The addition of mammalian cell culture medium impacts nanoparticle toxicity in zebrafish.

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.

PAH bioremediation with Rhodococcus rhodochrous ATCC 21198: Impact of cell immobilization and surfactant use on PAH treatment and post-remediation toxicity.

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.

Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

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.

Deep autoencoder-based behavioral pattern recognition outperforms standard statistical methods in high-dimensional zebrafish studies.

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.

3' RNA-seq is superior to standard RNA-seq in cases of sparse data but inferior at identifying toxicity pathways in a model organism.

Introduction: The application of RNA-sequencing has led to numerous breakthroughs related to investigating gene expression levels in complex biological systems. Among these are knowledge of how organisms, such as the vertebrate model organism zebrafish (Danio rerio), respond to toxicant exposure. Recently, the development of 3' RNA-seq has allowed for the determination of gene expression levels with a fraction of the required reads compared to standard RNA-seq. While 3' RNA-seq has many advantages, a comparison to standard RNA-seq has not been performed in the context of whole organism toxicity and sparse data. Methods and results: Here, we examined samples from zebrafish exposed to perfluorobutane sulfonamide (FBSA) with either 3' or standard RNA-seq to determine the advantages of each with regards to the identification of functionally enriched pathways. We found that 3' and standard RNA-seq showed specific advantages when focusing on annotated or unannotated regions of the genome. We also found that standard RNA-seq identified more differentially expressed genes (DEGs), but that this advantage disappeared under conditions of sparse data. We also found that standard RNA-seq had a significant advantage in identifying functionally enriched pathways via analysis of DEG lists but that this advantage was minimal when identifying pathways via gene set enrichment analysis of all genes. Conclusions: These results show that each approach has experimental conditions where they may be advantageous. Our observations can help guide others in the choice of 3' RNA-seq vs standard RNA sequencing to query gene expression levels in a range of biological systems.

Comparative analysis between zebrafish and an automated live-cell assay to classify developmental neurotoxicant chemicals.

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.

Advances in PAH mixture toxicology enabled by zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds produced by a variety of petrogenic and pyrogenic sources. PAHs inherently occur in the environment in complex mixtures. The early life-stage zebrafish model is a valuable tool for high-throughput screening (HTS) for toxicity of complex chemical mixtures due to its rapid development, high fecundity, and superb sensitivity to chemical insult. Zebrafish are amenable to exposure to surrogate mixtures as well as extracts of environmental samples and effect-directed analysis. In addition to its utility to HTS, the zebrafish has proven an excellent model for assessing chemical modes of action and identifying molecular initiating and other key events in an Adverse Outcome Pathway framework. Traditional methods of assessing PAH mixture toxicity prioritize carcinogenic potential and lack consideration of non-carcinogenic modes of action, assuming a similar molecular initiating event for all PAHs. Recent work in zebrafish has made it clear that while PAHs belong to the same chemical class, their modes of action can be divergent. Future research should use zebrafish to better classify PAHs by their bioactivity and modes of action to better understand mixture hazards.

Chronic Vitamin E Deficiency Dysregulates Purine, Phospholipid, and Amino Acid Metabolism in Aging Zebrafish Skeletal Muscle.

Muscle wasting occurs with aging and may be a result of oxidative stress damage and potentially inadequate protection by lipophilic antioxidants, such as vitamin E. Previous studies have shown muscular abnormalities and behavioral defects in vitamin E-deficient adult zebrafish. To test the hypothesis that there is an interaction between muscle degeneration caused by aging and oxidative damage caused by vitamin E deficiency, we evaluated long-term vitamin E deficiency in the skeletal muscle of aging zebrafish using metabolomics. Zebrafish (55 days old) were fed E+ and E- diets for 12 or 18 months. Then, skeletal muscle samples were analyzed using UPLC-MS/MS. Data were analyzed to highlight metabolite and pathway changes seen with either aging or vitamin E status or both. We found that aging altered purines, various amino acids, and DHA-containing phospholipids. Vitamin E deficiency at 18 months was associated with changes in amino acid metabolism, specifically tryptophan pathways, systemic changes in the regulation of purine metabolism, and DHA-containing phospholipids. In sum, while both aging and induced vitamin E deficiency did have some overlap in altered and potentially dysregulated metabolic pathways, each factor also presented unique alterations, which require further study with more confirmatory approaches.

