Profiling environmental chemicals for activity in the antioxidant response element signaling pathway using a high throughput screening approach - PubMed (original) (raw)
Profiling environmental chemicals for activity in the antioxidant response element signaling pathway using a high throughput screening approach
Sunita J Shukla et al. Environ Health Perspect. 2012 Aug.
Abstract
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.
Conflict of interest statement
This document has been reviewed by the National Health and Environmental Effects Research Laboratory of the U.S. EPA and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute the endorsement of recommendation for use.
The authors declare they have no actual or potential competing financial interests.
Figures
Figure 1
Schematics of ARE-bla and ARE-luc reporter gene assays. The ARE-bla reporter harbors three AREs derived from the human NQO1 gene upstream of a basic (minimal) promoter that drives the expression of b-lactamase. The ARE-luc reporter gene harbors seven multimerized inverted consensus AREs upstream of a synthetic basic (minimal) promoter, which contains only Nrf2 binding sequences and CCAAT and TATA boxes that drive the expression of firefly luciferase.
Figure 2
Compounds from cluster 1 with selective activity in the ARE-bla (A) and ARE-luc (B) assays. Compounds shown were chosen for follow-up studies and tested across the two ARE reporter gene assays. Each concentration response curve and EC50 value represents the mean ± SD response of ARE-bla (n = 5) or ARE-luc (n = 3) determinations.
Figure 3
Compounds from cluster 5 with selective activity in the ARE-bla assay. Compounds shown were chosen for follow-up studies and tested across ARE reporter gene assays. Each concentration response curve and EC50 value represents the mean ± SD response of ARE-bla assay determinations (n = 5). Compounds were inactive in the ARE-luc assay (data not shown.)
Figure 4
Compounds from cluster 7 with selective activity in the ARE-bla (A) and ARE-luc (B) assays. Compounds shown were chosen for follow-up studies and tested across ARE reporter gene assays. Each concentration response curve and EC50 value represents the mean ± SD response of ARE-bla (n = 5) or ARE-luc (n = 3) determinations.
Figure 5
Compounds from cluster 8 with selective activity in the ARE-bla (A) and ARE-luc (B) assays. Compounds shown were chosen for follow-up studies and tested across ARE reporter gene assays. Each concentration response curve and EC50 value represents the mean ± SD response of ARE-bla (n = 5) or ARE-luc (n = 3) determinations.
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