UDP-glucuronosyltransferase Isoforms Research Articles

Cellular Asymmetric Catalysis by UDP-glucuronosyltransferase 1A8 Shows Functional Localization to the Basolateral Plasma Membrane

UDP-glucuronosyltransferases (UGTs) are highly expressed in liver, intestine and kidney, and catalyze the glucuronic acid conjugation of both endogenous compounds and xenobiotics. Using recombinant human UGT isoforms, we show that glucuronic acid conjugation of the model substrate, (-)-epicatechin, is catalyzed mainly by UGT1A8 and UGT1A9. In HepG2 cells, pretreatment with polyunsaturated fatty acids increased substrate glucuronidation. In the intestinal Caco-2/HT29-MTX co-culture model, overall relative glucuronidation rates were much higher than in HepG2 cells, and (-)-epicatechin was much more readily conjugated when applied to the basolateral side of the cell monolayer. Under these conditions, 95% of the conjugated product was effluxed back to the site of application, and none of the other phase 2-derived metabolites followed this distribution pattern. HT29-MTX cells contained >1000-fold higher levels of UGT1A8 mRNA than Caco-2 or HepG2 cells. Gene expression of UGT1A8 increased after treatment of cells with docosahexaenoic acid, as did UGT1A protein levels. Immunofluorescence staining and Western blotting showed the presence of UGT1A in the basal and lateral parts of the plasma membrane of HT29-MTX cells. These results suggest that some of the UGT1A8 enzyme is not residing in the endoplasmic reticulum but spans the plasma membrane, resulting in increased accessibility to compounds outside the cell. This facilitates more efficient conjugation of substrate and is additionally coupled with rapid efflux by functionally associated basolateral transporters. This novel molecular strategy allows the cell to carry out conjugation without the xenobiotic entering into the interior of the cell.

Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes

1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established.2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs.3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 μM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 μM.4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug–drug interactions.

Enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7.

UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0 μM and 31.1 μM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7 μM and 3.1 μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism.

The UDP-glucuronosyltransferases of the blood-brain barrier: their role in drug metabolism and detoxication.

UDP-glucuronosyltransferases (UGTs) form a multigenic family of membrane-bound enzymes expressed in various tissues, including brain. They catalyze the formation of β-D-glucuronides from structurally unrelated substances (drugs, other xenobiotics, as well as endogenous compounds) by the linkage of glucuronic acid from the high energy donor, UDP-α-D-glucuronic acid. In brain, UGTs actively participate to the overall protection of the tissue against the intrusion of potentially harmful lipophilic substances that are metabolized as hydrophilic glucuronides. These metabolites are generally inactive, except for important pharmacologically glucuronides such as morphine-6-glucuronide. UGTs are mainly expressed in endothelial cells and astrocytes of the blood brain barrier (BBB). They are also associated to brain interfaces devoid of BBB, such as circumventricular organ, pineal gland, pituitary gland and neuro-olfactory tissues. Beside their key-role as a detoxication barrier, UGTs play a role in the steady-state of endogenous compounds, like steroids or dopamine (DA) that participate to the function of the brain. UGT isoforms of family 1A, 2A, 2B and 3A are expressed in brain tissues to various levels and are known to present distinct but overlapping substrate specificity. The importance of these enzyme species with regard to the formation of toxic, pharmacologically or physiologically relevant glucuronides in the brain will be discussed.

