Cytochrome P450 1A2 Messenger RNA is a More Reliable Marker than Cytochrome P450 1A2 Activity, Phenacetin O-Deethylation, for Assessment of Induction Potential of Drug-Metabolizing Enzymes Using HepaRG Cells

Drug Metab Lett. 2018;12(1):14-23. doi: 10.2174/1872312812666180119114013.

Abstract

Background: The HepaRG cells have key drug metabolism functionalities comparable to those of primary human hepatocytes. Many studies have reported that this cell line can be used as a reliable in vitro model for human drug metabolism studies, including the assessment of cytochrome P450 (CYP) induction.

Objectives: The objective of this study is to determine whether CYP mRNA level measurement is superior to the CYP enzyme activity measurement as a convenient high-throughput method for evaluating CYP induction potential using HepaRG cells.

Methods: QuantiGene Plex 2.0 Assay and LC/MS/MS. mRNA expression levels and enzyme activities of CYP1A2, CYP2B6, and CYP3A in HepaRG cells treated with prototypical inducers of each CYP isoform [omeprazole (OME) for CYP1A2, phenobarbital (PB) for CYP2B6, and rifampicin (RIF) for CYP3A] were evaluated.

Results: Although the activities of CYP2B6 and CYP3A were induced by treatment with PB and RIF, we found that the activity of phenacetin O-deethylase (PHOD), which is known as a marker of the activity of CYP1A2, was also enhanced by treatment with these non-CYP1A2 inducers in HepaRG cells. Based on previously published reports, we hypothesized that the expression ratio of CYP3A to CYP1A2 is much higher in HepaRG cells than in human hepatocytes; this may result in a nonnegligible contribution of CYP3A to the PHOD reaction in HepaRG cells. Studies using CYP3A inhibitor and pregnane X receptor-knockout HepaRG cells supported this hypothesis.

Conclusion: The measurement of mRNA serves as a higher reliable indicator for the evaluation of CYP induction potential when using HepaRG cells.

Keywords: CYP induction; HepaRG cells; PXR-knockout HepaRG cells; QuantiGene Plex 2.0 assay; drug–drug interaction; hepatocytes..

Publication types

  • Validation Study

MeSH terms

  • Biomarkers / analysis
  • Cell Line
  • Cytochrome P-450 CYP1A2 / genetics
  • Cytochrome P-450 CYP1A2 / metabolism*
  • Cytochrome P-450 CYP2B6 / metabolism
  • Cytochrome P-450 CYP3A / metabolism
  • Cytochrome P-450 Enzyme Inducers / pharmacology*
  • Enzyme Induction / drug effects
  • Hepatocytes
  • Humans
  • Metabolic Clearance Rate / drug effects*
  • Omeprazole / pharmacology
  • Phenacetin / metabolism
  • Phenobarbital / pharmacology
  • RNA, Messenger / analysis*
  • Reproducibility of Results
  • Rifampin / pharmacology

Substances

  • Biomarkers
  • Cytochrome P-450 Enzyme Inducers
  • RNA, Messenger
  • CYP1A2 protein, human
  • CYP2B6 protein, human
  • CYP3A protein, human
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 CYP3A
  • Phenacetin
  • Omeprazole
  • Rifampin
  • Phenobarbital