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.

Leveraging Embryonic Zebrafish To Prioritize ToxCast Testing

“…Within vertebrates, abamectin activates γ-aminobutyric acid (GABA)-gated chloride channels and induces paralysis, 19,22 whereas butafenacil inhibits protoporphyrinogen oxidase (PPOX) and abolishes hemoglobin and red blood cell production. 21 As seen in our findings in early stage zebrafish embryos, abamectin and butafenacil exposure resulted in behavioral and hematologic abnormalities, respectively, in adult rodent toxicity studies required for registration. 23,24 As ToxCast data were publicly available for the same Phase I chemicals screened within our high-content screening assays, the objective of this study was (1) to determine whether highthroughput in vitro screening data from the ToxCast program would have prioritized abamectin and butafenacil for further testing and (2) to determine whether a single three-day zebrafish embryo assay is a strong predictor of ToxPi scores derived from a large battery of ToxCast assays.…”

15 years of zebrafish chemical screening

“…High-content imaging, for example, is becoming a powerful tool when combined with translucent zebrafish larvae. Using high-content screening researchers have identified butafenacil as a potent inducer of anemia [28], stimulators of pancreatic beta-cell proliferation [29], enhancers of FGF signaling [30], and the novel compound ‘lenaldekar,’ which selectively kills leukemic cells [9]. On the data analysis side, phenotypic ‘barcoding,’ where a complex endpoint such as a behavioral response is divided into discrete numeric units enabling comparisons of the phenotypes produced by thousands of chemicals to one another [31].…”

“…For example, the creation of transgenic fish containing inducible oncogenes under the control of the heat shock promoter ( hsp ) has facilitated the discovery of cancer suppressing pathways, such as a COX/β-catenin-dependent signaling pathway in the case of an acute myelogenous leukemia (AML)-ETO suppressor screen [19] and the MAPK/ERK and AKT/S6K1 pathways in an HRAS G12V suppressor screen [32]. Another example of two useful genetic tools is the vasculature marker lines Tg(fli:EGFP) and Tg(flk1:EGPF); their importance is highlighted by their use in no less than six chemical screens for angiogenesis modulators [28, 33–37]. Some screens have become quite sophisticated.…”

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“…Man-made environ mental endocrine disrupters called xenobiotics or xenoestrogens have also been reported to alter sex determination and differentiat ion in fishes because they primarily act either estrogen agonist or androgen antagonist in fish Whitehead, 2006;Filby et al, 2007;Leet at al., 2014;Wood et al, 2015). Gro wing concerns have been raised that the estrogenic environmental endocrine disrupters may have dramatic effect on sex determination and differentiation in wild life due to the fact that the applications of exogenous steroids can change the direction of phenotypic sex in fishes, (Co lborn and Clement, 1992;Curtis and Skaar, 2002;Leino et al, 2005;Wood et al, 2015).…”

“…To date, small fresh water fishes including fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes) (Ankley and Johnson, 2004) and zebrafish (Danio rerio) Leet et al, 2014) are widely used as toxicological research models to test the effects of toxic chemicals. A mong these fresh water fishes, the fathead minnow, a member o f the ecologically important Cyprin idae family and native to both lotic and lenthic environ ments across North America (Isaak, 1961;Held and Petarka, 1974;Ankley and Villeneuve, 2006;Leet et al, 2014;Villeneuve et al, 2014) is a common ly used test organism in toxicological studies (Duda and Butner, 1993;Ankley and Villeneuve, 2006;Villeneuve et al, 2014). Although, the pattern of gonadal sex differentiat ion was studied in fathead minnows (Ugu z, 2008), the effects of exogenous steroids on sex differentiat ion and determination has not been studied in this species of fish.…”