Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity.

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

Modernizing persistence-bioaccumulation-toxicity (PBT) assessment with high throughput animal-free methods.

The assessment of persistence (P), bioaccumulation (B), and toxicity (T) of a chemical is a crucial first step at ensuring chemical safety and is a cornerstone of the European Union's chemicals regulation REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals). Existing methods for PBT assessment are overly complex and cumbersome, have produced incorrect conclusions, and rely heavily on animal-intensive testing. We explore how new-approach methodologies (NAMs) can overcome the limitations of current PBT assessment. We propose two innovative hazard indicators, termed cumulative toxicity equivalents (CTE) and persistent toxicity equivalents (PTE). Together they are intended to replace existing PBT indicators and can also accommodate the emerging concept of PMT (where M stands for mobility). The proposed "toxicity equivalents" can be measured with high throughput in vitro bioassays. CTE refers to the toxic effects measured directly in any given sample, including single chemicals, substitution products, or mixtures. PTE is the equivalent measure of cumulative toxicity equivalents measured after simulated environmental degradation of the sample. With an appropriate panel of animal-free or alternative in vitro bioassays, CTE and PTE comprise key environmental and human health hazard indicators. CTE and PTE do not require analytical identification of transformation products and mixture components but instead prompt two key questions: is the chemical or mixture toxic, and is this toxicity persistent or can it be attenuated by environmental degradation? Taken together, the proposed hazard indicators CTE and PTE have the potential to integrate P, B/M and T assessment into one high-throughput experimental workflow that sidesteps the need for analytical measurements and will support the Chemicals Strategy for Sustainability of the European Union.

HumanMetagenomeDB: a public repository of curated and standardized metadata for human metagenomes.

Metagenomics became a standard strategy to comprehend the functional potential of microbial communities, including the human microbiome. Currently, the number of metagenomes in public repositories is increasing exponentially. The Sequence Read Archive (SRA) and the MG-RAST are the two main repositories for metagenomic data. These databases allow scientists to reanalyze samples and explore new hypotheses. However, mining samples from them can be a limiting factor, since the metadata available in these repositories is often misannotated, misleading, and decentralized, creating an overly complex environment for sample reanalysis. The main goal of the HumanMetagenomeDB is to simplify the identification and use of public human metagenomes of interest. HumanMetagenomeDB version 1.0 contains metadata of 69 822 metagenomes. We standardized 203 attributes, based on standardized ontologies, describing host characteristics (e.g. sex, age and body mass index), diagnosis information (e.g. cancer, Crohn's disease and Parkinson), location (e.g. country, longitude and latitude), sampling site (e.g. gut, lung and skin) and sequencing attributes (e.g. sequencing platform, average length and sequence quality). Further, HumanMetagenomeDB version 1.0 metagenomes encompass 58 countries, 9 main sample sites (i.e. body parts), 58 diagnoses and multiple ages, ranging from just born to 91 years old. The HumanMetagenomeDB is publicly available at https://webapp.ufz.de/hmgdb/.

Retinoic acid-dependent regulation of miR-19 expression elicits vertebrate axis defects.

Retinoic acid (RA) is involved in multifarious and complex functions necessary for vertebrate development. RA signaling is reliant on strict enzymatic regulation of RA synthesis and metabolism. Improper spatiotemporal expression of RA during development can result in vertebrate axis defects. microRNAs (miRNAs) are also pivotal in orchestrating developmental processes. While mechanistic links between miRNAs and axial development are established, the role of miRNAs in regulating metabolic enzymes responsible for RA abundance during axis formation has yet to be elucidated. Our results uncovered a role of miR-19 family members in controlling RA metabolism through the regulation of CYP26A1 during vertebrate axis formation. Global miRNA expression profiling showed that developmental RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. A reporter assay confirmed that cyp26a1 is a bona fide target of miR-19 in vivo. Transient knockdown of miR-19 phenocopied axis defects caused by RA exposure. Exogenous miR-19 rescued the axis defects induced by RA exposure. Taken together, these results indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 expression and subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development via regulation of RA signaling.

