Grantee Research Project Results

2014 Progress Report: Multi-Sensor Reporter Cell Technology to Assess Hazard Involving Endocrine Signaling Pathways

EPA Grant Number: R835165
Title: Multi-Sensor Reporter Cell Technology to Assess Hazard Involving Endocrine Signaling Pathways
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Aja, Hayley
Project Period: March 1, 2012 through February 28, 2016 (Extended to February 28, 2017)
Project Period Covered by this Report: March 1, 2014 through February 28,2015
Project Amount: $950,507
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 high-throughput evaluation of toxicity pathways is an emerging paradigm for future toxicity characterization. In order to meet the goals of this paradigm, methods are needed to assess toxicity pathways using as few assays as possible. We propose the use of multi-sensor cell-based reporter assays to meet this need. These assays will utilize both bioluminescence resonance energy transfer (BRET) and reporter gene transcription assays to evaluate interactions of individual chemicals and chemical mixtures along nuclear receptor-mediated signaling pathways. Methods are being developed for the evaluation of chemical effects on five ligand-responsive signaling pathways that regulate aspects of lipid/glucose metabolism and reproductive development.

Progress Summary:

Aim (1) Construct a next-generation, multi-sensor reporter assay for the detection of chemical interactions with the vertebrate RXR:PPAR signaling pathway. The number of signaling pathways and endpoints was increased. We now have assays for the evaluation of human PPARα:RXRα:SRC1, human PPARγ:RXRα:SRC1, human RXRα:RXRα:SRC1, daphnid MET:SRC, and daphnid E75:HR3.

Aim (2) Functionally validate the assays using multiple chemicals that impact different components of the signaling pathways. Several classes of chemicals were evaluated for their interactions with human PPARα:RXRα:SRC1, human PPARγ:RXRα:SRC1, human RXRα:RXRα:SRC1, and daphnid MET:SRC. The following was determined.

Xenobiotics have diverse structures are capable of activating the PPARα and PPARγ signaling pathways.
RXR ligands typically stimulate receptor assembly; while, PPAR ligands typically activate existing assembled receptors.
PPARα and PPARγ exhibit different specificities towards xenobiotic ligands.
PPARα and PPARγ signaling pathways can be differentially inhibited and activated by the same ligand.
Some xenobiotics can activate PPAR signaling pathways by activating RXRα.
RXRα ligands can activate one PPAR signaling pathway while having no effect on the other.
Chemicals shown to interact with the PPAR signaling network include organophosphates, brominated flame retardants, phthalates, insecticides and some natural products.
Exogenous ligands of the MET:SRC signaling pathway stimulate subunit dimerization in addition to receptor activation. The use of dimerization as an endpoint in chemical screening assays with this and possible other receptor pathways is both cost and time efficient.
The MET:SRC signaling pathway has thus far been shown to be responsive only to some insect growth regulating insecticides.
The HR3:E75 receptor complex spontaneously dimerizes which results in the inhibition of the gene regulatory activity of HR3.

Conclusions:

Exogenous ligands to PPARs and RXRs can elicit multiple effects on the PPAR signaling pathway with probable consequences on lipid and glucose homeostasis. The screening paradigm developed in this program will provide insight in the interactive effects of chemicals on this network that will facilitate the predictive screening of chemical mixtures.

BRET assays provide further insight into the specific mode of action of environmental chemicals on receptor signaling pathways.

The BRET assay has time and costs advantages over conventional reporter assays for screening chemical activity towards some receptors such as MET:SRC and human RXRα:RXRα.

Journal Articles:

No journal articles submitted with this report: View all 26 publications for this project

Supplemental Keywords:

multi-sensor reporter cell technology, endocrine disruptors, ligands, PPARs, signaling pathways

Progress and Final Reports:

Original Abstract

The 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.