Grantee Research Project Results
2012 Progress 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 Period Covered by this Report: June 1, 2012 through May 31,2013
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 is 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 is 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 will test our central hypothesis and accomplish 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.
Progress Summary:
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.
HCS Assay for Identification of Chemicals Impacting Cardiovascular Function in Zebrafish Embryos
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 percent and 42-74 percent, respectively. Criteria were then 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.
HCS 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 percent and 93.3-100 percent, 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.
Future Activities:
For Year 2 of this project, we will 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. Based on data available from previous teratogenesis screens using zebrafish embryos, acute toxicity benchmarks (AC50) for chemicals within the EPA’s ToxCast™ Phase-I chemical library will first be ranked from most to least potent based on survival and gross malformations present at 6 dpf following a 8-hpf to 5-dpf exposure. Using the HCS assay summarized in Figure 1, we will then screen the most potent chemicals in concentration-response format (0.05-50 μM) to evaluate the ability of these assays to detect targeted effects on cardiovascular function. Following identification of lead candidates (‘hits’) from our pilot screens, we will then test whether targeted effects on the cardiovascular or nervous system are mitigated (or exacerbated) in the presence of a selective GPER antagonist (G-15). As part of this sub-aim, we will screen seven GPER-positive reference chemicals and five GPER-negative reference chemicals in the presence or absence of G-15 to test whether GPER inhibition partially or fully blocks reference chemical-induced cardiovascular toxicity or developmental neurotoxicity. Finally, during Year 2, we will develop and optimize a third HCS assay focused on quantification of neurite outgrowth within fixed and antibody-labeled zebrafish embryos.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 28 publications | 8 publications in selected types | All 8 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
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) |
Exit Exit Exit |
Supplemental Keywords:
Zebrafish, high-content screening assay, cardiovascular toxicity, developmental neurotoxicity, G-protein-coupled estrogen receptorProgress 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.