Induction of CYP1A2, 2B6, and 3A In Fresh Human Hepatocytes
This assay is used to assess the potential of a test compound to induce CYP1A2, CYP2B6, and CYP3A in fresh human hepatocytes..
- Either a minimum of 600 µL of test compound at 10 mM in DMSO, or 10 mg of powder
- The molecular mass (exact mass) of test compound and its salt form
- The MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.
- Table of MTS absorbance values (an index of cell viability) at each concentration of test compound and in controls
- Tables of CYP1A2, CYP2B6, and CYP3A mRNA expression for each treatment group
- Percentage of induction by each concentration of the test compound relative to the solvent and positive controls
- Test compound dissolved in DMSO, acetonitrile, or methanol
- Plated fresh human hepatocytes from 3 individual donors
- Three concentrations of test compound, typically 1, 10 and 100 µM
- Triplicate incubations (N=3) at each concentration of test compound, positive controls, and negative controls
- Positive control compounds are known inducers of each CYP
- Negative control is the vehicle only
- Incubate hepatocytes with test compound, vehicle, and positive controls in parallel wells for 72 hours at 37°C, replacing with fresh compound-containing medium daily
- Determine mRNA expression of each CYP in test compound, vehicle and positive control wells
- In wells treated with positive control, CYP activity/mRNA expression >2-fold the activity in negative control
- Determine cell viability in the same wells used to determine CYP activity, using conversion of a tetrazolium salt (MTS) to the corresponding formazan product
- CYP-specific inducers as positive controls
|CYP Isoform||Positive Control (CYP Inducer)|
|1A2||Omeprazole, 50 µM|
|2B6||Phenobarbital, 1000 µM|
|3A||Rifampicin, 50 µM|
- Cell viability is determined by measuring the concentration of formazan dye converted from MTS by metabolically active hepatocytes based on absorbance at 492 nm.