title: A cell-based reporter gene assay for determining induction of CYP3A4 in a high-volume system.
abstract: Assessing the inducibility of CYP3A4 by various xenobiotics can predict potential drug interactions. In the present investigation, human hepatoma cells were stably integrated with either the CYP3A4 enhancer region and a luciferase reporter gene or the CYP3A4-luciferase construct and the human pregnane X receptor (PXR). Several colonies containing one to three copies of luciferase per cell were identified by Southern blot analysis. Those transformants producing high luciferase activity in response to rifampicin were used to standardize a 96-well plate screening system with minimal inter- and intraplate variability. Standardization also consisted of assessing viability of cells cultured in medium containing various serum concentrations. In cells maintained for 48 h in medium with less than 5% serum, a significant (p < 0.01) decline was observed in viability accompanied by altered induction. A defined serum-free medium also produced less viable cells but did not alter the inductive response. Treatment of transformants with various concentrations of rifampicin produced a dose-response curve with maximal induction at 10 microM (5.6 +/- 0.18- and 2.1 +/- 0.3-fold above dimethyl sulfoxide (DMSO)-treated cells in transformants with and without PXR, respectively). Of additional agents examined for their ability to induce CYP3A4, omeprazole (200 microM) was the most potent inducer (12.8 +/- 1.9- and 2.4 +/- 0.2-fold above DMSO-treated cells in transformants with and without PXR, respectively). Mifepristone and mevastatin produced modest induction (approximately 3-fold) in the cell line containing exogenous PXR, but produced less than 1.2-fold increases in cells lacking PXR. Thus, only potent inducers can be identified in the cell line without PXR. In contrast, cells containing the receptor can be used to rank CYP3A4 induction. Because a high volume of chemicals can be readily and accurately screened for their ability to induce CYP3A4 with this format, such a system could be valuable in the initial stages of preclinical drug development.
authors.affiliation: Puracyp, Inc., San Diego, California 92121, USA.
authors.forename: Judy
authors.initials: J
authors.lastname: Raucy
authors.forename: Lyndon
authors.initials: L
authors.lastname: Warfe
authors.forename: Mei-Fei
authors.initials: MF
authors.lastname: Yueh
authors.forename: Scott W
authors.initials: SW
authors.lastname: Allen
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000502:physiology*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D004790:Enzyme Induction; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008148:Lovastatin / Q000031:analogs & derivatives* / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics*; D015735:Mifepristone / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics*; D009853:Omeprazole / Q000494:pharmacology; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011987:Receptors, Steroid / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection / Q000379:methods
chemical_list: D017931:DNA Primers; D000072317:Polychlorinated Dibenzodioxins; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; C012258:mevastatin; D015735:Mifepristone; D003577:Cytochrome P-450 Enzyme System; D008148:Lovastatin; D006899:Mixed Function Oxygenases; D008156:Luciferases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009853:Omeprazole; D012293:Rifampin