Grantee Research Project Results
Final Report: Imaging Assessment of G-protein-coupled Estrogen Receptor Activation
EPA Grant Number: R835169Title: Imaging Assessment of G-protein-coupled Estrogen Receptor Activation
Investigators: Volz, David C.
Institution: University of South Carolina at Columbia
EPA Project Officer: Aja, Hayley
Project Period: June 1, 2012 through May 31, 2015
Project Amount: $1,063,460
RFA: Developing High-Throughput Assays for Predictive Modeling of Reproductive and Developmental Toxicity Modulated Through the Endocrine System or Pertinent Pathways in Humans and Species Relevant to Ecological Risk Assessment (2011) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
The overall objective of this study was to develop high-content screening (HCS) assays to rapidly identify chemicals that exhibit aberrant G-protein-coupled estrogen receptor (GPER)-mediated developmental toxicity. Using zebrafish as a model for vertebrate embryogenesis, published findings from our laboratory show that (1) GPER is expressed as early as 1 hpf and localized to the brain and heart during embryogenesis; (2) continuous exposure to a selective GPER agonist (G-1) – but not a structurally similar selective GPER antagonist (G-15) – results in gross abnormalities and disruption of axial muscles; and (3) G-1-induced effects are blocked by co-exposure to G-15, suggesting that aberrant GPER activation alone is responsible for G-1-induced developmental toxicity. Therefore, our working hypothesis was that xenobiotic-induced activation of GPER results in targeted effects on the cardiovascular and/or nervous system, leading to indirect adverse effects on muscle development within zebrafish larvae. We tested our central hypothesis and accomplished the overall objective of this application by pursuing the following two research objectives:
- Develop HCS assays to assess the potential impacts of reference GPER agonists on cardiovascular or nervous system development during zebrafish embryogenesis.
- Screen EPA’s teratogenic ToxCast Phase-I chemical library to identify and classify chemicals that mediate developmental toxicity via a GPER-dependent adverse outcome pathway (AOP).
Summary/Accomplishments (Outputs/Outcomes):
Research Objective 1: During Year 1 of this project, we accomplished Research Objective 1 by developing and optimizing two HCS assays that rapidly identify chemicals impacting cardiovascular or nervous system function in the absence of effects on survival and growth. Abstracts summarizing each these assays are below.
High-Content Screening Assay for Identification of Chemicals Impacting Cardiovascular Function
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 HCS assay that enables us to screen and identify chemicals affecting cardiovascular function at sub-lethal, non-teratogenic concentrations. Following static exposure of one embryo per well from 5-72 hours post-fertilization (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 endpoint across four independent control plates containing 384 initial embryos per plate. Survival and imaging success rates across these plates ranged from 93-99% and 42-74%, respectively. Criteria then were 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 non-teratogenic concentrations.
High-Content Screening Assay for Identification of Chemicals Impacting Spontaneous Activity
Although cell-based assays exist, rapid and cost-efficient HCS assays within intact organisms are needed to support prioritization for developmental neurotoxicity (DNT) testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (~19 hours post-fertilization, 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 concentration-response format. Following static exposure of one embryo per well from 5-25 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 87.5-100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the 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.
Research Objective 2: During Years 2 and 3 of this project, we accomplished Research Objective 2 by (1) screening the most acutely toxic chemicals within EPA’s ToxCast Phase-I library in concentration-response format using our HSC assay for cardiovascular toxicity; (2) identifying chemicals that may mediate cardiovascular toxicity via a GPER-dependent AOP; (3) conducting hypothesis-driven, follow-up studies that uncover mechanisms of toxicity for novel ‘hits’ – specifically, abamectin and butafenacil – identified from our screens during Years 1 and 2; and (4) determining whether high-throughput in vitro screening data from the ToxCast program would have prioritized abamectin and butafenacil for further testing. Abstracts summarizing each of these projects are below.
High-Content Screening in Zebrafish Embryos Identifies Butafenacil as a Potent Inducer of Anemia
After ranking developmental toxicity data generated from the 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 our HCS assay for cardiovascular toxicity. 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 a herbicide that inhibits protoporphyrinogen oxidase (PPOX) – 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 PPOX 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 PPOX 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.
