Effect of polyethylene glycol 400 on the pharmacokinetics and tissue distribution of baicalin by intravenous injection based on the enzyme activity of UGT1A8/1A9.

Baicalin (BG) is a bioactive flavonoid extracted from the dried root of the medicinal plant, Scutellaria radix (SR) (dicotyledonous family, Labiatae), and has several biological activities. Polyethylene glycol 400 (PEG400) has been used as a suitable solvent for several traditional Chinese medicines (TCM) and is often used as an excipient for the compound preparation of SR. However, the drug-excipient interactions between BG and PEG400 are still unknown. Herein, we evaluated the effect of a single intravenous PEG400 administration on the BG levels of rats using pharmacokinetic and tissue distribution studies. A liver microsome and recombinant enzyme incubation system were used to further confirm the interaction mechanism between PEG400 and UDP-glucuronosyltransferases (UGTs) (UGT1A8 and UGT1A9). The pharmacokinetic study demonstrated that following the co-intravenous administration of PEG400 and BG, the total clearance (CLz) of BG in the rat plasma decreased by 101.60% (p < 0.05), whereas the area under the plasma concentration-time curve (AUC)0-t and AUC0-inf increased by 144.59% (p < 0.05) and 140.05% (p < 0.05), respectively. Additionally, the tissue distribution study showed that the concentration of BG and baicalein-6-O-ß-D-glucuronide (B6G) in the tissues increased, whereas baicalein (B) in the tissues decreased, and the total amount of BG and its metabolites in tissues altered following the intravenous administration of PEG400. We further found that PEG400 induced the UGT1A8 and UGT1A9 enzyme activities by affecting the maximum enzymatic velocity (Vmax) and Michaelis-Menten constant (Km) values of UGT1A8 and UGT1A9. In conclusion, our results demonstrated that PEG400 interaction with UGTs altered the pharmacokinetic behaviors and tissue distribution characteristics of BG and its metabolites in rats.

Glabrone as a specific UGT1A9 probe substrate and its application in discovering the inhibitor glycycoumarin.

UDP-glucuronosyltransferase 1A9 (UGT1A9) is one of the most important UGT isoforms, and plays an important role in the metabolic elimination of therapeutic drugs via glucuronidation. Herbal medicines affecting the activity of UGT1A9 may influence the metabolism of related drugs, thus causing herb-drug interactions and even adverse effects. However, few methods are available to evaluate the activity of UGT1A9. In this study, a natural product glabrone was discovered as an isoform-specific probe substrate for UGT1A9. The Vmax and Km values of glabrone were 362.6 nmol/min/mg protein and 17.2 µM for human liver microsomes (HLMs), and 382.3 nmol/min/mg protein and 16.6 µM for recombinant human UGT1A9, respectively. Glabrone 7-O-glucuronide, the UGT1A9 metabolite of glabrone, was prepared by using a plant glucuronosyltransferase UGT88D1, and the structure was identified by NMR spectroscopy. Using glabrone as a probe, we established a rapid HPLC method to screen UGT1A9 inhibitors from 54 natural products isolated from the Chinese herbal medicine licorice. Among them, glycycoumarin was found as a potent UGT1A9 inhibitor with an IC50 value of 6.04 µM. In rats, the pretreatment of glycycoumarin (4 mg/kg, i.p.) for 3 days could remarkably increase the plasma concentrations of dapagliflozin while decrease the concentrations of dapagliflozin-O-glucuronide after administration of dapagliflozin (1 mg/kg, i.v.), which is mainly metabolized by UGT1A9. The results indicated the potential risk of herb-drug interactions between licorice and UGT1A9-metabolizing drugs.

Biotransformation of phytoestrogens from soy in enzymatically characterized liver microsomes and primary hepatocytes of Atlantic salmon.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Pterostilbene supplements carry the risk of drug interaction via inhibition of UDP-glucuronosyltransferases (UGT) 1A9 enzymes.