Investigation of Peroxisome Proliferator-Activated Receptor Genes as Requirements for Visual Startle Response Hyperactivity in Larval Zebrafish Exposed to Structurally Similar Per- and Polyfluoroalkyl Substances (PFAS).

BACKGROUND: Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. OBJECTIVES: The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. METHODS: Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to 0.43-7.86µM PFOS, 7.87-120µM PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration-response (BMCT) modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to 7.86µM PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed. RESULTS: Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, BMCT estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. DISCUSSION: This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. https://doi.org/10.1289/EHP13667.

New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project.

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

Toxicological disruption of signaling homeostasis: tyrosine phosphatases as targets.

The protein tyrosine phosphatases (PTPs) consist of a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTPs have critical roles in maintaining signaling quiescence in resting cells and in restoring homeostasis by effecting signal termination. Interest in these enzymes has increased in recent years following the discovery that the activity of PTPs is modulated through redox mechanisms during signaling. The molecular features that enable redox regulation of PTPs during physiological signaling also render them highly susceptible to oxidative and electrophilic inactivation by a broad spectrum of structurally disparate xenobiotic compounds. The loss of PTP activity results in a profound disregulation of protein phosphotyrosine metabolism, leading to widespread and persistent activation of signaling cascades in the cell.

MicroRNAs control neurobehavioral development and function in zebrafish.

microRNAs (miRNAs) have emerged as regulators of a broad spectrum of neurodevelopmental processes, including brain morphogenesis, neuronal differentiation, and survival. While the role of miRNAs in establishing and maintaining the developing nervous system is widely appreciated, the developmental neurobehavioral role of miRNAs has yet to be defined. Here we show that transient disruption of brain morphogenesis by ethanol exposure results in behavioral hyperactivity in larval zebrafish challenged with changes in lighting conditions. Aberrations in swimming activity persist in juveniles that were developmentally exposed to ethanol. During early neurogenesis, multiple gene expression profiling studies revealed widespread changes in mRNA and miRNA abundance in ethanol-exposed embryos. Consistent with a role for miRNAs in neurobehavioral development, target prediction analyses identified multiple miRNAs misexpressed in the ethanol-exposed cohorts that were also predicted to target inversely expressed transcripts known to influence brain morphogenesis. In vivo knockdown of miR-9/9* or miR-153c persistently phenocopied the effect of ethanol on larval and juvenile swimming behavior. Structural analyses performed on adults showed that repression of miR-153c during development impacts craniofacial skeletal development. Together, these data support an integral role for miRNAs in the establishment of vertebrate neurobehavioral and skeletal systems.

The AnimalAssociatedMetagenomeDB reveals a bias towards livestock and developed countries and blind spots in functional-potential studies of animal-associated microbiomes.

BACKGROUND: Metagenomic data can shed light on animal-microbiome relationships and the functional potential of these communities. Over the past years, the generation of metagenomics data has increased exponentially, and so has the availability and reusability of data present in public repositories. However, identifying which datasets and associated metadata are available is not straightforward. We created the Animal-Associated Metagenome Metadata Database (AnimalAssociatedMetagenomeDB - AAMDB) to facilitate the identification and reuse of publicly available non-human, animal-associated metagenomic data, and metadata. Further, we used the AAMDB to (i) annotate common and scientific names of the species; (ii) determine the fraction of vertebrates and invertebrates; (iii) study their biogeography; and (iv) specify whether the animals were wild, pets, livestock or used for medical research. RESULTS: We manually selected metagenomes associated with non-human animals from SRA and MG-RAST.  Next, we standardized and curated 51 metadata attributes (e.g., host, compartment, geographic coordinates, and country). The AAMDB version 1.0 contains 10,885 metagenomes associated with 165 different species from 65 different countries. From the collected metagenomes, 51.1% were recovered from animals associated with medical research or grown for human consumption (i.e., mice, rats, cattle, pigs, and poultry). Further, we observed an over-representation of animals collected in temperate regions (89.2%) and a lower representation of samples from the polar zones, with only 11 samples in total. The most common genus among invertebrate animals was Trichocerca (rotifers). CONCLUSION: Our work may guide host species selection in novel animal-associated metagenome research, especially in biodiversity and conservation studies. The data available in our database will allow scientists to perform meta-analyses and test new hypotheses (e.g., host-specificity, strain heterogeneity, and biogeography of animal-associated metagenomes), leveraging existing data. The AAMDB WebApp is a user-friendly interface that is publicly available at https://webapp.ufz.de/aamdb/ .