In vitro glucuronidation of the primary metabolite of 10-chloromethyl-11-demethyl-12-oxo-calanolide A by human liver microsomes and its interactions with UDP-glucuronosyltransferase substrates

F18 (10-chloromethyl-11-demethyl-12-oxo-calanolide), an analog of (+)-Calanolide A, is a novel small-molecule nonnucleoside reverse transcriptase inhibitor for the therapy of human immunodeficiency virus (HIV) infection. M3, the most abundant primary metabolite of F18 in human liver microsomes (HLMs) and rat liver microsomes (RLMs), is mainly excreted in bile as a glucuronide conjugate in rats after oral administration. The aim of this study was to identify the UDP glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of M3 by HLMs and recombinant human UGTs and investigate the metabolic interactions of M3 with the substrates of UGTs in HLMs. As a result, UGT1A1 was the major isozyme responsible for the glucuronidation of M3, followed by UGT1A4, UGT1A9 and UGT2B7. M3 exhibited significant inhibition against UGT1A9 and UGT2B7 in both HLMs and recombinant human UGTs. In addition, M3 inhibited UGT1A9 catalyzed mycophenolic acid (MPA) glucuronidation with Ki of 0.39 μM, and M3 also inhibited the glucuronidation of 3′-azido-3′-deoxythymidine (AZT) by a “mixed-type” mechanism with Ki of 16.8 μM. The results suggest that UGT1A1 provides the major contribution to M3 glucuronidation in vitro and M3 has the potential to interact with xenobiotics and endogenous chemicals that are UGT1A9 and UGT 2B7 substrates.

UDP-glucuronosyltransferase (UGT) 1A1 mainly contributes to the glucuronidation of trovafloxacin

Identification of drug-metabolizing enzyme(s) responsible for the metabolism of drugs is an important step to understand not only interindividual variability in pharmacokinetics but also molecular mechanisms of metabolite-related toxicity. While it was reported that the major metabolic pathway of trovafloxacin, which is an antibiotic, was glucuronidation, the UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the trovafloxacin glucuronidation has not been identified yet. In the present study, among the functional human UGT members, UGT1A1, UGT1A3, and UGT1A9 exhibited higher trovafloxacin acyl-glucuronidation activities. While other UGT members such as UGT1A8, UGT2B7, and UGT2B15 showed glucuronidation activity toward trovafloxacin, the metabolic velocity was extremely low. In human liver microsomes, trovafloxacin acyl-glucuronidation followed the Hill equation with S50 value of 95 μM, Vmax value of 243 pmol/min per mg, and a Hill coefficient of 2.0, while the UGT1A1-expressing system displayed Michaelis–Menten kinetics with a substrate inhibition, with Km value of 759 μM and Vmax value of 1160 pmol/min per mg. In human liver microsomes prepared from poor metabolizers (UGT1A1*28/*28), significantly reduced trovafloxacin acyl-glucuronide formation activity was observed, indicating that UGT1A1 mainly, while other UGT members such as UGT1A3 and UGT1A9 partially, contributes to the glucuronidation of trovafloxacin.

Regulation profile of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) components towards UDP-glucuronosyltransferases (UGTs) isoforms

1. Endogenous compounds have been reported to be the regulators of UDP-glucuronosyltransferases (UGTs) isoforms. This study aims to investigate the regulatory effects of the activity of UGT isoforms by two important lipid components phosphatidylcholine (PC) and lysophosphatidylcholines (LPC) using in vitro incubation system.2. UGTs supersomes-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as the probe reaction to evaluate the inhibition of compounds towards UGT isoforms except UGT1A4, and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation reaction was utilized to phenotype the activity of UGT1A4.3. About 50 μM of LPC15:0, LPC16:0, LPC17:0, LPC18:0, LPC18:1 and PC16:0, 2:0 exhibited inhibition towards more than 90% activity of UGT isoforms, and other LPC and PC components showed negligible inhibitory potential towards all the UGT isoforms. UGT1A6 and UGT1A8 were identified to be the most sensitive UGT isoforms susceptible for the inhibition by LPC15:0, LPC16:0, LPC17:0, LPC18:0, LPC18:1 and PC16:0, 2:0, indicating the strong influence of these LPC and PC components towards UGT1A6 and UGT1A8-catalyzed metabolic reaction when the concentrations of these components increased.

An orphan esterase ABHD10 modulates probenecid acyl glucuronidation in human liver.