Pterostilbene (PT) is a natural stilbene common in small berries and food supplements, possessing numerous pharmacological activities. However, whether PT can affect the activities of UDP-glucuronosyltransferases (UGT) enzymes remains unclear. The aim of the present study was to investigate the effect of PT on UGT activities and to quantitatively evaluate the food-drug interaction potential due to UGT inhibition. Our data indicated that PT exhibited potent inhibition against HLM, UGT1A6, UGT1A9, UGT2B7, and UGT2B15, moderate inhibition against UGT1A1, UGT1A3, UGT1A8, and UGT2B4, negligible inhibition against UGT1A4, UGT1A7, UGT1A10, and UGT2B17. Further kinetic investigation demonstrated that PT exerted potent noncompetitive inhibition 4-MU glucuronidation by UGT1A9, with IC50 and Ki values of 0.92 µM and 0.52 ± 0.04 µM, respectively. Quantitative prediction study suggested that coadministration of PT supplements at 100 mg/day or higher doses may result in at least a 50% increase in the AUC of drugs predominantly cleared by UGT1A9. Thus, the coadministration of PT supplements and drugs primarily cleared by UGT1A9 may result in potential drug interaction, and precautions should be taken when coadministration of PT supplements and drugs metabolized by UGT1A9.

Epigenetic regulation of UDP-Glucuronosyltransferase by microRNA-200a/-183: implications for responses to sorafenib treatment in patients with hepatocellular carcinoma.

Patients receiving sorafenib treatment for hepatocellular carcinoma (HCC) experience different treatment efficacy. Personalized sorafenib treatment should be achieved through the identification of predictors of therapeutic response. In the current study, we found that high UGT1A9 expression indicated better prognosis for HCC patients treated with sorafenib after surgery. In silico analysis predicted microRNA-200a/-183 as potential regulators of the UGT1A gene family via binding to the shared UGT1A9 3'-UTR. A significant inverse correlation between microRNA-200a/-183 and UGT1A9 mRNA level was observed in a panel of HCC specimens. Direct binding was further demonstrated by luciferase reporter gene vector carrying wild-type or binding site truncated UGT1A9 3'-UTR. MicroRNA-200a/-183 downregulated UGT1A9 expression in a dose-dependent manner and significantly reduced sorafenib ß-D-glucuronide formation in HCC cells. These data indicated that UGT1A9, under epigenetic regulation of microRNA-200a/-183, could predict patients who might benefit from adjuvant sorafenib treatment after surgery.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro.

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Identification of human UDP-glucuronosyltransferase isoforms involved in the isofraxidin glucuronidation and assessment of the species differences of the reaction.

Isofraxidin, 7-Hydroxy-6.8-dimethoxy-2H-1-benzopyran-2-one, is a major active component of Acanthopanax senticosus, which has been used as Acanthopanax (Ciwujia) injection to treat cardiovascular and cerebrovascular diseases in China for more than thirty years. The purpose of this study was to identify the roles of human UDP-glucuronosyltransferases (UGTs) in isofraxidin glucuronidation in the liver and intestinal microsomes and to reveal the potential species differences by comparing the liver microsomal glucuronidation from different experimental animals. One metabolite was biosynthesized and characterized as isofraxidin-7-O-glucuronide by liquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). The intrinsic clearances in human liver and intestinal microsomes were 63.8 and 16.4µL/min/mg, respectively. Human liver microsomes displays higher potential for isofraxidin elimination than human intestinal microsomes. The reaction phenotyping analysis was conducted using cDNA-expressed human UGTs and chemical inhibitors. The results indicated that UGT1A1 and UGT1A9 were the main isoforms involved in the formation of isofraxidin-7-O-glucuronide. The isofraxidin glucuronidation in liver microsomes from human (HLM), rat (RLM), mouse (MLM), dog (DLM), monkey (CyLM), minipig (PLM), and guinea pig (GpLM) followed the Michealis-Menten model. The isofraxidin glucuronidation displays species differences in terms of catalytic activities. GpLM had the highest clearance with the CLint value of 152µL/min/mg. CyLM, RLM and MLM exhibit similar catalytic activities in isofraxidin glucuronidation with the intrinsic clearance values of 54.6, 58.0 and 50.2µL/min/mg, respectively, which are higher than those of PLM and DLM (23.9 and 37.7µL/min/mg, respectively). Rat exhibits the most similar intrinsic metabolic clearance (CLint) to human.

