Characterization of hyperglycemia due to sub-chronic administration of red ginseng extract via comparative global proteomic analysis.

Ginseng (Panax ginseng Meyer) is commonly used as an herbal remedy worldwide. Few studies have explored the possible physiological changes in the liver although patients often self-medicate with ginseng preparations, which may lead to exceeding the recommended dose for long-term administration. Here, we analyzed changes in the hepatic proteins of mouse livers using quantitative proteomics after sub-chronic administration of Korean red ginseng (KRG) extract (control group and 0.5, 1.0, and 2.0 g/kg KRG) using tandem mass tag (TMT) 6-plex technology. The 1.0 and 2.0 g/kg KRG groups exhibited signs of liver injury, including increased levels of aspartate transaminase (AST) and alanine aminotransferase (ALT) in the serum. Furthermore, serum glucose levels were significantly higher following KRG administration compared with the control group. Based on the upregulated proteins found in the proteomic analysis, we found that increased cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) levels promoted greater hydrogen sulfide (H2S) synthesis in the liver. This investigation provides novel evidence that sub-chronic administration of KRG can elevate H2S production by increasing protein expression of CBS and CSE in the liver.

Characterization of red ginseng-drug interaction by CYP3A activity increased in high dose administration in mice.

Ginseng (Panax ginseng Meyer) is a popular traditional herbal medicine used worldwide. Patients often take ginseng preparations with other medicines where the ginseng dose could exceed the recommended dose during long-term administration. However, ginseng-drug interactions at high doses of ginseng are poorly understood. This study showed the possibility of herb-drug interactions between the Korean red ginseng (KRG) extract and cytochrome P450 (CYP) substrates in higher administration in mice. The CYP activities were determined in vivo after oral administration of KRG extract doses of 0.5, 1.0, and 2.0 g/kg for 2 or 4 weeks by monitoring the concentration of five CYP substrates/metabolites in the blood. The area under the curve for OH-midazolam/midazolam catalysed by CYP3A was increased significantly by the administration of 2.0 g/kg KRG extract for 2 and 4 weeks. CYP3A-catalysed midazolam 1'-hydroxylation also increased significantly in a dose- and time-dependent manner in the S9 fraction of mouse liver which was not related to induction by transcription. Whereas CYP2D-catalysed dextromethorphan O-deethylation decreased in a dose- and time-dependent manner in vivo. In conclusion, interactions were observed between KRG extract and CYP2D and CYP3A substrates at subchronic-high doses of KRG administration in mice.

Characterization of in vitro and in vivo metabolism of leelamine using liquid chromatography-tandem mass spectrometry.

Leelamine is a diterpene compound found in the bark of pine trees and has garnered considerable interest owing to its potent anticancer properties. The aim of the present study was to investigate the metabolic profile of leelamine in human liver microsomes (HLMs) and mice using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that leelamine undergoes only Phase I metabolism, which generates one metabolite that is mono-hydroxylated at the C9 carbon of the octahydrophenanthrene ring (M1) both in vitro and in vivo. The structure and metabolic pathway of M1 were determined from the MSn fragmentation obtained by collision-induced dissociation using LC-MS/MS in HLMs. Cytochrome p450 (CYP) 2D6 was found to be the dominant CYP enzyme involved in the biotransformation of leelamine to its hydroxylated metabolite, whereas CYP2C19, CYP1A1, and CYP3A4 contributed to some extent. Moreover, we identified only one metabolite M1, in the urine, but none in the feces. In conclusion, leelamine was metabolized to a mono-hydroxyl metabolite by CYP2D6 and mainly excreted in the urine.

Correction to: Development of a simultaneous LC-MS/MS method to predict in vivo drug-drug interaction in mice.

The author would like to change conflict of interest statement of the online published article.

Investigation of nonalcoholic fatty liver disease-induced drug metabolism by comparative global toxicoproteomics.

