CYTOCHROME P450 INDUCTION IN RAT HEPATOCYTES ASSESSED BY QUANTITATIVE REAL-TIME REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION AND THE RNA INVASIVE CLEAVAGE ASSAY MICHAEL E. BURCZYNSKI, MICHAEL MCMILLIAN, JAMES B. PARKER, STEWART BRYANT, ANGELIQUE LEONE, ELFRIDA R. GRANT, JACQUELINE M. THORNE, ZHONG ZHONG, ROBERT A. ZIVIN, AND MARK D. JOHNSON Departments of Pre-Clinical Drug Metabolism and Toxicology (M.E.B., M.M., J.B.P., S.B., A.L., M.D.J.) and Exploratory Technologies (E.R.G., J.M.T., Z.Z., R.A.Z.), The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey (Received March 1, 2001; accepted May 7, 2001) This paper is available online at http://dmd.aspetjournals.org ABSTRACT: The acceleration of drug discovery due to combinatorial chemistry and high-throughput screening methods has increased the num- bers of candidate pharmaceuticals entering the drug development phase, and the capability to accurately predict whether drug can- didates will induce various members of the drug-metabolizing cy- tochrome P450 (CYP) enzyme superfamily is currently of great interest to the pharmaceutical industry. In the present study, we describe the rapid and reliable analysis of CYP induction in a readily obtained model system (cultured rat hepatocytes) using both real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR) and the RNA invasive cleavage assay. The levels of members in the three primary inducible rat CYP subfamilies (CYP1A1, CYP2B1/2, and CYP3A1) were analyzed in untreated and induced (-naphthoflavone, phenobarbital, and hy- drocortisone) hepatocyte cultures under various media conditions to screen for optimal CYP induction profiles. The fold inductions measured by real-time RT-PCR and the RNA invasive cleavage assay were also compared with enzyme activity measurements in parallel cultures using liquid chromatography/double mass spec- trometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these tech- niques, various culture conditions were examined for optimizing induction of the three CYP subfamily members. Both real-time RT-PCR and the RNA invasive cleavage assay prove to be effective methods for determining the effects of drugs on specific CYPs in primary rat hepatocytes. The cytochromes P450 (CYPs 1 ) belong to a superfamily of en- zymes involved in a diverse array of physiological and xenobiotic metabolic pathways. Accordingly, the CYPs account for a substantial fraction of drug metabolism in humans. An area of concern in the pharmaceutical industry is the potential induction of various CYPs by pharmaceutical candidates, which if undetected can lead to toxicity or reduced efficacy due to drug-drug interactions (Park et al., 1996). Although species differences in metabolism exist between rat and humans (Kocarek et al., 1994; Xu et al., 2000), primary rat hepato- cytes cultured in vitro provide a reproducible and relatively accurate reflection of CYP induction in vivo. Progressively, primary hepato- cyte culture technology has implemented hormone supplementation (Dickins and Peterson, 1980; Dich et al., 1988; Sidhu and Omiecinski, 1995), substratum attachment (Jauregui et al., 1986; Schuetz et al., 1988; LeCluyse et al., 1994), enriched medium formulations (Wax- man et al., 1990; LeCluyse et al., 1999), and extracellular matrix (ECM) overlay approaches (Sidhu et al., 1994; Brown et al., 1995; Silva et al., 1998) in an attempt to recapitulate the whole organ responses to CYP inducers: these efforts have met with considerable success. Modern screening methodologies for monitoring gene expression, such as quantitative real-time reverse transcription-polymerase chain reaction (real time RT-PCR), are finding utility in clinical settings (Emig et al., 1999; Eckert et al., 2000) and are poised for mainstream use in drug development. Real-time RT-PCR quantitation of nucleic acid is based on detection of amplified products at the end of each cycle, which in turn permits a quantitative calculation of target RNA in an unknown sample by comparison with the kinetics of PCR product accumulation in samples of known quantity. Real-time RT-PCR fluorescence is typically monitored during am- plification by the hybridization of additional gene-specific oligonu- cleotide(s) that are fluorescently labeled to allow detection during PCR (Heid et al., 1996). In the TaqMan assay (Applied Biosystems, Foster City, CA), detection occurs following Taq-driven release of a 5' reporter dye from a 3' quencher molecule on a single hybridized probe during polymerase extension (Livak et al., 1995). In the Light- Cycler method (Roche Diagnostics, Indianapolis, IN), detection oc- curs during the annealing phase via a fluorescence resonance energy transfer (FRET) mechanism when an acceptor probe and a donor probe hybridize to a region of the amplified product in proximity 1 Abbreviations used are: CYP, cytochrome P450; ECM, extracellular matrix; RT-PCR, reverse transcription-polymerase chain reaction; PCR, polymerase chain reaction; FRET, fluorescence-resonance energy transfer; LC/MS/MS, liquid chromatography/double mass spectrometry; -NF, -naphthoflavone; HC, hy- drocortisone; PB, phenobarbital; T m , melting point temperature; DEX, dexameth- asone. Address correspondence to: Mark D. Johnson, Pre-Clinical Drug Metabolism and Molecular Toxicology, R. W. Johnson Pharmaceutical Research Institute, Ortho McNeil Pharmaceutical Building, Room 2286, Route 202, P.O. Box 300, Raritan, NJ 08869. E-mail: mjohnson@prius.jnj.com 0090-9556/01/2909-1243–1250$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 9 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 377/923364 DMD 29:1243–1250, 2001 Printed in U.S.A. 1243 by guest on December 6, 2013 dmd.aspetjournals.org Downloaded from