Wedelolactone metabolism in rats through regioselective glucuronidation catalyzed by uridine diphosphate-glucuronosyltransferases 1As (UGT1As).

BACKGROUND: Wedelolactone (WEL), a medicinal plant-derived coumestan, has been reported to exhibit a diverse range of pharmacological activities. However, the metabolism and disposition of WEL remain unexplored. PURPOSE: The present study aims to investigate the metabolism of WEL in rats and identify the enzymes responsible for forming major WEL metabolites. METHODS: Plasma, urine, feces, and bile samples were collected before and after 50 mg/kg WEL was orally administered to rats. Metabolites were profiled by ultrahigh performance liquid chromatography/quadrupole time-of-flight mass spectrometry and identified by high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy. The in vitro WEL glucuronidation activities of human liver microsomes, human kidney microsomes, human intestine microsomes, and 12 recombinant human uridine diphosphate-glucuronosyltransferase (UGT) isoforms were screened. Molecular docking simulation of the interaction between WEL and UGT1A9 was conducted. RESULTS: WEL underwent extensive metabolism, and 17 metabolites were identified. The major metabolic pathways observed were glucuronidation and methylation. Glucuronic acid was preferentially introduced into 5-OH, whereas no obvious regioselectivity was observed in the methylation of 11-OH and 12-OH. Multiple UGTs, including UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10, were involved in forming WEL glucuronides and O-methylated WEL glucuronides. CONCLUSION: The extensive glucuronidation and methylation is responsible for the low oral bioavailability of WEL in rats. UGT1A1 and UGT1A9 were the major enzymes involved in the glucuronidation of WEL and O-methylated WEL. Molecular docking studies revealed that 5-OH was accessible to the catalytic domain of UGT1As; therefore, 5-OH exhibited a high probability of glucuronidation.

Regioselective glucuronidation of gingerols by human liver microsomes and expressed UDP-glucuronosyltransferase enzymes: reaction kinetics and activity correlation analyses for UGT1A9 and UGT2B7.

OBJECTIVES: To determine the reaction kinetics for regioselective glucuronidation of gingerols (i.e. 6-, 8- and 10-gingerol) by human liver microsomes and expressed UDP-glucuronosyltransferase (UGT) enzymes, and to identify the main UGT enzymes involved in regioselective glucuronidation of gingerols. METHODS: The rates of glucuronidation were determined by incubating the gingerols with uridine diphosphoglucuronic acid-supplemented microsomes. Kinetic parameters were derived by fitting an appropriate model to the data. Activity correlation analyses were performed to identify the main UGT enzymes contributing to hepatic metabolism of gingerols. KEY FINDINGS: Glucuronidation at the 4'-OH group was much more favoured than that at 5-OH. The degree of position preference was compound-dependent; the catalytic efficiency ratios of 4'-O- to 5-O-glucuronidation were 9.1, 19.7 and 2.9 for 6-, 8- and 10-gingerol, respectively. UGT1A8 (an intestinal enzyme), UGT1A9 and UGT2B7 were the enzymes showing the highest activity towards gingerols. Formation of 5-O-glucuronide was mainly catalysed by UGT1A9. UGT2B7 was the only enzyme that generated glucuronides at both 4'-OH and 5-OH sites, although a strong position preference was observed with 4'-OH (≥80.2%). Further, activity correlation analyses indicated that UGT2B7 and UGT1A9 were primarily responsible for 4'-O-glucuronidation and 5-O-glucuronidation of gingerols in the liver, respectively. CONCLUSIONS: Gingerols were metabolized by multiple hepatic and gastrointestinal UGT enzymes. Also, UGT1A9 and 2B7 were the main contributors to regioselective glucuronidation of gingerols in the liver.

Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes.

Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far. Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one. Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CLint) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities. Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.

Comparison of inhibition capability of scutellarein and scutellarin towards important liver UDP-glucuronosyltransferase (UGT) isoforms.

