Induction of Drug Metabolism Enzymes and MDR1 Using a Novel Human Hepatocyte Cell Line

Abstract

Induction of drug-metabolizing enzymes and transporters can cause drug-drug interactions and loss of efficacy. In vitro induction studies traditionally use primary hepatocyte cultures and enzyme activity with selected marker compounds. We investigated the use of a novel human hepatocyte clone, the Fa2N-4 cell line, as an alternative reagent, which is readily available and provides a consistent, reproducible system. We used the Invader assay to monitor gene expression in these cells. This assay is a robust, yet simple, high-throughput system for quantification of mRNA transcripts. CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1 transcripts were quantified from total RNA extracts from Fa2N-4 cells treated with a panel of known inducers and compared with vehicle controls. In addition, we used enzyme activity assays to monitor the induction of CYP1A2, CYP2C9, and CYP3A4. The Fa2N-4 cells responded in a similar manner as primary human hepatocytes. Treatment with 10 M rifampin resulted in increases in CYP3A4 mRNA (17-fold) and activity (6-␤-hydroxytestoterone formation, 9-fold); and in CYP2C9 mRNA (4-fold) and activity (4Ј-hydroxydiclofenac formation, 2-fold). Treatment with 50 M ␤-naphthoflavone resulted in increases in CYP1A2 mRNA (15-fold) and activity (7-ethoxyresorufin O-dealkylation, 27-fold). UGT1A mRNA was induced by ␤-naphthoflavone (2-fold), and MDR1 (P-glycoprotein) mRNA was induced by rifampin (3-fold). These preliminary data using a few prototypical inducers show that Fa2N-4 cells can be a reliable surrogate for primary human hepatocytes, and, when used in conjunction with the Invader technology, could provide a reliable assay for assessment of induction of drug-metabolizing enzymes and transporters.

Shear stress activation of nuclear receptor PXR in endothelial detoxification

“…1A). Next, we asked whether shear stress increased the expression of an endogenous PXR target gene MDR1, which is a known PXR target encoding p-glycoprotein or ATP-binding cassette B1 (ABCB1), a transporter responsible for drug efflux (12,13). Quantitative reverse-transcriptase PCR (qRT-PCR) showed that MDR1 expression was induced by LSS, but not OSS, in human umbilical vein ECs (HUVECs) ( Fig.…”

A mechanism-based pharmacokinetic/pharmacodynamic model for CYP3A1/2 induction by dexamethasone in rats

“…The CYP3A1/2 enzyme activities were evaluated by the testosterone substrate assay [21] . The separation and detection of testosterone and its metabolites were performed using a Bisep™-1100 HPLC system (Unimicro Technologies Inc, USA), according to a previously described method [22] .…”

Application of Embryonic Stem Cells as a Novel Tool in Drug Screening

“…However, the sensitivity of hepatotoxicity using primary human hepatocytes or the HepG2 cell line cannot predict effects in early development and toxicological differences, which depend on the state of differentiation in hepatocytes (Knasmuller et al, 2004;Xu et al, 2004). In addition to, primary cells such as hepatocytes in particular and many transformed human hepatocyte-derived cell lines (immortalized cultures, i.e Fa2-N4 cells, HepaRG cells) have limitations in their life span and can have donor-dependent variations (Mills et al, 2004). However, they have disadvantages such as discontinuous phenotypic characteristics, functional properties and genetic instability.…”