Innovative tools and methods for toxicity testing within PARC work package 5 on hazard assessment.

New approach methodologies (NAMs) have the potential to become a major component of regulatory risk assessment, however, their actual implementation is challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) was designed to address many of the challenges that exist for the development and implementation of NAMs in modern chemical risk assessment. PARC's proximity to national and European regulatory agencies is envisioned to ensure that all the research and innovation projects that are initiated within PARC agree with actual regulatory needs. One of the main aims of PARC is to develop innovative methodologies that will directly aid chemical hazard identification, risk assessment, and regulation/policy. This will facilitate the development of NAMs for use in risk assessment, as well as the transition from an endpoint-based animal testing strategy to a more mechanistic-based NAMs testing strategy, as foreseen by the Tox21 and the EU Chemical's Strategy for Sustainability. This work falls under work package 5 (WP5) of the PARC initiative. There are three different tasks within WP5, and this paper is a general overview of the five main projects in the Task 5.2 'Innovative Tools and methods for Toxicity Testing,' with a focus on Human Health. This task will bridge essential regulatory data gaps pertaining to the assessment of toxicological prioritized endpoints such as non-genotoxic carcinogenicity, immunotoxicity, endocrine disruption (mainly thyroid), metabolic disruption, and (developmental and adult) neurotoxicity, thereby leveraging OECD's and PARC's AOP frameworks. This is intended to provide regulatory risk assessors and industry stakeholders with relevant, affordable and reliable assessment tools that will ultimately contribute to the application of next-generation risk assessment (NGRA) in Europe and worldwide.

Bioinformatics Resource Manager v2.3: an integrated software environment for systems biology with microRNA and cross-species analysis tools.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNAs that direct post-transcriptional regulation of protein coding genes. Recent studies have shown miRNAs are important for controlling many biological processes, including nervous system development, and are highly conserved across species. Given their importance, computational tools are necessary for analysis, interpretation and integration of high-throughput (HTP) miRNA data in an increasing number of model species. The Bioinformatics Resource Manager (BRM) v2.3 is a software environment for data management, mining, integration and functional annotation of HTP biological data. In this study, we report recent updates to BRM for miRNA data analysis and cross-species comparisons across datasets. RESULTS: BRM v2.3 has the capability to query predicted miRNA targets from multiple databases, retrieve potential regulatory miRNAs for known genes, integrate experimentally derived miRNA and mRNA datasets, perform ortholog mapping across species, and retrieve annotation and cross-reference identifiers for an expanded number of species. Here we use BRM to show that developmental exposure of zebrafish to 30 uM nicotine from 6-48 hours post fertilization (hpf) results in behavioral hyperactivity in larval zebrafish and alteration of putative miRNA gene targets in whole embryos at developmental stages that encompass early neurogenesis. We show typical workflows for using BRM to integrate experimental zebrafish miRNA and mRNA microarray datasets with example retrievals for zebrafish, including pathway annotation and mapping to human ortholog. Functional analysis of differentially regulated (p<0.05) gene targets in BRM indicates that nicotine exposure disrupts genes involved in neurogenesis, possibly through misregulation of nicotine-sensitive miRNAs. CONCLUSIONS: BRM provides the ability to mine complex data for identification of candidate miRNAs or pathways that drive phenotypic outcome and, therefore, is a useful hypothesis generation tool for systems biology. The miRNA workflow in BRM allows for efficient processing of multiple miRNA and mRNA datasets in a single software environment with the added capability to interact with public data sources and visual analytic tools for HTP data analysis at a systems level. BRM is developed using Java™ and other open-source technologies for free distribution (http://www.sysbio.org/dataresources/brm.stm).