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand-Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP-glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (Ki ) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the Ki values were calculated to be 0.02, 5.0, 5.8 and 35.9 µM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro-in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components.

Regioselective glucuronidation of oxyresveratrol, a natural hydroxystilbene, by human liver and intestinal microsomes and recombinant UGTs.

Oxyresveratrol (OXY) is a natural hydroxystilbene that shows similar bioactivity but better water solubility than resveratrol. This study aims to characterize its glucuronidation kinetics in human liver (HLMs) and intestinal (HIMs) microsomes and identify the main UDP-glucuronosyltransferase (UGT) isoforms involved. Three and four mono-glucuronides of OXY were generated in HIMs and HLMs, respectively, with oxyresveratrol-2-O-ß-D-glucuronosyl (G4) as the major metabolite in both organs. The kinetics of G4 formation fit a sigmoidal model in HLMs and biphasic kinetics in HIMs. Multiple UGT isoforms catalyzed G4 formation with the highest activity observed with UGT1A9 followed by UGT1A1. G4 formation by both isoforms followed substrate inhibition kinetics. Propofol (UGT1A9 inhibitor) effectively blocked G4 generation in HLMs (IC50 63.7 ± 11.6 µM), whereas the UGT1A1 inhibitor bilirubin only produced partial inhibition in HLMs and HIMs. These findings shed light on the metabolic mechanism of OXY and arouse awareness of drug interactions.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the glucuronidation of fraxetin.

Fraxetin, a major constituent of the traditional medicine plant Fraxinus rhynchophylla Hance (Oleaceae), has been found to possess multiple bioactivities. However, the metabolic pathway(s) of fraxetin in human tissues has not been reported yet. This study aimed to characterize the glucuronidation pathway(s) of fraxetin in human tissues. Fraxetin could be metabolized to two glucuronides in human liver microsomes (HLMs). These two glucuronides were biosynthesized and characterized as 7-O-glucuronide (7-O-G) and 8-O-glucuronide (8-O-G). UGT1A1, -1A6, -1A7, -1A8, -1A9 and -1A10 participated in the formation of 7-O-G, while the formation of 8-O-G was catalyzed selectively by UGT1A6 and UGT1A9. UGT1A9 showed the highest catalytic activities in the formation of 7-O-G and 8-O-G. Both kinetic characterization and inhibition assays demonstrated that UGT1A9 played important roles in fraxetin glucuronidations in HLMs, especially in the formation of the major metabolite 8-O-G. Furthermore, the intrinsic clearance of fraxetin in both human liver microsomes and UGT1A9 was greater than that of 7,8-dihydroxylcoumarin, revealing that the addition of a C-6 methoxy group led to the higher metabolic clearance. In summary, the glucuronidation pathways of fraxetin in human liver microsomes were well-characterized, and UGT1A9 was the major isoform responsible for the glucuronidations of fraxetin.

Deglycosylation of liquiritin strongly enhances its inhibitory potential towards UDP-glucuronosyltransferase (UGT) isoforms.

The detailed mechanisms on licorice-drug interaction remain to be unclear. The aim of the present study is to investigate the inhibition of important UGT isoforms by two important ingredients of licorice, liquiritin, and liquiritigenin. The results showed that liquiritigenin exhibited stronger inhibition towards all the tested UGT isoforms than liquiritin. Data fitting using Dixon and Lineweaver-Burk plots demonstrated the competitive inhibition of liquiritigenin towards UGT1A1 and UGT1A9-mediated 4-MU glucuronidation reaction. The inhibition kinetic parameters (Ki ) were calculated to be 9.1 and 3.2 µM for UGT1A1 and UGT1A9, respectively. Substrate-dependent inhibition behaviour was also observed for UGT1A1 in the present study. All these results will be helpful for understanding the deep mechanism of licorice-drug interaction. However, when translating these in vitro parameters into in vivo situations, more complex factors should be considered, such as substrate-dependent inhibition of UGT isoforms, the contribution of UGT1A1 and UGT1A9 towards the metabolism of drugs, and many factors affecting the abundance of ingredients in the licorice.

Modulation of CYP1A and genotoxic effects in European seabass (Dicentrarchus labrax) exposed to weathered oil: a mesocosm study.