Identification of Chemicals Mediating GPER-dependent Cardiovascular Toxicity
We relied on a 12-chemical training set that consisted of seven reference GPER agonists with increasing potency to human GPER (GPER-positive chemicals), as well as five reference chemicals that represent varying chemical classes and, to our knowledge, do not bind GPER nor initiate GPER-mediated AOPs (GPER-negative chemicals). All reference chemicals exhibited sufficient hydrophobicity (log Kow > 2) to ensure adequate chemical movement across the chorion during embryogenesis. The selective GPER antagonist (G-15) was also used to test whether cardiovascular toxicity was mitigated (or exacerbated) in the presence of G-15; the predicted log Kow and IC50 (for human GPER) for G-15 are 5.49 and 20 nM, respectively. With the exception of G-1 (a selective GPER agonist) and TCDD (a potent AHR agonist and well known cardiotoxicant), none of the GPER-positive and GPER-negative chemicals that we screened impacted cardiovascular development and function in the absence of effects on survival and body length. Despite these negative findings, we proceeded to test whether TCDD-induced cardiotoxicity was partially mediated via activation of GPER, as evidence in the literature suggests that TCDD-induced toxicity may, in part, be mediated by activation of non-genomic signaling. Unfortunately, by the time we were ready to conduct this follow-up study, our existing G-15 stocks were expired and G-15 is no longer sold in the United States by our original vendor. Therefore, we purchased new G-15 from an alternative vendor and, unlike G-15 obtained from our original vendor, found that G-15 from this alternative vendor was insoluble in 1X embryo media (EM) – a reverse osmosis (RO) water-based media we routinely use for rearing zebrafish embryos. However, since G-15 was soluble in RO water alone (rather than 1X EM), we reared zebrafish embryos in RO water to address this unexpected issue and, as a positive control, exposed embryos to G-1 in the presence or absence of G-15. While rearing control embryos in RO water alone resulted in no adverse effects on embryonic development (compared to rearing in 1X EM), G-15 was unable to block G-1-induced toxicity, suggesting that G-15 from this alternative vendor lacked GPER antagonist activity. As a result, we discontinued this project, as we no longer have access to a reliable source of G-15.
Abamectin Induces Rapid and Reversible Hypoactivity within Early Zebrafish Embryos
During early zebrafish embryogenesis, spontaneous tail contractions represent the first sign of locomotion and result from innervation of primary motoneuron axons to target axial muscles. Based on a high-content screen, we previously demonstrated that exposure of zebrafish embryos to abamectin – an avermectin insecticide – from 5-25 hours post-fertilization (hpf) abolished spontaneous activity in the absence of effects on survival and gross morphology. Therefore, the objective of this study was to begin investigating the mechanism of abamectin-induced hypoactivity in zebrafish. Similar to 384-well plates, static exposure of embryos to abamectin from 5-25 hpf in glass beakers resulted in elimination of activity at low micromolar concentrations. However, abamectin did not affect neurite outgrowth from spinal motoneurons and, compared with exposure from 5-25 hpf, embryos were equally susceptible to abamectin-induced hypoactivity when exposures were initiated at 10 and 23 hpf. Moreover, immersion of abamectin-exposed embryos in clean water resulted in complete recovery of spontaneous activity relative to vehicle controls, suggesting that abamectin reversibly activated ligand-gated chloride channels and inhibited neurotransmission. To test this hypothesis, we pretreated embryos to vehicle or non-toxic concentrations of fipronil or endosulfan – two insecticides that antagonize the g-aminobutyric acid (GABA) receptor – from 5-23 hpf, and then exposed embryos to vehicle or abamectin from 23-25 hpf. Interestingly, activity levels within abamectin-exposed embryos pretreated with either antagonist were similar to embryos exposed to vehicle alone. Using quantitative PCR and phylogenetic analysis, we then confirmed the presence of GABA receptor α1 and β2 subunits at 5, 10, and 23 hpf, and demonstrated that zebrafish GABA receptor subunits are homologous to mammalian GABA receptor subunits. Overall, our data collectively suggest that abamectin induces rapid and reversible hypoactivity within early zebrafish embryos, an effect that may be mediated through the GABA receptor.
Butafenacil: A positive Control for Identifying Anemia- and Porphyria-inducing Chemicals
Butafenacil is a herbicide that inhibits protoporphyrinogen oxidase (PPOX), an enzyme that catalyzes oxidation of protoporphyrinogen IX to protoporphyrin IX during chlorophyll and heme biosynthesis. Based on a high-content screen, we previously identified butafenacil as a potent inducer of anemia in zebrafish embryos. Therefore, the objective of this study was to begin investigating the utility of butafenacil as a positive control for identifying anemia- and porphyria-inducing chemicals. Static exposure to butafenacil from 5-72 hours post-fertilization (hpf) in glass beakers resulted in a concentration-dependent decrease in arterial circulation at low micromolar concentrations. At 72 hpf, the magnitude of butafenacil-induced anemia was similar when embryos were exposed in the presence or absence of light, whereas protoporphyrin accumulation and acute toxicity were significantly lower or absent when embryos were exposed under dark conditions. To identify sensitive developmental windows, we treated embryos to butafenacil from 5, 10, 24, or 48 hpf to 72 hpf in the presence of light, and found that anemia and protoporphyrin accumulation were present at 72 hpf following initiation of exposure at 5 and 10 hpf. On the contrary, protoporphyrin accumulation – but not anemia – was present following initiation of exposure at 24 hpf. Lastly, protoporphyrin accumulation at 72 hpf after exposure from 24-48 hpf suggests that protoporphyrins were not eliminated over a 24-h recovery period. Collectively, our data suggest that butafenacil may be a reliable positive control for identifying anemia- and porphyria-inducing chemicals, and that pharyngula (24-48 hpf) may be a critical window of susceptibility for butafenacil exposure.
