title: 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 microM rifampin resulted in increases in CYP3A4 mRNA (17-fold) and activity (6-beta-hydroxytestoterone formation, 9-fold); and in CYP2C9 mRNA (4-fold) and activity (4'-hydroxydiclofenac formation, 2-fold). Treatment with 50 microM beta-naphthoflavone resulted in increases in CYP1A2 mRNA (15-fold) and activity (7-ethoxyresorufin O-dealkylation, 27-fold). UGT1A mRNA was induced by beta-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.
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA.
authors.forename: Jessica B
authors.initials: JB
authors.lastname: Mills
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Rose
authors.forename: Nalini
authors.initials: N
authors.lastname: Sadagopan
authors.forename: Jasminder
authors.initials: J
authors.lastname: Sahi
authors.forename: Sonia M F
authors.initials: SM
authors.lastname: de Morais
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D002478:Cells, Cultured; D002648:Child; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction; D005260:Female; D022781:Hepatocytes / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C403209:MEF1 protein, human; D014157:Transcription Factors; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human