BaP were associated with increased taxonomic diversity, exposure was associated with unweighted UniFrac distance, and microbiome diversity and exposure predicted larval behavior. Further, a gnotobiotic zebrafish experiment clarified whether microbiome presence was associated with BaP exposure response and behavioral changes. We found that gut microbiome state altered the relationship between BaP exposure concentration and behavioral response. These results support the idea that the zebrafish gut microbiome is a determinant of the developmental neurotoxicity that results from chemical exposure.
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Comportamento Animal , Benzo(a)pireno , Microbioma Gastrointestinal , Larva , Peixe-Zebra , Animais , Peixe-Zebra/microbiologia , Benzo(a)pireno/toxicidade , Microbioma Gastrointestinal/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/microbiologiaRESUMO
Engineered nanomaterials (ENMs) are ubiquitous in contemporary applications, yet their environmental and human health impacts remain inadequately understood. This study addresses the challenge of identifying potential risks associated with ENM exposure by highlighting the significant variability in existing research methodologies. Without a systematic collection of toxicological data that encompasses standardized materials, relevant platforms, and assays, the task of identifying potential risks linked to ENM exposure becomes an intricate challenge. In vitro assessments often use media rich in ionic species, such as RPMI and fetal bovine serum (FBS). Zebrafish embryos, known to develop normally in low-ionic environments, were exposed to Cerium Oxide, Zinc Oxide, and Graphene Oxides in different media at varying concentrations. Here, we discovered that zebrafish embryos tolerated a mix of 80 % RPMI, 2 % FBS, and 1 % antibiotic cocktail. The results revealed that adverse effects observed in zebrafish with certain nanomaterials in Ultra-Pure (UP) water were mitigated in cell culture medium, emphasizing the importance of revisiting previously considered non-toxic materials in vitro. The zebrafish results underscore the importance of utilizing a multidimensional in vivo platform to gauge the biological activity of nanomaterials accurately.
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Polycyclic aromatic hydrocarbons (PAHs) are prevalent environmental contaminants that are harmful to ecological and human health. Bioremediation is a promising technique for remediating PAHs in the environment, however bioremediation often results in the accumulation of toxic PAH metabolites. The objectives of this research were to demonstrate the cometabolic treatment of a mixture of PAHs by a pure bacterial culture, Rhodococcus rhodochrous ATCC 21198, and investigate PAH metabolites and toxicity. Additionally, the surfactant Tween ® 80 and cell immobilization techniques were used to enhance bioremediation. Total PAH removal ranged from 70-95% for fluorene, 44-89% for phenanthrene, 86-97% for anthracene, and 6.5-78% for pyrene. Maximum removal was achieved with immobilized cells in the presence of Tween ® 80. Investigation of PAH metabolites produced by 21198 revealed a complex mixture of hydroxylated compounds, quinones, and ring-fission products. Toxicity appeared to increase after bioremediation, manifesting as mortality and developmental effects in embryonic zebrafish. 21198's ability to rapidly transform PAHs of a variety of molecular structures and sizes suggests that 21198 can be a valuable microorganism for catalyzing PAH remediation. However, implementing further treatment processes to address toxic PAH metabolites should be pursued to help lower post-remediation toxicity in future studies.
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Biodegradação Ambiental , Células Imobilizadas , Hidrocarbonetos Policíclicos Aromáticos , Rhodococcus , Tensoativos , Peixe-Zebra , Rhodococcus/metabolismo , Tensoativos/toxicidade , Tensoativos/química , Tensoativos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Animais , Células Imobilizadas/metabolismo , Polissorbatos/toxicidade , Polissorbatos/química , Poluentes Ambientais/toxicidade , Poluentes Ambientais/metabolismo , Poluentes Ambientais/química , Fenantrenos/toxicidade , Fenantrenos/metabolismo , Fenantrenos/química , Embrião não Mamífero/efeitos dos fármacosRESUMO
Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.
