Efficient assessment of the utility of immortalized Fa2N-4 cells for cytochrome P450 (CYP) induction studies using multiplex quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and substrate cassette methodologies

Abstract

Induction of cytochrome P450 (CYP) 1A2, CYP2B6, and CYP3A4 by 22 prototypical inducers was evaluated in the Fa2N-4 immortalized human hepatic cell line. To facilitate this a duplex one-step quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for CYP1A2 and CYP3A4 and a substrate cassette allowing simultaneous monitoring of CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activity were developed. CYP1A2 messenger RNA (mRNA) and activity were induced by the prototypical aryl hydrocarbon receptor (AhR) ligand β-naphthoflavone (E_max = 217- and 11-fold, respectively, and EC₅₀ = 8 µM). CYP3A4 mRNA and activity were induced by the prototypical pregnane X receptor (PXR) ligands, rifampicin (E_max = 36- and 6-fold, respectively, and EC₅₀ = 4 µM) and phenobarbital (E_max = 12- and 4-fold, respectively, and EC₅₀ = 205 µM). No induction of CYP2B6 was detected with several prototypical constitutive androstane receptor (CAR) ligands. A large mRNA–activity E_max ratio was observed for some time-dependent inhibitors of CYP3A4, whereas EC₅₀ determinations appeared to be independent of the endpoint. In conclusion, Fa2N-4 cells are a good surrogate for primary human hepatocytes for assessing AhR and PXR-mediated CYP1A2 and CYP3A4 induction, respectively, but not for CAR-mediated CYP2B6 induction. The sensitive and selective methodologies presented in this paper afford maximal data generation and enhanced throughput capability and are readily transferable to primary human hepatocytes or alternate cellular systems.

Keywords::

• Cytochrome P450 (CYP) induction
• cytochrome P450
• drug–drug interactions
• Fa2N-4 cells
• time-dependent inhibition