Monoassociation with bacterial isolates reveals the role of colonization, community complexity and abundance on locomotor behavior in larval zebrafish.

BACKGROUND: Across taxa, animals with depleted intestinal microbiomes show disrupted behavioral phenotypes. Axenic (i.e., microbe-free) mice, zebrafish, and fruit flies exhibit increased locomotor behavior, or hyperactivity. The mechanism through which bacteria interact with host cells to trigger normal neurobehavioral development in larval zebrafish is not well understood. Here, we monoassociated zebrafish with either one of six different zebrafish-associated bacteria, mixtures of these host-associates, or with an environmental bacterial isolate. RESULTS: As predicted, the axenic cohort was hyperactive. Monoassociation with three different host-associated bacterial species, as well as with the mixtures, resulted in control-like locomotor behavior. Monoassociation with one host-associate and the environmental isolate resulted in the hyperactive phenotype characteristic of axenic larvae, while monoassociation with two other host-associated bacteria partially blocked this phenotype. Furthermore, we found an inverse relationship between the total concentration of bacteria per larvae and locomotor behavior. Lastly, in the axenic and associated cohorts, but not in the larvae with complex communities, we detected unexpected bacteria, some of which may be present as facultative predators. CONCLUSIONS: These data support a growing body of evidence that individual species of bacteria can have different effects on host behavior, potentially related to their success at intestinal colonization. Specific to the zebrafish model, our results suggest that differences in the composition of microbes in fish facilities could affect the results of behavioral assays within pharmacological and toxicological studies.

Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles.

Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

Molecular signaling networks that choreograph epimorphic fin regeneration in zebrafish - a mini-review.

This short review provides a current synopsis of caudal fin regeneration in zebrafish with an emphasis on the molecular signaling networks that dictate epimorphic regeneration. At the outset, the fundamentals of caudal fin architecture and the stages of epimorphic regeneration are described. This is followed by a detailed look at the main networks implicated in fin regeneration, namely the Wnt, fibroblast growth factor, activin-betaA, retinoic acid and hedgehog signaling pathways. Throughout this mini-review, these molecular networks are examined through the lens of wound healing, blastema formation or regenerative outgrowth, three of the main stages of epimorphic regeneration. Next, the emerging role of noncoding RNAs as regulators of regeneration and mechanisms of regenerative termination are discussed. Finally, the implications for future research and the broader field of regenerative medicine are examined.

Exploring interactions between xenobiotics, microbiota, and neurotoxicity in zebrafish.

Susceptibility to xenobiotic exposures is variable. One factor that might account for this is the microbiome, which encompasses all microorganisms, their encoded genes, and associated functions that colonize a host organism. Microbiota harbor the capacity to affect the toxicokinetics and toxicodynamics of xenobiotic exposures. The neurotoxicological effects of environmental chemicals may be modified by intestinal microbes via the microbiota-gut-brain axis. This is a complex, bi-directional signaling pathway between intestinal microbes and the host nervous system. As a model organism, zebrafish are extremely well-placed to illuminate mechanisms by which microbiota modify the developmental neurotoxicity of environmental chemicals. The goal of this review article is to examine the microbiota-gut-brain axis in a toxicological context, specifically focusing on the strengths and weaknesses of the zebrafish model for the investigation of interactions between xenobiotic agents and host-associated microbes. Previous studies describing the relationship between intestinal microbes and host neurodevelopment will be discussed. From a neurotoxicological perspective, studies utilizing zebrafish to assess links between neurotoxicological outcomes and the microbiome are emphasized. Overall, there are major gaps in our understanding the mechanisms by which microbiota interact with xenobiotics to cause or modify host neurotoxicity. In this review, we demonstrate that zebrafish are an ideal model system for studying the complex relationship between chemical exposures, microorganisms, and host neurotoxicological outcomes.