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Caenorhabditis elegans is a valuable model to study organ, tissue, and cell-type responses to external cues. However, the nematode comprises multiple syncytial tissues with spatial coordinates corresponding to distinct nuclear transcriptomes. Here, we present a single-nucleus RNA sequencing (snRNA-seq) protocol that aims to overcome difficulties encountered with single-cell RNA sequencing in C. elegans. We describe steps for isolating C. elegans nuclei for downstream applications including snRNA-seq applied to the context of alcohol exposure. For complete details on the use and execution of this protocol, please refer to Truong et al. (2023).1.
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Caenorhabditis elegans , Núcleo Celular , Animais , Caenorhabditis elegans/genética , Análise de Sequência de RNA , Sequência de Bases , Núcleo Celular/genética , RNA Nuclear PequenoRESUMO
Zebrafish have become an essential tool in screening for developmental neurotoxic chemicals and their molecular targets. The success of zebrafish as a screening model is partially due to their physical characteristics including their relatively simple nervous system, rapid development, experimental tractability, and genetic diversity combined with technical advantages that allow for the generation of large amounts of high-dimensional behavioral data. These data are complex and require advanced machine learning and statistical techniques to comprehensively analyze and capture spatiotemporal responses. To accomplish this goal, we have trained semi-supervised deep autoencoders using behavior data from unexposed larval zebrafish to extract quintessential "normal" behavior. Following training, our network was evaluated using data from larvae shown to have significant changes in behavior (using a traditional statistical framework) following exposure to toxicants that include nanomaterials, aromatics, per- and polyfluoroalkyl substances (PFAS), and other environmental contaminants. Further, our model identified new chemicals (Perfluoro-n-octadecanoic acid, 8-Chloroperfluorooctylphosphonic acid, and Nonafluoropentanamide) as capable of inducing abnormal behavior at multiple chemical-concentrations pairs not captured using distance moved alone. Leveraging this deep learning model will allow for better characterization of the different exposure-induced behavioral phenotypes, facilitate improved genetic and neurobehavioral analysis in mechanistic determination studies and provide a robust framework for analyzing complex behaviors found in higher-order model systems.
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Modern toxicology's throughput has dramatically increased due to alternative models, laboratory automation, and machine learning. This has enabled comparative studies across species and assays to prioritize chemical hazard potential and to understand how different model systems might complement one another. However, such comparative studies of high-throughput data are still in their infancy, with more groundwork needed to firmly establish the approach. Therefore, this study aimed to compare the bioactivity of the NIEHS Division of Translational Toxicology's (DTT) 87-compound developmental neurotoxicant (DNT) library in zebrafish and an in vitro high-throughput cell culture system. The early life-stage zebrafish provided a whole animal approach to developmental toxicity assessment. Chemical hits for abnormalities in embryonic zebrafish morphology, mortality, and behavior (ZBEscreen™) were compared with chemicals classified as high-risk by the Cell Health Index (CHI™), which is an outcome class probability from a machine learning classifier using 12 parameters from the SYSTEMETRIC® Cell Health Screen (CHS). The CHS was developed to assess human toxicity risk using supervised machine learning to classify acute cell stress phenotypes in a human leukemia cell line (HL60 cells) following a 4-h exposure to a chemical of interest. Due to the design of the screen, the zebrafish assays were more exhaustive, yielding 86 total bioactive hits, whereas the SYSTEMETRIC® CHS focusing on acute toxicity identified 20 chemicals as potentially toxic. The zebrafish embryonic and larval photomotor response assays (EPR and LPR, respectively) detected 40 of the 47 chemicals not found by the zebrafish morphological screen and CHS. Collectively, these results illustrate the advantages of using two alternative models in tandem for rapid hazard assessment and chemical prioritization and the effectiveness of CHI™ in identifying toxicity within a single multiparametric assay.
