title: High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.
abstract: 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.
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Leet
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: Casey D
authors.initials: CD
authors.lastname: Lindberg
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Bassett
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Isales
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: Krystle L
authors.initials: KL
authors.lastname: Yozzo
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: Tara D
authors.initials: TD
authors.lastname: Raftery
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: David C
authors.initials: DC
authors.lastname: Volz
mesh_terms: D000740:Anemia / Q000139:chemically induced / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D002319:Cardiovascular System / Q000187:drug effects* / Q000473:pathology; D004625:Embryo, Nonmammalian / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000473:pathology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000473:pathology; D004785:Environmental Pollutants / Q000633:toxicity; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000633:toxicity*; D011743:Pyrimidines / Q000633:toxicity*; D014481:United States; D015027:Zebrafish
chemical_list: D004785:Environmental Pollutants; D006845:Hydrocarbons, Fluorinated; D011743:Pyrimidines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C487162:butafenacil
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