Review of the zebrafish as a model to investigate per- and polyfluoroalkyl substance toxicity.

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.

A CRISPR-Cas9 mutation in sox9b long intergenic noncoding RNA (slincR) affects zebrafish development, behavior, and regeneration.

The role of long noncoding RNAs (lncRNAs) regulators of toxicological responses to environmental chemicals is gaining prominence. Previously, our laboratory discovered an lncRNA, sox9b long intergenic noncoding RNA (slincR), that is activated by multiple ligands of aryl hydrocarbon receptor (AHR). Within this study, we designed a CRISPR-Cas9-mediated slincR zebrafish mutant line to better understand its biological function in presence or absence of a model AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The slincRosu3 line contains an 18 bp insertion within the slincR sequence that changes its predicted mRNA secondary structure. Toxicological profiling showed that slincRosu3 is equally or more sensitive to TCDD for morphological and behavioral phenotypes. Embryonic mRNA-sequencing showed differential responses of 499 or 908 genes in slincRosu3 in absence or presence of TCDD Specifically, unexposed slincRosu3 embryos showed disruptions in metabolic pathways, suggesting an endogenous role for slincR. slincRosu3 embryos also had repressed mRNA levels of sox9b-a transcription factor that slincR is known to negatively regulate. Hence, we studied cartilage development and regenerative capacity-both processes partially regulated by sox9b. Cartilage development was disrupted in slincRosu3 embryos both in presence and absence of TCDD. slincRosu3 embryos also displayed a lack of regenerative capacity of amputated tail fins, accompanied by a lack of cell proliferation. In summary, using a novel slincR mutant line, we show that a mutation in slincR can have widespread impacts on gene expression and structural development endogenously and limited, but significant impacts in presence of AHR induction that further highlights its importance in the developmental process.

Coupling Environmental Whole Mixture Toxicity Screening with Unbiased RNA-Seq Reveals Site-Specific Biological Responses in Zebrafish.

Passive sampling device (PSD) extracts paired with developmental toxicity assays in Danio Rerio (zebrafish) are excellent sensors for whole mixture toxicity associated with the bioavailable non-polar organics at environmental sites. We expand this concept by incorporating RNA-Seq in 48-h post fertilization zebrafish statically exposed to PSD extracts from two Portland Harbor Superfund Site locations: river mile 6.5W (RM 6.5W) and river mile 7W (RM 7W). RM 6.5W contained higher concentrations of polycyclic aromatic hydrocarbons (PAHs), but the diagnostic ratios of both extracts indicated similar PAH sourcing and composition. Developmental screens determined RM 6.5W to be more toxic with the most sensitive endpoint being a "wavy" notochord malformation. Differential gene expression from exposure to both extracts was largely parallel, although more pronounced for RM 6.5W. When compared to the gene expression associated with individual chemical exposures, PSD extracts produced some gene signatures parallel to PAHs but were more closely matched by oxygenated-PAHs. Additionally, differential expression, reminiscent of the wavy notochord phenotype, was not accounted for by either class of chemical, indicating the potential of other contaminants driving mixture toxicity. These techniques offer a compelling method for non-targeted hazard characterization of whole mixtures in an in vivo vertebrate system without requiring complete chemical characterization.

The Superfund Research Program Analytics Portal: linking environmental chemical exposure to biological phenotypes.

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 .

11-Cl-BBQ, a select modulator of AhR-regulated transcription, suppresses lung cancer cell growth via activation of p53 and p27Kip1.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.

Defining the environmental determinants of dysbiosis at scale with zebrafish.

The gut microbiome, critical to maintaining vertebrate homeostasis, is susceptible to a various exposures. In some cases, these exposures induce dysbiosis, wherein the microbiome changes into a state conducive to disease progression. To better prevent, manage, and treat health disorders, we need to define which exposures induce dysbiosis. Contemporary methods face challenges due to the immense diversity of the exposome and the restricted throughput of conventional experimental tools used for dysbiosis evaluation. We propose integrating high-throughput model systems as an augment to traditional techniques for rapid identification of dysbiosis-inducing agents. Although high-throughput screening tools revolutionized areas such as pharmacology and toxicology, their incorporation in gut microbiome research remains limited. One particularly powerful high-throughput model system is the zebrafish, which affords access to scalable in vivo experimentation involving a complex gut microbiome. Numerous studies have employed this model to identify potential dysbiosis triggers. However, its potential could be further harnessed via innovative study designs, such as evaluation of synergistic effects from combined exposures, expansions to the methodological toolkit to discern causal effects of microbiota, and efforts to assess and improve the translational relevance of the model. Ultimately, this burgeoning experimental resource can accelerate the discovery of agents that underlie dysbiotic disorders.

