Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants.

Targeted assays are needed to better evaluate effects of chemicals on organogenesis and begin classification of chemicals by toxicologically relevant modes-of-action. Using transgenic zebrafish (fli1:egfp) that stably express eGFP within vascular endothelial cells, we have developed and optimized a 384-well-based high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular function at sublethal, nonteratogenic concentrations. Following static exposure of one embryo per well from 5 to 72 h postfertilization (hpf), automated image acquisition procedures and custom image analysis protocols are used to quantify body length, circulation, heart rate, pericardial area (a biomarker for cardiac looping defects), and intersegmental vessel area within freshly hatched live embryos. After optimizing 72 hpf anesthetization procedures, we evaluated each end point across four independent control plates containing 384 initial embryos per plate. Survival and imaging success rates across these plates ranged from 93 to 99% and 42 to 74%, respectively. Criteria were then defined for assay success and analysis of treatments, and 10 chemicals were screened for targeted effects on cardiovascular function. Compared to existing zebrafish-based assays, this method provides a comprehensive discovery platform with (1) increased sample sizes; (2) broad concentration-response format; and (3) the ability to identify chemicals that target cardiovascular function at nonteratogenic concentrations.

Using zebrafish as a model, we previously reported that developmental exposure to triphenyl phosphate (TPP) – a high-production volume organophosphate-based flame retardant – results in dioxin-like cardiac looping impairments that are independent of the aryl hydrocarbon receptor. Using a pharmacologic approach, the objective of this study was to investigate the potential role of retinoic acid receptor (RAR) – a nuclear receptor that regulates vertebrate heart morphogenesis – in mediating TPP-induced developmental toxicity in zebrafish. We first revealed that static exposure of zebrafish from 5-72 hours post-fertilization (hpf) to TPP in the presence of non-toxic concentrations of an RAR antagonist (BMS493) significantly enhanced TPP-induced toxicity (relative to TPP alone), even though identical non-toxic BMS493 concentrations mitigated retinoic acid (RA)-induced toxicity. BMS493-mediated enhancement of TPP toxicity was not a result of differential TPP uptake or metabolism, as internal embryonic doses of TPP and diphenyl phosphate (DPP) – a primary TPP metabolite - were not different in the presence or absence of BMS493. Using real-time PCR, we then quantified the relative change in expression of cytochrome P450 26a1 (cyp26a1) – a major target gene for RA-induced RAR activation in zebrafish – and found that RA and TPP exposure resulted in a ∼5-fold increase and decrease in cyp26a1 expression, respectively, relative to vehicle-exposed embryos. To address whether TPP may interact with human RARs, we then exposed Chinese hamster ovary cells stably transfected with chimeric human RARα-, RARβ-, or RARγ to TPP in the presence of RA, and found that TPP significantly inhibited RA-induced luciferase activity in a concentration-dependent manner. Overall, our findings suggest that zebrafish RARs may be involved in mediating TPP-induced developmental toxicity, a mechanism of action that may have relevance to humans.

Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05–50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO) – an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors – flumioxazin – resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models.

Mitochondria, an integral component of cellular energy metabolism and other key functions, are extremely vulnerable to damage by environmental stressors. Although methods to measure mitochondrial function in vitro exist, sensitive, medium- to high-throughput assays that assess respiration within physiologically-relevant whole organisms are needed to identify drugs and/or chemicals that disrupt mitochondrial function, particularly at sensitive early developmental stages. Consequently, we have developed and optimized an assay to measure mitochondrial bioenergetics in zebrafish larvae using the XFe24 Extracellular Flux Analyzer. To prevent larval movement from confounding oxygen consumption measurements, we relied on MS-222-based anesthetization. We obtained stable measurement values in the absence of effects on average oxygen consumption rate and subsequently optimized the use of pharmacological agents for metabolic partitioning. To confirm assay reproducibility we demonstrated that triclosan, a positive control, significantly decreased spare respiratory capacity. We then exposed zebrafish from 5 hours post-fertilization (hpf) – 6 days post-fertilization (dpf) to three polycyclic aromatic hydrocarbons (PAHs) – benzo(a)pyrene (BaP), phenanthrene (Phe), and fluoranthene (FL) – and measured various fundamental parameters of mitochondrial respiratory chain function, including maximal respiration, spare respiratory capacity, mitochondrial and non-mitochondrial respiration. Exposure to all three PAHs decreased spare respiratory capacity and maximal respiration. Additionally, Phe exposure increased non-mitochondrial respiration and FL exposure decreased mitochondrial respiration and increased non-mitochondrial respiration. Overall, this whole organism-based assay provides a platform for examining mitochondrial dysfunction in vivo at critical developmental stages. It has important implications in biomedical sciences, toxicology and ecophysiology, particularly to examine the effects of environmental chemicals and/or drugs on mitochondrial bioenergetics.