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.

Several locations in the Elizabeth River, VA, USA are highly contaminated with polycyclic aromatic hydrocarbons (PAHs), due to the release of creosote mixtures from wood treatment facilities. Interestingly, some populations of Atlantic killifish (Fundulus heteroclitus) inhabiting the Elizabeth River (ER) are resistant to PAH-induced teratogenesis. However, evolutionary resistance to PAHs due to chronic PAH exposure is associated with reduced fitness and increased susceptibility to other environmental stressors in at least one PAH-resistant ER killifish population. More specifically, wild-caught and first generation PAH-resistant juvenile killifish have altered metabolic demands when compared to non-resistant fish. Herein, we investigated this association further by examining a previously under-studied population captured from the creosote-contaminated site Republic Creosoting (Rep). We assessed PAH toxicity and effects on energy metabolism in Rep killifish in comparison with killifish from the reference site Kings Creek (KC). Following exposures to simple and complex PAH mixtures, Rep killifish exhibited several phenotypes associated with PAH resistance including decreased incidences of developmental cardiovascular deformities and recalcitrant cytochrome P450 1A (CYP1A) activity. We evaluated bioenergetics in killifish embryos throughout development and found elevated basal oxygen consumption rates in Rep embryos relative to KC embryos. Furthermore, juvenile F1 Rep fish had significantly lower maximal metabolic rates and aerobic scopes than KC juveniles. These results suggest that populations of killifish that have adapted or evolved to withstand the toxicity associated with PAHs consequently have altered energetic metabolism or demands. Such consequences could result in an enhanced vulnerability to other environmental and anthropogenic stressors in PAH-resistant killifish.

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