Probenecid, a widely used uricosuric agent, is mainly metabolized to probenecid acyl glucuronide (PRAG), which is considered a causal substance of severe allergic or anaphylactoid reactions. PRAG can be hydrolyzed (deglucuronidated) to probenecid. The purpose of this study was to identify enzymes responsible for probenecid acyl glucuronidation and PRAG deglucuronidation in human livers and to examine the effect of deglucuronidation in PRAG formation. In human liver homogenates (HLHs), the intrinsic clearance (CLint) of PRAG deglucuronidation was much greater (497-fold) than that of probenecid acyl glucuronidation. Evaluation of PRAG formation by recombinant UDP-glucuronosyltransferase (UGT) isoforms and an inhibition study using HLHs as an enzyme source demonstrated that multiple UGT isoforms, including UGT1A1, UGT1A9, and UGT2B7, catalyzed probenecid acyl glucuronidation. We found that recombinant α/β hydrolase domain containing 10 (ABHD10) substantially catalyzed PRAG deglucuronidation activity, whereas carboxylesterases did not. Similar inhibitory patterns by chemicals between HLHs and recombinant ABHD10 supported the major contribution of ABHD10 to PRAG deglucuronidation in human liver. Interestingly, it was demonstrated that the CLint value of probenecid acyl glucuronidation in HLHs was increased by 1.7-fold in the presence of phenylmethylsulfonyl fluoride, which potently inhibited ABHD10 activity. In conclusion, we found that PRAG deglucuronidation catalyzed by ABHD10 suppressively regulates PRAG formation via multiple UGT enzymes in human liver. The balance of activities by these enzymes is important for the formation of PRAG, which may be associated with the adverse reactions observed after probenecid administration.

In vitro assay of six UDP-glucuronosyltransferase isoforms in human liver microsomes, using cocktails of probe substrates and liquid chromatography-tandem mass spectrometry.

UDP-glucuronosyltransferase (UGT)-mediated drug-drug interactions are commonly evaluated during drug development. We present a validated method for the simultaneous evaluation of drug-mediated inhibition of six major UGT isoforms, developed in human liver microsomes through the use of pooled specific UGT probe substrates (cocktail assay) and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The six probe substrates used in this assay were estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-hydroxyindole (UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). In a cocktail incubation, UGT1A1, UGT1A9, and UGT2B7 activities were substantially inhibited by other substrates. This interference could be eliminated by dividing substrates into two incubations: one containing estradiol, trifluoperazine, and 4-hydroxyindole, and the other containing chenodeoxycholic acid, propofol, and naloxone. Incubation mixtures were pooled for the simultaneous analysis of glucuronyl conjugates in a single LC-MS/MS run. The optimized cocktail method was further validated against single-probe substrate assays using compounds known to inhibit UGTs. The degree of inhibition of UGT isoform activities by such known inhibitors in this cocktail assay was not substantially different from that in single-probe assays. This six-isoform cocktail assay may be very useful in assessing the UGT-based drug-interaction potential of candidates in a drug-discovery setting.

Glucuronidation of fimasartan, a new angiotensin receptor antagonist, is mainly mediated by UGT1A3

1. Fimasartan is an angiotensin receptor II antagonist used to treat patients with hypertension. This drug is mainly excreted into bile as either the parent compound or a glucuronide conjugate. In this study, we examined the glucuronidation of fimasartan and characterized the UDP-glucuronosyltransferases (UGTs) responsible for the glucuronidation.2. Only one type of fimasartan glucuronide was observed after incubation with pooled human liver microsomes (HLMs) and was identified as an N2-glucuronide based on comparison with an authentic standard.3. Among the 12 UGT isoforms tested, UGT1A1, UGT1A3 and UGT2B7 showed catalytic activity toward fimasartan glucuronidation. The intrinsic clearance (CLint) of UGT1A3 was 68.5- and 21.4-fold higher than that of UGT1A1 and UGT2B7, respectively, and the estimated relative contribution of UGT1A3 in human liver was 94.1%. Both chemical inhibition and correlation studies demonstrated that fimasartan glucuronidation activity in HLMs was significantly related with UGT1A3 activity. Fimasartan glucuronide was identified as a substrate for P-glycoprotein (Pgp) and breast cancer response protein (BCRP).4. These findings collectively indicate that UGT1A3 is the major UGT isoform responsible for the glucuronidation of fimasartan, and this glucuronide is excreted from hepatocytes via MDR1 and BCRP.

