Profiling Environmental Chemicals for Activity in the Antioxidant Response Element Signaling Pathway Using a High Throughput Screening Approach

NIH Chemical Genomics Center, National Institutes of Health, Department of Health and Human Services, Rockville, Maryland, USA.
Environmental Health Perspectives (Impact Factor: 7.98). 05/2012; 120(8):1150-6. DOI: 10.1289/ehp.1104709
Source: PubMed


Background: Oxidative stress has been implicated in the pathogenesis of a variety of diseases ranging from cancer to neurodegeneration, highlighting the need to identify chemicals that can induce this effect. The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of oxidative stress. Thus, assays that detect the up-regulation of this pathway could be useful for identifying chemicals that induce oxidative stress.
Objectives: We used cell-based reporter methods and informatics tools to efficiently screen a large collection of environmental chemicals and identify compounds that induce oxidative stress.
Methods: We utilized two cell-based ARE assay reporters, β-lactamase and luciferase, to screen a U.S. National Toxicology Program 1,408-compound library (NTP 1408, which contains 1,340 unique compounds) for their ability to induce oxidative stress in HepG2 cells using quantitative high throughput screening (qHTS).
Results: Roughly 3% (34 of 1,340) of the unique compounds demonstrated activity across both cell-based assays. Based on biological activity and structure–activity relationship profiles, we selected 50 compounds for retesting in the two ARE assays and in an additional follow-up assay that employed a mutated ARE linked to β-lactamase. Using this strategy, we identified 30 compounds that demonstrated activity in the ARE-bla and ARE-luc assays and were able to determine structural features conferring compound activity across assays.
Conclusions: Our results support the robustness of using two different cell-based approaches for identifying compounds that induce ARE signaling. Together, these methods are useful for prioritizing chemicals for further in-depth mechanism-based toxicity testing.

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    • "addition to the AREc32 assay because it is based on a different cell line ( HepG2 liver cells ) which might reveal cell specific differences in the response . Addi - tionally , different reporter gene constructs may lead to a different responsiveness of assays depending on promoter / enhancer con - struction , ARE orientation , and other factors ( Shukla et al . , 2012 ) . The p53 - bla assay ( Yeh et al . , 2014 ) , derived from HCT - 116 hu - man colon carcinoma cells , was applied because activation of p53 has been discussed as marker for genotoxic properties of chemicals ( Duerksen - Hughes et al . , 1999 ) . P53 can be stabilized and thus activated by a variety of stress events , such as genotoxi"
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