Profiling environmental chemicals for activity in the antioxidant response element signaling pathway using a high throughput screening approach.

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, ß-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 ß-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.

Identification of clinically used drugs that activate pregnane X receptors.

The pregnane X receptor (PXR) binds xenobiotics and regulates the expression of several drug-metabolizing enzymes and transporters. Human PXR (hPXR) activation and CYP3A4 induction can be involved in drug-drug interactions, resulting in reduced efficacy or increased toxicity. However, there are known species-specific differences with regard to PXR activation that should be taken into account when animal PXR data are extrapolated to humans. We profiled 2816 clinically used drugs from the National Institutes of Health Chemical Genomics Center Pharmaceutical Collection for their ability to activate hPXR and rat PXR (rPXR) at the cellular level, induce human CYP3A4 at the cellular level, and bind human PXR at the protein level. From 6 to 11% of drugs were identified as active across the four assays, which included assay-specific and pan-active compounds. The lowest concordance was observed between the hPXR and rPXR assays, and many compounds active in both assays nonetheless demonstrated significant potency differences between species. Analysis based on clustering potency values demonstrated the greatest activity correlation between the hPXR activation and CYP3A4 induction assays. Structure-activity relationship analysis identified chemical scaffolds that were pan-active (e.g., dihydropyridine calcium channel blockers) and others that were uniquely active in individual assays (e.g., steroids and fatty acids). These results provide important information on PXR activation by clinically used drugs, highlight the species specificity of PXR activation by xenobiotics, and provide a means of prioritizing compounds for follow-up studies and optimization efforts.