Translational Toxicology in Zebrafish.

A major goal of translational toxicology is to identify adverse chemical effects and determine whether they are conserved or divergent across experimental systems. Translational toxicology encompasses assessment of chemical toxicity across multiple life stages, determination of toxic mode-of-action, computational prediction modeling, and identification of interventions that protect or restore health following toxic chemical exposures. The zebrafish is increasingly used in translational toxicology because it combines the genetic and physiological advantages of mammalian models with the higher-throughput capabilities and genetic manipulability of invertebrate models. Here, we review recent literature demonstrating the power of the zebrafish as a model for addressing all four activities of translational toxicology. Important data gaps and challenges associated with using zebrafish for translational toxicology are also discussed.

Evaluation of Developmental Toxicity, Developmental Neurotoxicity, and Tissue Dose in Zebrafish Exposed to GenX and Other PFAS.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are a diverse class of industrial chemicals with widespread environmental occurrence. Exposure to long-chain PFAS is associated with developmental toxicity, prompting their replacement with short-chain and fluoroether compounds. There is growing public concern over the safety of replacement PFAS. OBJECTIVE: We aimed to group PFAS based on shared toxicity phenotypes. METHODS: Zebrafish were developmentally exposed to 4,8-dioxa-3H-perfluorononanoate (ADONA), perfluoro-2-propoxypropanoic acid (GenX Free Acid), perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid (PFESA1), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluoro-n-octanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), or 0.4% dimethyl sulfoxide (DMSO) daily from 0-5 d post fertilization (dpf). At 6 dpf, developmental toxicity and developmental neurotoxicity assays were performed, and targeted analytical chemistry was used to measure media and tissue doses. To test whether aliphatic sulfonic acid PFAS cause the same toxicity phenotypes, perfluorobutanesulfonic acid (PFBS; 4-carbon), perfluoropentanesulfonic acid (PFPeS; 5-carbon), PFHxS (6-carbon), perfluoroheptanesulfonic acid (PFHpS; 7-carbon), and PFOS (8-carbon) were evaluated. RESULTS: PFHxS or PFOS exposure caused failed swim bladder inflation, abnormal ventroflexion of the tail, and hyperactivity at nonteratogenic concentrations. Exposure to PFHxA resulted in a unique hyperactivity signature. ADONA, PFESA1, or PFOA exposure resulted in detectable levels of parent compound in larval tissue but yielded negative toxicity results. GenX was unstable in DMSO, but stable and negative for toxicity when diluted in deionized water. Exposure to PFPeS, PFHxS, PFHpS, or PFOS resulted in a shared toxicity phenotype characterized by body axis and swim bladder defects and hyperactivity. CONCLUSIONS: All emerging fluoroether PFAS tested were negative for evaluated outcomes. Two unique toxicity signatures were identified arising from structurally dissimilar PFAS. Among sulfonic acid aliphatic PFAS, chemical potencies were correlated with increasing carbon chain length for developmental neurotoxicity, but not developmental toxicity. This study identified relationships between chemical structures and in vivo phenotypes that may arise from shared mechanisms of PFAS toxicity. These data suggest that developmental neurotoxicity is an important end point to consider for this class of widely occurring environmental chemicals. https://doi.org/10.1289/EHP5843.

Triclosan-Selected Host-Associated Microbiota Perform Xenobiotic Biotransformations in Larval Zebrafish.

