Grantee Research Project Results

2013 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, 2013 through February 28,2014
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 two-hybrid reporter gene 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 four 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. Effort during the first year focused upon Aim 1. This aim was 100% completed during the previous project period.

Aim (2) Functionally validate the assay using multiple chemicals that impact different components of the RXR:PPAR signaling pathway. Several classes of chemicals were evaluated for their interactions with human PPARa:RXRa:SRC1, human PPARa:RXRa:SRC1, human RXRa:RXRa:SRC1, and daphnid MET:SRC. The following was determined.

Xenobiotics have diverse structures are capable of activating the PPARa and PPARa signaling pathways.
PPARa and PPARa exhibit different specificities towards xenobiotic ligands.
PPARa and PPARa signaling pathways can be differentially inhibited and activated by the same ligand. Some xenobiotics can activate PPAR signaling pathways by activating RXRa.
RXRa ligands can activate one PPAR signaling pathway while having no effect on the other.
Simultaneous occupancy of a ligand on both PPAR and RXR does not appear to enhance activation of the PPAR:RXR complex over that observed by occupancy of a single subunit.
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.

Conclusions:

Exogenous ligands to PPARs and RXRs can elicit multiple effects on the PPAR signaling pathway with probably 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 with relevant chemicals and chemical mixtures are currently underway to provide further insight into the specific mode of action of environmental chemicals on this signaling pathway.

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

Journal Articles:

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

Supplemental Keywords:

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

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.