Abstract
The DPX-2 cell line, a derivative of HepG2 cells, harbors human PXR and a luciferase-linked CYP3A4 promoter. These cells were used in a panel of cell-based assays for a parallel assessment of CYP3A4 induction, metabolism, and inhibition at the cellular level. CYP3A4 induction in the DPX-2 cell line by various agents was monitored in 96-well plates by a luciferase-based transcriptional activation assay. Of the prototypical CYP3A4 inducers examined, all exhibited elevated luciferase activity in DPX-2 cells. CYP3A4 enzyme activity in noninduced and rifampicin-induced DPX-2 cells was also assessed using Vivid fluorogenic substrates. Significantly elevated CYP3A4 activity levels (2.8-fold +/- 0.2-fold above DMSO-treated cells) were found in DPX-2 cells after 48 hours of exposure to rifampicin, but were undetectable in parental HepG2 cells. Rifampicin-induced activity levels were found to be suitable for assessing the inhibitory potential of new chemical entities in downstream CYP3A4 inhibition assays. The elevated CYP3A4 activity was inhibited 85% by 10 microM ketoconazole. In addition, a cytotoxicity assay to correct for possible toxic effects of compounds at the cellular level was applied. The comparative data obtained with a combination of the above assays suggests that the application of several independent in vitro technologies used in DPX-2 cells is the best possible strategy for the assessment of the complex phenomena of CYP3A4 induction and inhibition.
MeSH terms
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Carcinoma, Hepatocellular / enzymology*
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Cell Line, Tumor / drug effects
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Cell Line, Tumor / enzymology
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Chromans / pharmacology
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Clotrimazole / pharmacology
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Cytochrome P-450 CYP1A2 / biosynthesis
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Cytochrome P-450 CYP1A2 / genetics
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Cytochrome P-450 CYP3A
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Cytochrome P-450 Enzyme Inhibitors
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Cytochrome P-450 Enzyme System / biosynthesis*
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Dexamethasone / pharmacology
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Dimethyl Sulfoxide / pharmacology
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Enhancer Elements, Genetic
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Enzyme Induction / drug effects
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Genes, Reporter
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Genes, Synthetic
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Humans
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Ketoconazole / pharmacology
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Liver Neoplasms / enzymology*
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Lovastatin / analogs & derivatives
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Lovastatin / pharmacology
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Luciferases / genetics
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Mifepristone / pharmacology
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Neoplasm Proteins / antagonists & inhibitors
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Neoplasm Proteins / biosynthesis
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Neoplasm Proteins / genetics
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Nifedipine / pharmacology
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Omeprazole / pharmacology
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Paclitaxel / pharmacology
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Phenytoin / pharmacology
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Pregnane X Receptor
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Receptors, Cytoplasmic and Nuclear / genetics
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Receptors, Steroid / genetics
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Recombinant Fusion Proteins / antagonists & inhibitors
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Recombinant Fusion Proteins / biosynthesis
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Rifampin / pharmacology
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Thiazolidinediones / pharmacology
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Transcription, Genetic / drug effects
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Troglitazone
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Troleandomycin / pharmacology
Substances
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Chromans
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Cytochrome P-450 Enzyme Inhibitors
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Neoplasm Proteins
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Pregnane X Receptor
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Receptors, Cytoplasmic and Nuclear
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Receptors, Steroid
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Recombinant Fusion Proteins
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Thiazolidinediones
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mevastatin
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Mifepristone
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Phenytoin
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Dexamethasone
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Cytochrome P-450 Enzyme System
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Lovastatin
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Troleandomycin
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Luciferases
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CYP1A2 protein, human
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CYP3A protein, human
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Cytochrome P-450 CYP1A2
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Cytochrome P-450 CYP3A
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CYP3A4 protein, human
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Clotrimazole
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Troglitazone
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Nifedipine
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Omeprazole
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Paclitaxel
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Ketoconazole
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Rifampin
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Dimethyl Sulfoxide