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Leucemia , Peixe-Zebra , Animais , Humanos , Bioensaio , Células HL-60 , LarvaRESUMO
The existence of thousands of per- and polyfluoroalkyl substances (PFAS) and evidence that some cause adverse health effects has created immense need to better understand PFAS toxicity and to move beyond one-chemical-at-a-time approaches to hazard assessment for this chemical class. The zebrafish model enables rapid assessment of large libraries of PFAS, powerful comparison of compounds in a single in vivo system, and evaluation across life stages and generations, and has led to significant advances in PFAS research in recent years. The focus of this review is to assess contemporary findings regarding PFAS toxicokinetics, toxicity and apical adverse health outcomes, and potential modes of action using the zebrafish model. Much of the peer-reviewed literature has focused on a small subset of PFAS structural subclasses, such as the perfluoroalkyl sulfonic acids and perfluoroalkyl carboxylic acids. However, recent data on more diverse PFAS structures are enabling prioritization of compounds of concern. Structure-activity comparisons and the utilization of modeling and 'omics technologies in zebrafish have greatly contributed to our understanding of the hazard potential for a growing number of PFAS and will surely inform our understanding and predictive capabilities for many more PFAS in the future.
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Ácidos Alcanossulfônicos , Fluorocarbonos , Animais , Peixe-Zebra , Fluorocarbonos/toxicidade , Ácidos Sulfônicos , Ácidos Carboxílicos , ToxicocinéticaRESUMO
Single-cell transcriptomic platforms provide an opportunity to map an organism's response to environmental cues with high resolution. Here, we applied single-nucleus RNA sequencing (snRNA-seq) to establish the tissue and cell type-resolved transcriptome of the adult C. elegans and characterize the inter- and trans-generational transcriptional impact of ethanol. We profiled the transcriptome of 41,749 nuclei resolving into 31 clusters, representing a diverse array of adult cell types including syncytial tissues. Following exposure to human-relevant doses of alcohol, several germline, striated muscle, and neuronal clusters were identified as being the most transcriptionally impacted at the F1 and F3 generations. The effect on germline clusters was confirmed by phenotypic enrichment analysis as well as by functional validation, which revealed a remarkable inter- and trans-generational increase in germline apoptosis, aneuploidy, and embryonic lethality. Together, snRNA-seq represents a valuable approach for the detailed examination of an adult organism's response to environmental exposures.
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Caenorhabditis elegans , Transcriptoma , Animais , Adulto , Humanos , Transcriptoma/genética , Perfilação da Expressão Gênica , Etanol/farmacologia , RNA Nuclear PequenoRESUMO
Fibroblast growth factor receptors (FGFRs) are transmembrane receptor tyrosine kinases that regulate multiple physiological processes. Aberrant activation of FGFR2 and FGFR3 has been linked to the pathogenesis of many tumor types, including cholangiocarcinoma and bladder cancer. Current therapies targeting the FGFR2/3 pathway exploiting small-molecule kinase inhibitors are associated with adverse events due to undesirable inhibition of FGFR1 and FGFR4. Isoform-specific FGFR2 and FGFR3 inhibitors that spare FGFR1 and FGFR4 could offer a favorable toxicity profile and improved therapeutic window to current treatments. Herein we disclose the discovery of dual FGFR2/FGFR3 inhibitors exploiting scaffold repurposing of a previously reported ALK2 tool compound. Structure-based drug design and structure-activity relationship studies were employed to identify selective and orally bioavailable inhibitors with equipotent activity toward wild-type kinases and a clinically observed gatekeeper mutant.
