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.