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Profiling Environmental Chemicals in the Antioxidant Response Element Pathway using Quantitative High Throughput Screening (qHTS)
Citation:
Shukla, S. J., S. Huang, S. SIMMONS, R. Tice, K. L. Witt, AND M. Xia. Profiling Environmental Chemicals in the Antioxidant Response Element Pathway using Quantitative High Throughput Screening (qHTS). Presented at Society of Toxicology (SOT) Annual Meeting, Washington, DC, March 06 - 10, 2011.
Impact/Purpose:
This presentation will highlight the advances made in developing high- throughput assays for toxicity screening based on cellular stress response pathways and how these assays are used to establish quantitative biological activity relationships to characterize toxicant mode of action.
Description:
The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of oxidative stress, which can contribute to a number of diseases, including cancer. We screened 1408 NTP-provided substances in 1536-well qHTS format at concentrations ranging from 0.59 nM to 92 uM for their ability to induce this pathway in ARE-bla HepG2 cells transfected with a beta-lactamase reporter gene under the control of ARE and in HepG2cells transfected with a luciferase reporter measuring Nrf2 specific ARE activation. Raw data were normalized relative to beta-napththoflavone (100%) and DMSO (basal, 0%). Concentration response curves were generated for each substance, with EC50 values and efficacy calculated for substances classified as positive in either assay. We identified 364 and 44 compounds that stimulated ARE in the bla and luciferase assays, respectively, with 34 of these compounds active in both assays. Based on either a positive response in both assays or a structure-activity relationship to an active compound, we re-tested 63 compounds in the two ARE assays and also for their ability to deplete glutathione, as a downstream assay. Fifty-six out of 63 compounds were confirmed for ARE activity and several (e.g., hydroquinone) additionally depleted glutathione levels. Our results indicate the robustness of these two different approaches for assessing the ability of much larger compound libraries to induce ARE. Supported by NIEHS Interagency Agreement Y2-ES-7020-01.(This is an abstract of a proposed presentation and does not necessarily reflect EPA policy).