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:

The Aryl hydrocarbon Receptor (AhR) plays a critical role in the development of cancer or neoplasm and its formation is most likely due to genetic mutations and the influence of environment. Exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAH), their halogenated derivatives and heterocyclic amines causes the induction of phase I and II enzymes. The transcriptional activation of CYP1A2 is directly regulated by AhR.

A cell based assay (1A2-DRE cells, developed by Puracyp, Inc., CA) was used to assess the activation of AhR. Human hepatoma cells were stably transfected with the dioxin response element (DRE) and CYP1A2 promoter fused to a luciferase gene. Increased luciferase activity can be used to identify the compounds that cause the induction of CYP1A2 and AhR activation.

Assay Protocol Summary:

1,200 cells in 5 uL/well were dispensed into white wall/solid bottom 1536-well plates using a Multidrop Combi (Thermo Scientific) dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, the plates were incubated 24 hr at 37 C and 5% CO2. 5 ul One-Glo (Promega) was then added to each well and following 30 min incubation at room temperature, luciferase activity was quantified on a ViewLux CCD-based 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.