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The OSU/PNNL Superfund Research Program (SRP) represents a longstanding collaboration to quantify Polycyclic Aromatic Hydrocarbons (PAHs) at various superfund sites in the Pacific Northwest and assess their potential impact on human health. To link the chemical measurements to biological activity, we describe the use of the zebrafish as a high-throughput developmental toxicity model that provides quantitative measurements of the exposure to chemicals. Toward this end, we have linked over 150 PAHs found at Superfund sites to the effect of these same chemicals in zebrafish, creating a rich dataset that links environmental exposure to biological response. To quantify this response, we have implemented a dose-response modelling pipeline to calculate benchmark dose parameters which enable potency comparison across over 500 chemicals and 12 of the phenotypes measured in zebrafish. We provide a rich dataset for download and analysis as well as a web portal that provides public access to this dataset via an interactive web site designed to support exploration and re-use of these data by the scientific community at http://srp.pnnl.gov .
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Exposição Ambiental , Hidrocarbonetos Policíclicos Aromáticos , Peixe-Zebra , Animais , Humanos , Exposição Ambiental/análise , Substâncias Perigosas/análise , Noroeste dos Estados Unidos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/análiseRESUMO
Individuals within genetically diverse populations display broad susceptibility differences upon chemical exposures. Understanding the role of gene-environment interactions (GxE) in differential susceptibility to an expanding exposome is key to protecting public health. However, a chemical's potential to elicit GxE is often not considered during risk assessment. Previously, we've leveraged high-throughput zebrafish (Danio rerio) morphology screening data to reveal patterns of potential GxE effects. Here, using a population genetics framework, we apportioned variation in larval behavior and gene expression in three different PFHxA environments via mixed-effect modeling to assess significance of GxE term. We estimated the intraclass correlation (ICC) between full siblings from different families using one-way random-effects model. We found a significant GxE effect upon PFHxA exposure in larval behavior, and the ICC of behavioral responses in the PFHxA exposed population at the lower concentration was 43.7%, while that of the control population was 14.6%. Considering global gene expression data, a total of 3746 genes showed statistically significant GxE. By showing evidence that heritable genetics are directly affecting gene expression and behavioral susceptibility of individuals to PFHxA exposure, we demonstrate how standing genetic variation in a heterogeneous population such as ours can be leveraged to test for potential GxE.
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Upregulation of the fibroblast growth factor receptor (FGFR) signaling pathway has been implicated in multiple cancer types, including cholangiocarcinoma and bladder cancer. Consequently, small molecule inhibition of FGFR has emerged as a promising therapy for patients suffering from these diseases. First-generation pan-FGFR inhibitors, while highly effective, suffer from several drawbacks. These include treatment-related hyperphosphatemia and significant loss of potency for the mutant kinases. Herein, we present the discovery and optimization of novel FGFR2/3 inhibitors that largely maintain potency for the common gatekeeper mutants and have excellent selectivity over FGFR1. A combination of meticulous structure-activity relationship (SAR) analysis, structure-based drug design, and medicinal chemistry rationale ultimately led to compound 29, a potent and selective FGFR2/3 inhibitor with excellent in vitro absorption, distribution, metabolism, excretion (ADME), and pharmacokinetics in rat. A pharmacodynamic study of a closely related compound established that maximum inhibition of downstream ERK phosphorylation could be achieved with no significant effect on serum phosphate levels relative to vehicle.
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Neoplasias , Inibidores de Proteínas Quinases , Receptores de Fatores de Crescimento de Fibroblastos , Animais , Ratos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Transdução de Sinais , Relação Estrutura-Atividade , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/efeitos dos fármacosRESUMO
There is a growing need to establish alternative approaches for mixture safety assessment of polycyclic aromatic hydrocarbons (PAHs). Due to limitations with current component-based approaches, and the lack of established methods for using whole mixtures, a promising alternative is to use sufficiently similar mixtures; although, an established framework is lacking. In this study, several approaches are explored to form sufficiently similar mixtures. Multiple data streams including environmental concentrations and empirically and predicted toxicity data for cancer and non-cancer endpoints were used to prioritize chemical components for mixture formations. Air samplers were analyzed for unsubstituted and alkylated PAHs. A synthetic mixture of identified PAHs was created (Creosote-Fire Mix). Existing toxicity values and chemical concentrations were incorporated to identify hazardous components in the Creosote-Fire Mix. Sufficiently similar mixtures of the Creosote-Fire Mix were formed based on (1) relative abundance; (2) toxicity values; and (3) a combination approach incorporating toxicity and abundance. Hazard characterization of these mixtures was performed using high-throughput screening in primary normal human bronchial epithelium (NHBE) and zebrafish. Differences in chemical composition and potency were observed between mixture formation approaches. The toxicity-based approach (Tox Mix) was the most potent mixture in both models. The combination approach (Weighted-Tox Mix) was determined to be the ideal approach due its ability to prioritize chemicals with high exposure and hazard potential.
