Imaging Assessment of G-protein-coupled Estrogen Receptor ActivationEPA Grant Number: R835169
Title: Imaging Assessment of G-protein-coupled Estrogen Receptor Activation
Investigators: Volz, David C.
Institution: University of South Carolina at Columbia
EPA Project Officer: Klieforth, Barbara I
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: Computational Toxicology , Endocrine Disruptors , Health , Ecosystems , Safer Chemicals
The overall objective of this study is to develop a targeted in vivo imaging assay to screen and identify chemicals that exhibit aberrant G-protein-coupled estrogen receptor (GPER)-mediated developmental toxicity. Using zebrafish as a model for vertebrate embryogenesis, recent 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.
First, we will develop a 96-well plate-based imaging assay to assess the potential impacts of reference GPER agonists on cardiac function, nervous system development, and muscle development within developing zebrafish. Following embryonic exposure to increasing concentrations of reference GPER agonists in the presence or absence of G-15, we will quantify heart rates within living 96-hpf fish and, using a high-content fixation and immunolabeling procedure, quantify structural alterations to the nervous system and musculature within fixed 96-hpf fish. In addition, we will screen EPA’s library of teratogenic ToxCast™ Phase-I chemicals in concentration-response format to (1) quantify functional effects on the heart; (2) quantify structural effects on the nervous system and musculature; and (3) identify whether developmental toxicity is mediated via aberrant GPER activation.
We expect to provide a targeted imaging assay that links chemical mechanism of toxicity to target organ effects and heart physiology within individual 96-hpf zebrafish. Importantly, with the utilization of selective, non-toxic agonists or antagonists for additional xenobiotic receptors, this assay can be easily adapted to identify other adverse outcome pathways (AOPs) during vertebrate embryogenesis. These results are consistent with and will have a positive impact on EPA’s mission, as abnormal ligand-induced GPER activation represents a potentially novel and understudied mechanism of toxicity for environmentally relevant chemicals that affect vertebrate embryo development. In addition, the proposed assay will enhance EPA’s existing ToxCast™ zebrafish embryo assays by (1) providing a complementary approach to evaluate targeted effects on organogenesis and physiology and (2) improving classification of chemicals by toxicologically relevant AOPs.