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Multi-Sensor Reporter Cell Technology to Assess Hazard Involving Endocrine Signaling PathwaysEPA 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: Klieforth, Barbara I
Project Period: March 1, 2012 through February 28, 2016
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: Computational Toxicology , Endocrine Disruptors , Health , Ecosystems , Safer Chemicals
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. The PIs propose using multi-sensor cell-based reporter assays to meet this need. The proposed assay will allow for the single-assay assessment of the impact of a chemical or chemical mixture on several processes along a defined signaling pathway.
This proposed developmental work will use the RXR:PPAR signaling pathway--i.e. a pathway in which two nuclear receptors (the retinoid X and peroxisome proliferator-activated receptors) interact--which contributes to the regulation of development, reproduction, and metabolism. The development of a high-throughtput assay that would identify specific toxicological targets within the RXR signaling network would provide a wealth of mechanistic information on the potential hazard of individual chemicals.
This mechanistic data could be used to model toxicity associated with chemical mixtures.Approach:
The assay will be developed, refined, and tested in four distinct aims:
- The first aim will be to construct the assay system. This assay will simultaneously quantify RXR-PPAR complex formation in response to chemical exposure; the recruitment of co-activator SRC-1 by this complex; and, the activation of gene transcription by the complex. Protein interactions will be monitored in cells, in real-time, using bioluminescence resonance energy transfer. Transactivation of gene transcription will be measured using a luciferase reporter gene. RXR, PPAR, and the coactivator SRC-1 proteins will be tagged with luminescent or fluorescent proteins. The production of photons by these tags at defined wavelengths will be dependent upon association of the tagged protein with a partner protein tagged with a protein that emits photons that specifically excite the partner protein. Thus, emission occurs only when a specific exciter is partnered with a specific emitter. Transactivation of gene expression will be monitored by the production of red fluorescence by the reporter gene product. The multiple endpoints (light emission at defined wavelengths) will be monitored simultaneously following addition of a test chemical or chemical mixture using a fluorescence/luminescence plate reader.
- The second aim will be to evaluate the assay using a variety of compounds with known or suspected activity towards various targets within the assay system. Compounds with no known interactions with this pathway also will be evaluated to increase the inventory of active chemicals for use in subsequent aims.
- The third aim will be to evaluate the responsiveness and accuracy of the assay in quantitatively describing responses to binary chemical combinations. Binary combinations will be selected in which each chemical targets a different component of the pathway.
- The final aim will be to predict outcome of complex mixtures of chemicals using our Integrated Addition and Interaction model with model inputs derived from results collected in Aim 3. These model predictions will be compared to assay results obtained with the chemical mixtures in the multisensor assay.
Results will define an experimental approach that can be used in a high-throughput format to evaluate the response of hormone signaling pathways and networks to individual chemicals or mixtures. The assay also will have application across species and would significantly reduce uncertainty related to species sensitivity differences.