James J. Cali, Dongping Ma
Promega Corporation, Madison, WI
Aryl hydrocarbons induce transcription of the cytochrome P450 1A1 gene (CYP1A1) through aryl hydrocarbon receptor (AHR) activation. CYP1A1 is therefore used as a biomarker for exposure to AHR agonists such as the environmental toxicant 2,3,7,8-tetrachlorodibenzodioxin (TCDD). CYP1A1 induction can be monitored at the transcription level by northern blot analysis, qPCR or gene reporter assay, and at the protein level by western blot or CYP1A1 enzyme activity measurement. While transcriptional analysis and western blotting are labor intensive, the present cell based CYP1A1 enzyme assay is rapid, native and portable, meaning it can potentially be used on any unmodified AH-responsive cell type.
Here we describe this rapid, nondestructive bioluminescent cell based CYP1A1 enzyme assay. The assay uses a cell permeable bioluminogenic CYP1A1 substrate. Bioluminogenic substrates are proluciferins that are converted by the enzymes of interest to a product that is detected in a bioluminescent reaction with luciferase, correlating light output with target enzyme activity. A certain, proprietary bioluminogenic compound showed exquisite CYP1A1 selectivity over all other CYPs from a panel of 21 that were tested. In hepatocytes and HepG2 cells the assay detected robust activations by TCDD in a 96 well format. The non-disruptive format of the bioluminogenc assays left cells intact for an additional viability assay so that cytotoxicity and AHR activity was assessed from single culture wells. This bioluminescent assay enables a high throughput cell-based method for screening of AHR agonists.