EA451

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..

 Required from Customer
  • 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.

Deliverables

  • 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

Substrate

  • Test compound dissolved in DMSO, acetonitrile, or methanol

Assay System

  • Plated fresh human hepatocytes from 3 individual donors

Assay Conditions

  • 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

Assay QC

  • 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

Notes

  • 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.

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