ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Identifying the inductive constituents of cytochrome P450 (CYP) enzymes is important in characterizing the safety of ethnopharmacological herbal preparations. AIM OF THE STUDY: This study provides a rapid and accurate method for screening CYP inducers in Shenmai injection (SMI), a traditional Chinese medicine. MATERIALS AND METHODS: We combined a pregnane X receptor (PXR) reporter gene assay and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis to screen ethanol and aqueous extracts of SMI for CYP-inducing constituents. RESULTS: The ethanol extract exhibited stronger PXR activity than the aqueous extract. Of the 29 chemical compounds identified, 7 compounds with high relative concentrations in the ethanol extract were further evaluated for PXR activity. The highest activity was exhibited by methyl ophiopogonanone B and ginsenoside F2, indicating that they are CYP inducers. CONCLUSIONS: The identification method applied in this study was rapid and accurate and is suitable for screening CYP inducers in herbal preparations.
Subject(s)
Cytochrome P-450 Enzyme Inducers/analysis , Drugs, Chinese Herbal/analysis , Genes, Reporter/genetics , Panax/genetics , Pregnane X Receptor/genetics , Tandem Mass Spectrometry/methods , Chromatography, Liquid/methods , Drug Combinations , Humans , Panax/chemistryABSTRACT
Cocktail substrates are useful in investigating drug-drug interactions (DDI) that can rapidly identify the cytochrome P450 (CYP) isoforms that interact with test drugs. In this study, we developed and validated five probe drugs for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A using LC-MS/MS to determine CYP activities in mice. The five probe substrates were caffeine (2 mg/kg), bupropion (30 mg/kg), omeprazole (4 mg/kg), dextromethorphan (40 mg/kg), and midazolam (2 mg/kg) for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A, respectively. The cocktail substrates were orally administered to male 5-week-old ICR mice over 0-240 min. The analytical method was validated; it showed high selectivity, linearity, and acceptable accuracy. We confirmed the lack of interaction of this cocktail in the control state (no effect of CYP inducer or inhibitor) and suggested AUCratio (metabolite/substrate) as a unit to evaluate DDI in vivo. In addition, the cocktail assay was applied for the determination of pharmacokinetic parameters against phenobarbital as a selective CYP2B inducer and ketoconazole as a strong CYP3A inhibitor. The concentration of cocktail substrates and the LC-MS/MS method were optimized. In conclusion, we developed a simultaneous and comprehensive analysis system for predicting potential DDI in mice.
Subject(s)
Cytochrome P-450 CYP3A Inhibitors/metabolism , Cytochrome P-450 Enzyme Inducers/metabolism , Drug Interactions/physiology , Tandem Mass Spectrometry/methods , Animals , Caffeine/administration & dosage , Caffeine/analysis , Caffeine/metabolism , Chromatography, Liquid/methods , Chromatography, Liquid/trends , Cytochrome P-450 CYP3A Inhibitors/administration & dosage , Cytochrome P-450 CYP3A Inhibitors/analysis , Cytochrome P-450 Enzyme Inducers/administration & dosage , Cytochrome P-450 Enzyme Inducers/analysis , Dextromethorphan/administration & dosage , Dextromethorphan/analysis , Dextromethorphan/metabolism , Forecasting , Male , Mice , Mice, Inbred ICR , Midazolam/administration & dosage , Midazolam/analysis , Midazolam/metabolism , Tandem Mass Spectrometry/trendsABSTRACT
EROD activity and induction cytochrome P4501A in liver and gills of Senegal sole, Solea senegalensis, from a heavy metal and PAH polluted estuary, was studied. Liver and gill CYP1A catalytic activity was assessed at the enzyme activity level-measured as 7-ethoxyresorufin-O-deethylase and cellular localization of CYP1A in the liver was studied using immunohistochemistry. Liver EROD was correlated with phenanthrene-type metabolites in liver and copper concentrations in water. Strong CYP1A occurrence was observed in acinar pancreatic cells, pancreatic duct epithelium and vascular system endothelium and negative/rare induction were observed in hepatocytes and sinusoidal endothelium. In gills, EROD activity showed a significant correlation with different fractions of heavy metals in sediment but no correlation was observed between EROD activity and PAHs. Strongly positive CYP1A associated staining of the vascular system endothelia and primary filament cells and a moderate staining of pillar cells in gills were observed. The results substantiated the utility of EROD activity and CYP1A induction measurement as biomarkers for use by aquatic toxicologists and indicate that catalytic assays and immunohistochemical assays appear to be sensitive to different kinds of pollutants being the use of both methods recommended for monitoring programs.