The aim of this study was to assess medium-term toxicity of weathered oil on European seabass. A mesocosm system reproducing an oil spill at sea was applied. Fish were collected after 48 h, 7, 30 and 60 days. Cyp1a gene transcription, EROD and UDPGT activities, bile PAHs metabolites and micronuclei frequency were investigated. A progressive disappearance of low molecular weight n-alkanes and PAHs in the water of the mesocosm occurred during the experimentation. Fishes exposed to oil displayed a significant increase of cyp1a expression and EROD activity during the entire experiment as well as higher concentrations of PAHs metabolites in bile. Micronulei frequency resulted significantly higher during all experiment in oil exposed sea bass compared to controls. The results highlight the environmental risk associated with the release of oil products at sea and confirm the adopted parameters as useful tools for studying the impact of accidental oil spills on fish.

UDP-glucuronosyltransferase (UGT) 1A9-overexpressing HeLa cells is an appropriate tool to delineate the kinetic interplay between breast cancer resistance protein (BRCP) and UGT and to rapidly identify the glucuronide substrates of BCRP.

The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low bioavailability for flavonoids. To investigate the simplest interplay between one UDP-glucuronosyltransferase isoform and one efflux transporter in flavonoid disposition, engineered HeLa cells stably overexpressing UGT1A9 were developed, characterized, and further applied to investigate the metabolism of two model flavonoids (genistein and apigenin) and excretion of their glucuronides. The results indicated that the engineered HeLa cells overexpressing UGT1A9 rapidly excreted the glucuronides of genistein and apigenin. The kinetic characteristics of genistein or apigenin glucuronidation were similar with the use of UGT1A9 overexpressed in HeLa cells or the commercially available UGT1A9. Small interfering (siRNA)-mediated UGT1A9 silencing resulted in a substantial decrease in glucuronide excretion (>75%, p < 0.01). Furthermore, a potent inhibitor of breast cancer resistance protein (BCRP), 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), caused, in a dose-dependent manner, a substantial and marked reduction of the clearance (74-94%, p < 0.01), and a substantial increase in the intracellular glucuronide levels (4-8-fold, p < 0.01), resulting in a moderate decrease in glucuronide excretion (19-59%, p < 0.01). In addition, a significant, albeit moderate, reduction in the fraction of genistein metabolized (f(met)) in the presence of Ko143 was observed. In contrast, leukotriene C4 and siRNA against multidrug resistance protein (MRP) 2 and MRP3 did not affect excretion of flavonoid glucuronides. In conclusion, the engineered HeLa cells overexpressing UGT1A9 is an appropriate model to study the kinetic interplay between UGT1A9 and BCRP in the phase II disposition of flavonoids. This simple cell model should also be very useful to rapidly identify whether a phase II metabolite is the substrate of BCRP.

Inhibition of intestinal and hepatic glucuronidation of mycophenolic acid by Ginkgo biloba extract and flavonoids.

Herb-drug interactions have received more attention in recent years because of the widespread popularity of herbal supplements. However, there are limited data on the effect of herbs on glucuronidation in humans. The goal of this work was to examine the effect of Ginkgo biloba extract and its main flavonoid and terpene lactone constituents on mycophenolic acid (MPA) 7-O-glucuronidation. Human liver (HLM) and intestinal (HIM) microsomes were incubated with MPA and G. biloba extract (unhydrolyzed or acid-hydrolyzed), quercetin, kaempferol, ginkgolide A, ginkgolide B, or bilobalide. MPA-7-O-glucuronide formation was inhibited in HLM and HIM incubations by unhydrolyzed [IC(50) = 84.3 (HLM) and 6.9 (HIM) microg/ml] and hydrolyzed [IC(50) = 20.9 (HLM) and 4.3 (HIM) microg/ml] G. biloba extracts, quercetin [IC(50) = 19.1 (HLM) and 5.8 (HIM) microM], and kaempferol [IC(50) = 23.1 (HLM) and 7.7 (HIM) microM]. Terpene lactones did not show inhibition of MPA glucuronidation. Quercetin was a mixed-type inhibitor in HLM and HIM incubations [K(i) = 11.3 (HLM) and 2.8 (HLM) microM], whereas kaempferol was a noncompetitive inhibitor in HLM (K(i) = 33.7 microM) and a mixed-type inhibitor in HIM (K(i) = 4.5 microM). These results indicate that G. biloba extract or quercetin- and kaempferol-rich supplements may inhibit intestinal and hepatic glucuronidation of MPA. Future studies are needed to evaluate the clinical significance of this interaction.