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Scope: Nitrate supplementation is a popular ergogenic aid that improves exercise performance by reducing oxygen consumption during exercise. We investigated the effect of nitrate exposure and exercise on metabolic pathways in zebrafish liver. Materials and methods: Fish were exposed to sodium nitrate (606.9 mg/L), or control water, for 21 days and analyzed at intervals during an exercise test. We utilized untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and measured gene expression of 24 genes central to energy metabolism and redox signaling. Results: We observed a greater abundance of metabolites involved in endogenous nitric oxide (NO) metabolism and amino acid metabolism in nitrate-treated liver at rest, compared to rested controls. In the absence of exercise, nitrate treatment upregulated expression of genes central to nutrient sensing (pgc1a), protein synthesis (mtor) and purine metabolism (pnp5a and ampd1) and downregulated expression of genes involved in mitochondrial fat oxidation (acaca and cpt2). Conclusion: Our data support a role for sub-chronic nitrate treatment in the improvement of exercise performance, in part, by improving NO bioavailability, sparing arginine, and modulating hepatic gluconeogenesis and glycolytic capacity in the liver.
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Ubiquitous anthropogenic contaminants of concern, per- and polyfluoroalkyl substances (PFAS) are frequently detected in the environment and human populations around the world. Diet is a predominate route of human exposure, and PFAS are frequently measured in food. Manufacturing trends have shifted from legacy PFAS to shorter-chain alternatives that are suggested to be safer, such as perfluorohexanoic acid (PFHxA). However, the current amount of data to support safety assessments of these alternatives is not yet sufficient. The present study investigated the effects of a 42-day dietary exposure to 1, 10, or 100 ng/g PFHxA in juvenile zebrafish. The zebrafish model was leveraged to interrogate morphometrics, fecundity, and numerous behavior endpoints across multiple generations. Dietary PFHxA exposure did not result in measurable body burden and did not affect growth, fecundity, adult social perception behavior, or associative learning. PFHxA exposure did induce abnormal adult anxiety behaviors in the F0 generation that persisted transgenerationally in the F1 and F2. Abnormal larval and juvenile behavior was observed in the F1 generation, but not in the F2. PFHxA juvenile dietary exposure induced subtle and multigenerational behavior effects that warrant further investigation of this and other alternative short-chain PFAS.
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Current attention is focused on determining the potential for per- and polyfluoroalkyl substances (PFAS) to adversely impact human health. Zebrafish are a popular biological model because they share early development pathways with humans. A dietary exposure paradigm is growing in popularity in the zebrafish model because the outcomes often translate to humans. To create a diet of known composition, it is crucial to understand background PFAS levels present in zebrafish diet. Background PFAS, if present, potentially confounds interpretation of toxicological data. To date, no studies document the PFAS background levels in laboratory fish diet and there is only limited information on some pet foods. The objective of this study was to develop and validate an analytical method for up to 50 target PFAS in high lipid and protein content laboratory fish diets and pet foods. Long-chain perfluoroalkyl carboxylic acids (C9-C13) and perfluorooctane sulfonate (PFOS) were quantified in 11 out of 16 laboratory fish diets and in three out of five pet fish foods. Foods for pet birds, lizards, and dogs were below the limit of detection for all PFAS. In two of the laboratory fish diets, PFOS concentrations were >1.3 ng/g and the total PFAS for the three laboratory fish diets exceeded 1.0 ng/g. Hundreds of biomedical laboratories across the world utilize these commercial laboratory fish diets, and these results indicate that numerous zebrafish colonies may be inadvertently receiving significant dietary PFAS exposures. In light of this new information, it is critical to design PFAS studies with appropriate controls with measured background PFAS concentrations in the diet and to urge caution when interpreting the results.