Utilizing a Population-Genetic Framework to Test for Gene-Environment Interactions between Zebrafish Behavior and Chemical Exposure.

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.

Leveraging Multiple Data Streams for Prioritization of Mixtures for Hazard Characterization.

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.

Concentration-response gene expression analysis in zebrafish reveals phenotypically-anchored transcriptional responses to retene.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants and are associated with human disease. Canonically, many PAHs induce toxicity via activation of the aryl hydrocarbon receptor (AHR) pathway. While the interaction between PAHs and the AHR is well-established, understanding which AHR-regulated transcriptional effects directly result in observable phenotypes and which are adaptive or benign is important to better understand PAH toxicity. Retene is a frequently detected PAH in environmental sampling and has been associated with AHR2-dependent developmental toxicity in zebrafish, though its mechanism of toxicity has not been fully elucidated. To interrogate transcriptional changes causally associated with retene toxicity, we conducted whole-animal RNA sequencing at 48 h post-fertilization after exposure to eight retene concentrations. We aimed to identify the most sensitive transcriptomic responses and to determine whether this approach could uncover gene sets uniquely differentially expressed at concentrations which induce a phenotype. We identified a concentration-response relationship for differential gene expression in both number of differentially expressed genes (DEGs) and magnitude of expression change. Elevated expression of cyp1a at retene concentrations below the threshold for teratogenicity suggested that while cyp1a expression is a sensitive biomarker of AHR activation, it may be too sensitive to serve as a biomarker of teratogenicity. Genes differentially expressed at only non-teratogenic concentrations were enriched for transforming growth factor-ß (TGF-ß) signaling pathway disruption while DEGs identified at only teratogenic concentrations were significantly enriched for response to xenobiotic stimulus and reduction-oxidation reaction activity. DEGs which spanned both non-teratogenic and teratogenic concentrations showed similar disrupted biological processes to those unique to teratogenic concentrations, indicating these processes were disrupted at low exposure concentrations. Gene co-expression network analysis identified several gene modules, including those associated with PAHs and AHR2 activation. One, Module 7, was strongly enriched for AHR2-associated genes and contained the strongest responses to retene. Benchmark concentration (BMC) of Module seven genes identified a median BMC of 7.5 µM, nearly the highest retene concentration with no associated teratogenicity, supporting the hypothesis that Module seven genes are largely responsible for retene toxicity.

Experimental methods modestly impact interpretation of the effect of environmental exposures on the larval zebrafish gut microbiome.

Rapidly growing fields, such as microbiome science, often lack standardization of procedures across research groups. This is especially the case for microbiome investigations in the zebrafish (Danio rerio) model system, which is quickly becoming a workhorse system for understanding the exposure-microbiome-physiology axis. To guide future investigations using this model system, we defined how various experimental decisions affect the outcomes of studies on the effects of exogenous exposure on the zebrafish gut microbiome. Using a model toxicant, benzo[a]pyrene (BaP), we assessed how each of two dissection methods (gut dissection vs. whole fish), three DNA extraction kits (Qiagen Blood & Tissue, Macherey-Nagel NucleoSpin, and Qiagen PowerSoil), and inclusion of PCR replicates (single vs. pooled triplicate reactions) affected our interpretation of how exposure influences the diversity and composition of the gut microbiome, as well as our ability to identify microbiome biomarkers of exposure. We found that inclusion of PCR replicates had the smallest effect on our final interpretations, and the effects of dissection method and DNA extraction kit had significant effects in specific contexts, primarily in the cases of identifying microbial biomarkers.

Nitrate exposure reprograms hepatic amino acid and nutrient sensing pathways prior to exercise: A metabolomic and transcriptomic investigation in zebrafish (Danio rerio).

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.

Dietary Perfluorohexanoic Acid (PFHxA) Exposures in Juvenile Zebrafish Produce Subtle Behavioral Effects across Generations.

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.