Microbiota regulate important physiologic processes during early host development. They also biotransform xenobiotics and serve as key intermediaries for chemical exposure. Antimicrobial agents in the environment may disrupt these complex interactions and alter key metabolic functions provided by host-associated microbiota. To examine the role of microbiota in xenobiotic metabolism, we exposed zebrafish larvae to the antimicrobial agent triclosan. Conventionally colonized (CC), microbe-free axenic (AX), or axenic colonized on day 1 (AC1) zebrafish were exposed to 0.16-0.30 µM triclosan or vehicle on days 1, 6, 7, 8, and 9 days post fertilization (dpf). After 6 and 10 dpf, host-associated microbial community structure and putative function were assessed by 16S rRNA gene sequencing. At 10 dpf, triclosan exposure selected for bacterial taxa, including Rheinheimera. Triclosan-selected microbes were predicted to be enriched in pathways related to mechanisms of antibiotic resistance, sulfonation, oxidative stress, and drug metabolism. Furthermore, at 10 dpf, colonized zebrafish contained 2.5-3 times more triclosan relative to AX larvae. Nontargeted chemical analysis revealed that, relative to AX larvae, both cohorts of colonized larvae showed elevations in 23 chemical features, including parent triclosan and putative triclosan sulfate. Taken together, these data suggest that triclosan exposure selects for microbes that harbor the capacity to biotransform triclosan into chemical metabolites with unknown toxicity profiles. More broadly, these data support the concept that microbiota modify the toxicokinetics of xenobiotic exposure.

Host Developmental Toxicity of BPA and BPA Alternatives Is Inversely Related to Microbiota Disruption in Zebrafish.

Host-associated microbiota can biotransform xenobiotics, mediate health effects of chemical exposure, and play important roles in early development. Bisphenol A (BPA) is a widespread environmental chemical that has been associated with adverse endocrine and neurodevelopmental effects, some of which may be mediated by microbiota. Growing public concern over the safety of BPA has resulted in its replacement with structurally similar alternatives. In this study, we evaluated whether BPA and BPA alternatives alter microbiota and modulate secondary adverse behavioral effects in zebrafish. Zebrafish were developmentally exposed to BPA, Bisphenol AF (BPAF), Bisphenol B (BPB), Bisphenol F (BPF), or Bisphenol S (BPS). At 10 days post fertilization (dpf), toxicity assessments were completed and 16S rRNA gene sequencing was performed to evaluate potential chemical-dependent shifts in microbial community structure and predicted function. A standard light/dark behavioral assay was used to assess locomotor activity. Based on developmental toxicity assessments at 10 dpf, a range of potencies was observed: BPAF > BPB > BPF ∼ BPA > BPS. Analysis of 16S rRNA gene sequencing data showed significant concentration-dependent disruption of microbial community structure and enrichment of putative microbial functions with exposure to BPS, BPA, or BPF, but not BPB or BPAF. Interestingly, microbial disruption was inversely related to host developmental toxicity and estrogenicity. Exposure to BP analogs did not cause behavioral effects at 10 dpf. Our findings indicate that some BP analogs disrupt host microbiota early in life and demonstrate novel chemical-microbiota interactions that may add important context to current hazard identification strategies.

Development of a Zebrafish S1500+ Sentinel Gene Set for High-Throughput Transcriptomics.

Sentinel gene sets have been developed with the purpose of maximizing the information from targeted transcriptomic platforms. We recently described the development of an S1500+ sentinel gene set, which was built for the human transcriptome, utilizing a data- and knowledge-driven hybrid approach to select a small subset of genes that optimally capture transcriptional diversity, correlation with other genes based on large-scale expression profiling, and known pathway annotation within the human genome. While this detailed bioinformatics approach for gene selection can in principle be applied to other species, the reliability of the resulting gene set depends on availability of a large body of transcriptomics data. For the model organism zebrafish, we aimed to create a similar sentinel gene set (Zf S1500+ gene set); however, there is insufficient standardized expression data in the public domain to train the gene correlation model. Therefore, our strategy was to use human-zebrafish ortholog mapping of the human S1500+ genes and nominations from experts in the zebrafish scientific community. In this study, we present the bioinformatics curation and refinement process to produce the final Zf S1500+ gene set, explore whole transcriptome extrapolation using this gene set, and assess pathway-level inference. This gene set will add value to targeted high-throughput transcriptomics in zebrafish for toxicogenomic screening and other research domains.