Leveraging Embryonic Zebrafish to Prioritize ToxCast Testing
Based on two pilot high-content screens of acutely toxic chemicals within the ToxCast Phase-I Library, we previously demonstrated that exposure of zebrafish embryos to (1) abamectin from 5-25 hours post-fertilization (hpf) abolished spontaneous activity – an indicator of developmental neurotoxicity – in the absence of effects on survival and gross morphology, and (2) butafenacil from 5-72 hpf resulted in severe anemia in the absence of effects on cardiovascular development. Therefore, the objective of this study was to (1) determine whether high-throughput in vitro screening data from the ToxCast program would have prioritized abamectin and butafenacil for further testing and (2) determine whether a single 3-day zebrafish embryo assay is a strong predictor of Toxicological Priority Index (ToxPi) scores derived from ToxCast data. Although ToxPi scores are useful metrics that integrate ToxCast assay hit rates and chemical potency, assay hit rate was the primary driver for generation of higher ToxPi scores. In addition, due to the absence of ToxCast assays that identify PPOX inhibitors and/or chemicals affecting red blood cell production, butafenacil may not have been prioritized by the ToxCast program for future testing even though this chemical was identified as a potent inducer of anemia in zebrafish embryos. Lastly, based on a single 3-day assay using one 384-well microplate, 72-hpf zebrafish embryo survival was a strong predictor of ToxPi scores derived from a large, complex battery of nearly 800 ToxCast assay endpoints. Overall, our findings suggest that embryonic zebrafish may be valuable for prioritizing ToxCast testing as well as addressing toxicity pathways that may not be represented by the ToxCast assay battery.
Conclusions:
In summary, this project has led to the development and application of two different zebrafish-based HCS assays that provide comprehensive discovery platforms with 1) increased sample sizes, 2) broad concentration-response format, and 3) the ability to identify chemicals that target cardiovascular and early nervous system function at non-teratogenic concentrations. Although the first year was dedicated to technology (assay) development, the latter 2 years were focused on the application of these assays for chemical library screening and hypothesis-driven, follow-up studies that uncover mechanisms of toxicity for novel ‘hits’ identified from our screens. Findings generated from this project have resulted in a total of 7 journal articles and 20 national-level presentations during the 3-year project period. Over the long-term, we anticipate that these assays will help federal and state agencies prioritize chemicals for hypothesis-driven mode-of-action research as well as uncover mechanisms of developmental toxicity for complex environmental mixtures and understudied high-production volume chemicals.
Journal Articles on this Report : 8 Displayed | Download in RIS Format
Other project views: | All 28 publications | 8 publications in selected types | All 8 journal articles |
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Leet JK, Lindberg CD, Bassett LA, Isales GM, Yozzo KL, Raftery TD, Volz DC. High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia. PLoS ONE 2014;9(8):e104190, 10 pp. |
R835169 (2013) R835169 (Final) |
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Leet JK, Hipszer RA, Volz DC. Butafenacil: a positive control for identifying anemia-and porphyria-inducing chemicals. Toxicology Reports 2015;2:976-983. |
R835169 (Final) |
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McGee SP, Konstantinov A, Stapleton HM, Volz DC. Aryl phosphate esters within a major PentaBDE replacement product induce cardiotoxicity in developing zebrafish embryos: potential role of the aryl hydrocarbon receptor. Toxicological Sciences 2013;133(1):144-156. |
R835169 (Final) |
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Raftery TD, Isales GM, Yozzo KL, Volz DC. High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos. Environmental Science & Technology 2014;48(1):804-810. |
R835169 (2012) R835169 (2013) R835169 (Final) |
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Raftery TD, Volz DC. Abamectin induces rapid and reversible hypoactivity within early zebrafish embryos. Neurotoxicology and Teratology 2015;49:10-18. |
R835169 (Final) |
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Volz DC, Hipszer RA, Leet JK, Raftery TD. Leveraging embryonic zebrafish to prioritize ToxCast testing. Environmental Science & Technology Letters 2015;2(7):171-176. |
R835169 (Final) |
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Yozzo KL, McGee SP, Volz DC. Adverse outcome pathways during zebrafish embryogenesis: a case study with paraoxon. Aquatic Toxicology 2013;126:346-354. |
R835169 (Final) |
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Yozzo KL, Isales GM, Raftery TD, Volz DC. High-content screening assay for identification of chemicals impacting cardiovascular function in zebrafish embryos. Environmental Science & Technology 2013;47(19):11302-11310. |
R835169 (Final) |
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Supplemental Keywords:
zebrafish, high-content screening assay, cardiovascular toxicity, developmental neurotoxicity, ToxCastProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.