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

M. E. Burczynski, M. McMillian, J. B. Parker, S. Bryant, A. Leone, E. R. Grant, J. M. Thorne, Z. Zhong, R. A. Zivin, M. D. Johnson

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The acceleration of drug discovery due to combinatorial chemistry and high-throughput screening methods has increased the numbers of candidate pharmaceuticals entering the drug development phase, and the capability to accurately predict whether drug candidates will induce various members of the drug-metabolizing cytochrome 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 (β-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 and the RNA invasive cleavage assay were also compared with enzyme activity measurements in parallel cultures using liquid chromatography/double mass spectrometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these techniques, 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.

Original languageEnglish
Pages (from-to)1243-1250
Number of pages8
JournalDrug Metabolism and Disposition
Volume29
Issue number9
StatePublished - Sep 11 2001

Fingerprint

RNA Cleavage
Polymerase chain reaction
Transcription
Cytochrome P-450 Enzyme System
Reverse Transcription
Rats
Hepatocytes
Assays
RNA
Polymerase Chain Reaction
Pharmaceutical Preparations
Real-Time Polymerase Chain Reaction
Cytochrome P-450 CYP2B1
High-Throughput Screening Assays
Culture Techniques
Cytochrome P-450 CYP1A1
Liquid chromatography
Drug Industry
Enzyme activity
Drug Discovery

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

Burczynski, M. E., McMillian, M., Parker, J. B., Bryant, S., Leone, A., Grant, E. R., ... Johnson, M. D. (2001). Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay. Drug Metabolism and Disposition, 29(9), 1243-1250.

Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay. / Burczynski, M. E.; McMillian, M.; Parker, J. B.; Bryant, S.; Leone, A.; Grant, E. R.; Thorne, J. M.; Zhong, Z.; Zivin, R. A.; Johnson, M. D.

In: Drug Metabolism and Disposition, Vol. 29, No. 9, 11.09.2001, p. 1243-1250.

Research output: Contribution to journalArticle

Burczynski, ME, McMillian, M, Parker, JB, Bryant, S, Leone, A, Grant, ER, Thorne, JM, Zhong, Z, Zivin, RA & Johnson, MD 2001, 'Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay', Drug Metabolism and Disposition, vol. 29, no. 9, pp. 1243-1250.
Burczynski, M. E. ; McMillian, M. ; Parker, J. B. ; Bryant, S. ; Leone, A. ; Grant, E. R. ; Thorne, J. M. ; Zhong, Z. ; Zivin, R. A. ; Johnson, M. D. / Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay. In: Drug Metabolism and Disposition. 2001 ; Vol. 29, No. 9. pp. 1243-1250.
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