Non-alcoholic fatty liver disease (NAFLD) includes conditions such as steatosis, non-alcoholic steatohepatitis, and ultimately hepatocellular carcinoma. Although the pathology of NAFLD is well-established, NAFLD-induced drug metabolism mediated by cytochrome P450 (CYP) in the liver has remained largely unexplored. Therefore, we investigated NAFLD-induced drug metabolism mediated by CYP by quantitative toxicoproteomics analysis. After administration of a methionine-choline deficient (MCD) diet to induce development of NAFLD, tandem mass tags-based liquid chromatography-tandem mass spectrometry analysis was conducted to investigate the dynamics of hepatic proteins. A total of 1295 proteins were identified, of which 934 were quantified by proteomic analysis. Among these proteins, 21 proteins were up-regulated and 51 proteins were down-regulated by the MCD diet. Notably, domain annotation enrichment using InterPro indicated that proteins related to CYPs were significantly decreased. When we investigated CYP activity using in vivo and in vitro CYP cocktail assays, most CYPs were significantly decreased, whereas CYP2D was not changed after administration of the MCD diet. In conclusion, we identified significantly altered levels of CYPs and their activities induced by the MCD diet and confirmed the NAFLD-induced drug metabolism by pharmacokinetic analysis.

Development of a simultaneous LC-MS/MS method to predict in vivo drug-drug interaction in mice.

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.

Mechanism Investigation of Rifampicin-Induced Liver Injury Using Comparative Toxicoproteomics in Mice.

Tuberculosis is one of the top causes of death among curable infectious diseases; it is an airborne infectious disease that killed 1.1 million people worldwide in 2010. Anti-tuberculosis drug-induced liver injury is the primary cause of drug-induced liver injury (DILI). Rifampicin is one of the most common anti-tuberculosis therapies and has well-known hepatotoxicity. To understand the mechanism of rifampicin-induced liver injury, we performed a global proteomic analysis of liver proteins by LC-MS/MS in a mouse model after the oral administration of 177 and 442.5 mg/kg rifampicin (LD10 and LD25) for 14 days. Based on the biochemical parameters in the plasma after rifampicin treatment, the hepatotoxic effect of rifampicin in the mouse liver was defined as a mixed liver injury. In the present study, we identified 1101 proteins and quantified 1038 proteins. A total of 29 and 40 proteins were up-regulated and 27 and 118 proteins were down-regulated in response to 177 and 442.5 mg/kg rifampicin, respectively. Furthermore, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to characterize the mechanism of rifampicin-induced hepatotoxicity. In the molecular function category, glutathione transferase activity was up-regulated and proteins related to arachidonic acid metabolism were down-regulated. In the KEGG pathway enrichment-based clustering analysis, the peroxisome proliferator-activated receptor-γ (PPARγ) signaling pathway, cytochrome P450, glutathione metabolism, chemical carcinogenesis, and related proteins increased dose-dependently in rifampicin-treated livers. Taken together, this study showed in-depth molecular mechanism of rifampicin-induced liver injury by comparative toxicoproteomics approach.

Identification of absolute conversion to geraldol from fisetin and pharmacokinetics in mouse.

Fisetin (3,3',4',7-tetrahydroxyflavone) is a flavonoid found in several fruits, vegetables, nuts, and wine and has anti-oxidant, anti-inflammatory, and anti-angiogenic properties. Geraldol is the 3'-methoxylated metabolite of fisetin (3,4',7-trihydroxy-3'-methoxyflavone). The concentration of fisetin and geraldol in mouse plasma was determined by LC-MS/MS, following direct protein precipitation. These concentrations were determined after administration of fisetin at doses of 2mg/kg (i.v.) and 100 and 200mg/kg (p.o.). The method was validated in terms of linearity, accuracy, precision, matrix effect, and stability. The pharmacokinetics parameters of fisetin and geraldol were successfully determined using a validated method in mice. Results indicated that fisetin was very rapidly methylated to geraldol in vivo. Following administration of fisetin, it was observed that the Cmax and AUC values for geraldol were higher than those of fisetin. The absolute bioavailability of fisetin was calculated as 7.8% and 31.7% after oral administration of 100 and 200mg/kg fisetin, respectively. This method was successfully applied to determine the pharmacokinetic parameters of fisetin and its main metabolite geraldol in mouse plasma. Geraldol was the dominant circulating metabolite after fisetin administration in vivo.