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.

Time-Related Alteration of Aqueous-Phase Anthracene and Phenanthrene Photoproducts in the Presence of TiO2 Nanoparticles.

Polycyclic aromatic hydrocarbons (PAHs) and titanium dioxide (TiO2) nanoparticles (NPs) are photoactive environmental pollutants that can contaminate aquatic environments. Aqueous-phase interactions between PAHs and TiO2-NPs are of interest due to their emerging environmental relevance, particularly with the deliberate application of TiO2-NPs to remediate pollution events (e.g., oil spills). Our objective was to investigate anthracene (ANT) and phenanthrene (PHE) photoproduct formation and transformation following ultraviolet A (UVA) irradiation in the presence and absence of TiO2-NPs. ANT and PHE solutions were prepared alone or in combination with TiO2-NPs, UVA-irradiated, and either exposed to larval zebrafish or collected for chemical analyses of diverse hydroxylated PAHs (OHPAHs) and oxygenated PAHs (OPAHs). The expression profiles of genes encoding for enzymes involved in PAH metabolism showed PAH-specific and time-dependent inductions that demonstrated changes in PAH and photoproduct bioavailability in the presence of TiO2-NPs. Chemical analyses of PAH/NP solutions in the absence of zebrafish larvae identified diverse photoproducts of differing size and ring arrangements, which suggested photodissociation, recombination, and ring re-arrangements of PAHs occurred either during or following UVA irradiation. Both ANT and PHE solutions showed heightened oxidative potential following irradiation, but TiO2-NP-related increases in oxidative potential were PAH-specific. The exploitation of multiple analytical methods provided novel insights into distinct PAH photoactivity, TiO2-NP influence on photoproduct formation in a PAH-specific manner, and the significant role time plays in photochemical processes.

Treatment with Nitrate, but Not Nitrite, Lowers the Oxygen Cost of Exercise and Decreases Glycolytic Intermediates While Increasing Fatty Acid Metabolites in Exercised Zebrafish.

BACKGROUND: Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood. OBJECTIVES: We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production. METHODS: We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise. RESULTS: Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., ß-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts. CONCLUSIONS: Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.

Diverse PFAS produce unique transcriptomic changes linked to developmental toxicity in zebrafish.

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.

Environmental significance of PAH photoproduct formation: TiO2 nanoparticle influence, altered bioavailability, and potential photochemical mechanisms.

Interactions between polycyclic aromatic hydrocarbons (PAHs) and titanium dioxide (TiO2) nanoparticles (NPs) can produce unforeseen photoproducts in the aqueous phase. Both PAHs and TiO2-NPs are well-studied and highly persistent environmental pollutants, but the consequences of PAH-TiO2-NP interactions are rarely explored. We investigated PAH photoproduct formation over time for benzo[a]pyrene (BaP), fluoranthene (FLT), and pyrene (PYR) in the presence of ultraviolet A (UVA) using a combination of analytical and computational methods including, identification of PAH photoproducts, assessment of expression profiles for gene indicators of PAH metabolism, and computational evaluation of the reaction mechanisms through which certain photoproducts might be formed. Chemical analyses identified diverse photoproducts, but all PAHs shared a primary photoproduct, 9,10-phenanthraquinone (9,10-PQ), regardless of TiO2-NP presence. The computed reaction mechanisms revealed the roles photodissociation and singlet oxygen chemistry likely play in PAH mediated photochemical processes that result in the congruent production of 9,10-PQ within this study. Our investigation of PAH photoproduct formation has provided substantial evidence of the many, diverse and congruent, photoproducts formed from physicochemically distinct PAHs and how TiO2-NPs influence bioavailability and time-related formation of PAH photoproducts.

Developmental, Behavioral and Transcriptomic Changes in Zebrafish Embryos after Smoke Dye Exposure.

(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.

Morphological and Behavioral Effects in Zebrafish Embryos after Exposure to Smoke Dyes.

Solvent Violet 47 (SV47) and Disperse Blue 14 (DB14) are two anthraquinone dyes that were previously used in different formulations for the production of violet-colored smoke. Both dyes have shown potential for toxicity; however, there is no comprehensive understanding of their effects. Zebrafish embryos were exposed to SV47 or DB14 from 6 to 120 h post fertilization (hpf) to assess the dyes' potential adverse effects on developing embryos. The potential ability of both dyes to cross the blood-brain barrier was also assessed. At concentrations between 0.55 and 5.23 mg/L, SV47 showed a dose-dependent increase in mortality, jaw malformation, axis curvature, and edemas. At concentrations between 0.15 and 7.54 mg/L, DB14 did not have this same dose-dependence but had similar morphological outcomes at the highest doses. Nevertheless, while SV47 showed significant mortality from 4.20 mg/L, there was no significant mortality on embryos exposed to DB14. Regardless, decreased locomotor movement was observed at all concentrations of DB14, suggesting an adverse neurodevelopmental effect. Overall, our results showed that at similar concentrations, SV47 and DB14 caused different types of phenotypic effects in zebrafish embryos.

Multidimensional in vivo hazard assessment using zebrafish.

There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies.