Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay

The acceleration of drug discovery due to combinatorial chemistry and high- throughput screening methods has increased the numbers of candidate pharmac euticals entering the drug development phase, and the capability to accurat ely predict whether drug candidates will induce various members of the drug -metabolizing cytochrome P450 (CYP) enzyme superfamily is currently of grea t interest to the pharmaceutical industry. In the present study, we describ e the rapid and reliable analysis of CYP induction in a readily obtained mo del system (cultured rat hepatocytes) using both real-time quantitative rev erse transcription-polymerase chain reaction (real-time RT-PCR) and the RNA invasive cleavage assay. The levels of members in the three primary induci ble rat CYP subfamilies (CYP1A1, CYP2B1/2, and CYP3A1) were analyzed in unt reated and induced (beta -naphthoflavone, phenobarbital, and hydrocortisone ) hepatocyte cultures under various media conditions to screen for optimal CYP induction profiles. The fold inductions measured by real-time RT-PCR an d the RNA invasive cleavage assay were also compared with enzyme activity m easurements in parallel cultures using liquid chromatography/double mass sp ectrometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these techniques, various cultur e conditions were examined for optimizing induction of the three CYP subfam ily members. Both real-time RT-PCR and the RNA invasive cleavage assay prov e to be effective methods for determining the effects of drugs on specific CYPs in primary rat hepatocytes.