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Ácidos Alcanossulfônicos , Fluorocarbonos , Peixe-Zebra , Ácidos Alcanossulfônicos/toxicidade , Animais , Dieta/veterinária , Exposição Dietética , Fluorocarbonos/toxicidade , Humanos , LaboratóriosRESUMO
Understanding the mechanisms behind chemical susceptibility differences is key to protecting sensitive populations. However, elucidating gene-environment interactions (GxE) presents a daunting challenge. While mammalian models have proven useful, problems with scalability to an enormous chemical exposome and clinical translation faced by all models remain; therefore, alternatives are needed. Zebrafish (Danio rerio) have emerged as an excellent model for investigating GxE. This study used a combined bioinformatic and experimental approach to probe the mechanisms underlying chemical susceptibility differences in a genetically diverse zebrafish population. Starting from high-throughput screening (HTS) data, a genome-wide association study (GWAS) using embryonic fish exposed to 0.6 µM Abamectin revealed significantly different effects between individuals. A hypervariable region with two distinct alleles-one with G at the SNP locus (GG) and one with a T and the 16 bp deletion (TT)-associated with differential susceptibility was found. Sensitive fish had significantly lower sox7 expression. Due to their location and the observed expression differences, we hypothesized that these sequences differentially regulate sox7. A luciferase reporter gene assay was used to test if these sequences, alone, could lead to expression differences. The TT allele showed significantly lower expression than the GG allele in MCF-7 cells. To better understand the mechanism behind these expression differences, predicted transcription factor binding differences between individuals were compared in silico, and several putative binding differences were identified. EMSA was used to test for binding differences in whole embryo protein lysate to investigate these TF binding predictions. We confirmed that the GG sequence is bound to protein in zebrafish. Through a competition EMSA using an untagged oligo titration, we confirmed that the GG oligo had a higher binding affinity than the TT oligo, explaining the observed expression differences. This study identified differential susceptibility to chemical exposure in a genetically diverse population, then identified a plausible mechanism behind those differences from a genetic to molecular level. Thus, an HTS-compatible zebrafish model is valuable and adaptable in identifying GxE mechanisms behind susceptibility differences to chemical exposure.
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(1) Background: Disperse Blue 14, Disperse Red 9, Solvent Red 169 and Solvent Yellow 33 have been used to color smoke; however, they have not been comprehensively assessed for their potential health hazards. (2) Methods: To assess the effects of these dyes, zebrafish embryos were exposed from 6 to 120 h post fertilization (hpf) to 10-55 µM Disperse Red 9, 1-50 µM Solvent Red 169, 7.5-13.5 µM Solvent Yellow 33 or 133-314 µM Disperse Blue 14. Embryos were monitored for adverse effects on gene expression at 48 hpf as well as for mortality, development and behavior at 120 hpf. The dyes were examined for their potential to cross the blood-brain barrier. (3) Results: Solvent Yellow 33 and Disperse Blue 14 impaired development and behavior at all concentrations. Disperse Red 9 impaired behavior at all concentrations and development at all concentrations except for 10 µM. Solvent Red 169 caused no effects. Mortality was only seen in Disperse Blue 14 at 261.5 and 314 µM. Gene expression indicated impacts on neurodevelopment and folate and retinol metabolism as potential mechanisms of toxicity. (4) Conclusions: Smoke dyes have a high potential for causing developmental changes and neurotoxicity and should be examined more closely using comprehensive approaches as used here.