National Center for Advancing Translational Sciences [NCATS]
NIH Chemical Genomics Center [NCGC]
U.S. Environmental Protection Agency [EPA]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

Assay Overview:

Oxidative stress has been implicated in the pathogenesis of a variety of diseases ranging from cancer to neurodegeneration. The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of oxidative stress. The CellSensor ARE-bla HepG2 cell line (Invitrogen) can be used for analyzing the Nrf2/antioxidant response signaling pathway. Nrf2 (NF-E2-related factor 2) and Nrf1 are transcription factors that bind to AREs and activate these genes. The CellSensor ARE-bla Hep G2 cell line contains a beta-lactamase reporter gene under control of the Antioxidant Response Element (ARE) stably integrated into HepG2 cells. The activation of the report gene under culture conditions can be detected by fluorescence intensity. This cell line can be used to screen chemicals in a 1536-well plate format and to identify agonists that induce oxidative stress.

Assay Protocol Summary:

The ARE-bla cells were dispensed at 2,000 cells/5 ul/well in 1536-well black wall/clear bottom plates using a Multidrop dispenser. After the assay plates were incubated at a 37 C/5% CO2 incubator for 5 hours, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate by a pin tool. The plates were incubated at 37 C for 16 hours. After 1 uL of LiveBLAzer B/G FRET substrate was added using a Flying Reagent Dispenser, the plates were incubated at room temperature for 2 hours, and fluorescence intensity was measured by an Envision plate reader.

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.