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Profiling Environmental Chemicals for Activity in the Antioxidant Response Element Signaling Pathway Using a High-Throughput Screening Approach
Citation:
Shukla, S., R. Huang, Steve Simmons, R. Tice, K. Witt, D. VanLeer, R. Ramabhadran, C. Austin, AND M. Xia. Profiling Environmental Chemicals for Activity in the Antioxidant Response Element Signaling Pathway Using a High-Throughput Screening Approach. ENVIRONMENTAL HEALTH PERSPECTIVES. National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, 120(8):1150-6, (2012).
Impact/Purpose:
This paper is the first from the Tox21 consortium to look comparatively at assays independently developed assays designed to measure the same biological process. The paper certainly lends credibility to the Agency's and their partners' introspection about the veracity of data quality generated by the HTS effort in Tox21. The paper also clearly shows that while there is some discordance between the assays, both provide unique insights into chemical inducers of oxidative stress.
Description:
1 ABSTRACT 2 3 BACKGROUND: Oxidative stress has been implicated in the pathogenesis of a variety 4 of diseases ranging from cancer to neurodegeneration, highlighti.ng the need to identify 5 chemicals that can induce this effect. The antioxidant response element (ARE) signaling 6 pathway plays an important role in the amelioration of oxidative stress. Thus, assays that 7 detect the up-regulation of this pathway could be useful for identifying such chemicals. 8 OBJECTIVES: To utilize cell-based reporter methods and bioinformatic tools to 9 efficiently screen a large collection of environmental chemicals for compounds that 10 induce oxidative stress. 11 METHODS: We utilized two cell-based ARE assay reporters, B-lactamase and 12 luciferase, to screen a U.S. National Toxicology Program 1408 (1353 unique) compound 13 library for their ability to induce oxidative stress in HepG2 cells using quantitative high 14 throughput screening (qHTS). 15 RESULTS: Roughly 3% (34 of 1342 unique) of compounds demonstrated activity across 16 both cell-based assays. Based on biological activity and structure-activity relationship 17 profiles, we selected 50 compounds for re-testing in the two ARE assays and in an 18 additional follow-up assay that employed a mutated ARE linked to B-lactamase. Based ori 19 this strategy, we identified 30 compounds that demonstrated activity in the ARE-bla and 20 ARE-luc assays and were able to determine structural features conferring compound 21 activity across assays. 22 CONCLUSIONS: Our results support the robustness of utilizing two different cell-based 23 approaches for identifying compounds that induce ARE signaling. These methods are 24 useful for prioritizing chemicals for further in-depth mechanism--based toxicity testing. 25 2