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line.

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 microM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.

Disposition of flavonoids via enteric recycling: enzyme stability affects characterization of prunetin glucuronidation across species, organs, and UGT isoforms.

We characterized the in vitro glucuronidation of prunetin, a prodrug of genistein that is a highly active cancer prevention agent. Metabolism studies were conducted using expressed human UGT isoforms and microsomes/S9 fractions prepared from intestine and liver of rodents and humans. The results indicated that human intestinal microsomes were more efficient than liver microsomes in glucuronidating prunetin, but rates of metabolism were dependent on time of incubation at 37 degrees C. Human liver and intestinal microsomes mainly produced metabolite 1 (prunetin-5- O-glucuronide) and metabolite 2 (prunetin-4'- O-glucuronide), respectively. Using 12 human UGT isoforms, we showed that UGT1A7, UGT1A8, and UGT1A9 were mainly responsible for the formation of metabolite 1, whereas UGT1A1, UGT1A8, and UGT1A10 were mainly responsible for the formation of metabolite 2. This isoform-specific metabolism was consistent with earlier results obtained using human liver and intestinal microsomes, as the former (liver) is UGT1A9-rich whereas the latter is UGT1A10-rich. Surprisingly, we found that the thermostability of the microsomes was isoform- and organ-dependent. For example, human liver microsomal UGT activities were much more heat-stable (37 degrees C) than intestinal microsomal UGT activities, consistent with the finding that human UGT1A9 is much more thermostable than human UGT1A10 and UGT1A8. The organ-specific thermostability profiles were also evident in rat microsomes and mouse S9 fractions, even though human intestinal glucuronidation of prunetin differs significantly from rodent intestinal glucuronidation. In conclusion, prunetin glucuronidation is species-, organ-, and UGT-isoform-dependent, all of which may be impacted by the thermostability of specific UGT isoforms involved in the metabolism.

Modification of N-acetyltransferases and glutathione S-transferases by coffee components: possible relevance for cancer risk.

Enzymes of xenobiotic metabolism are involved in the activation and detoxification of carcinogens and can play a pivotal role in the susceptibility of individuals toward chemically induced cancer. Differences in such susceptibility are often related to genetically predetermined enzyme polymorphisms but may also be caused by enzyme induction or inhibition through environmental factors or in the frame of chemopreventive intervention. In this context, coffee consumption, as an important lifestyle factor, has been under thorough investigation. Whereas the data on a potential procarcinogenic effect in some organs remained inconclusive, epidemiology has clearly revealed coffee drinkers to be at a lower risk of developing cancers of the colon and the liver and possibly of several other organs. The underlying mechanisms of such chemoprotection, modifications of xenobiotic metabolism in particular, were further investigated in rodent and in vitro models, as a result of which several individual chemoprotectants out of the >1000 constituents of coffee were identified as well as some strongly metabolized individual carcinogens against which they specifically protected. This chapter discusses the chemoprotective effects of several coffee components and whole coffee in association with modifications of the usually protective glutathione-S-transferase (GST) and the more ambivalent N-acetyltransferase (NAT). A key role is played by kahweol and cafestol (K/C), two diterpenic constituents of the unfiltered beverage that were found to reduce mutagenesis/tumorigenesis by strongly metabolized compounds, such as 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine, 7,12-dimethylbenz[a]anthracene, and aflatoxin B(1), and to cause various modifications of xenobiotic metabolism that were overwhelmingly beneficial, including induction of GST and inhibition of NAT. Other coffee components such as polyphenols and K/C-free coffee are also capable of increasing GST and partially of inhibiting NAT, although to a somewhat lesser extent.