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

1. Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far.2. 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.3. 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.4. 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.

Herb–drug interaction prediction based on the high specific inhibition of andrographolide derivatives towards UDP-glucuronosyltransferase (UGT) 2B7

Herb–drug interaction strongly limits the clinical application of herbs and drugs, and the inhibition of herbal components towards important drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reasons. The present study aims to investigate the inhibition potential of andrographolide derivatives towards one of the most important phase II DMEs UDP-glucuronosyltransferases (UGTs). Recombinant UGT isoforms (except UGT1A4)-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation were employed to firstly screen the andrographolide derivatives' inhibition potential. High specific inhibition of andrographolide derivatives towards UGT2B7 was observed. The inhibition type and parameters (Ki) were determined for the compounds exhibiting strong inhibition capability towards UGT2B7, and human liver microsome (HLMs)-catalyzed zidovudine (AZT) glucuronidation probe reaction was used to furtherly confirm the inhibition behavior. In combination of inhibition parameters (Ki) and in vivo concentration of andrographolide and dehydroandrographolide, the potential in vivo inhibition magnitude was predicted. Additionally, both the in vitro inhibition data and computational modeling results provide important information for the modification of andrographolide derivatives as selective inhibitors of UGT2B7. Taken together, data obtained from the present study indicated the potential herb–drug interaction between Andrographis paniculata and the drugs mainly undergoing UGT2B7-catalyzed metabolic elimination, and the andrographolide derivatives as potential candidates for the selective inhibitors of UGT2B7.

Identification and preliminary characterization of UDP-glucuronosyltransferases catalyzing formation of ethyl glucuronide

Ethyl glucuronide (EtG), a minor metabolite of ethanol, is used as a marker of alcohol consumption in a variety of clinical and forensic settings. At present there are very few studies of UDP-glucuronosyltransferases (UGT), responsible for catalyzing EtG formation, and the possible effect of nutritional components, e.g. flavonoids, which are extensively glucuronidated, on EtG formation has not been addressed at all. The following incubation conditions were optimized with regard to previously published conditions: buffer, substrate concentration, and incubation time. Isolation of EtG from the incubation mixture was also optimized. Recombinant UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15) were screened for their activity towards ethanol, and kinetic data were then established for all enzymes. It was decided to study the effect of the flavonoids quercetin and kaempferol on glucuronidation of ethanol. Isolation was by solid-phase extraction (SPE) to minimize matrix effects. Analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS-MS), with EtG-d5 as the internal standard. SPE was vital to avoid severe ion suppression after direct injection of the incubation solution. EtG formation was observed for all enzymes under investigation; their kinetics followed the Michaelis-Menten model, meaning the maximum reaction rate achieved at saturating substrate concentrations (V(max)) and the substrate concentration at which the reaction rate is half of V(max) (Michaelis-Menten constant, K(m)) could be calculated. The highest rate of glucuronidation was observed with UGT1A9 and 2B7. After co-incubation with both flavonoids, formation of EtG was significantly reduced for all enzymes except for UGT2B15, whose activity did not seem to be affected. Results reveal that multiple UGT isoforms are capable of catalyzing glucuronidation of ethanol; nevertheless, the effect of UGT polymorphism on glucuronidation of ethanol needs further study. Formation of EtG is inhibited by the flavonoids under investigation. Obviously, nutritional components affect conversion of ethanol to EtG. This observation may serve as a partial explanation of its variable formation in man.

Hepatic Glucuronidation of Isoneochamaejasmin A from the Traditional Chinese MedicineStellera ChamaejasmeL. Root

Isoneochamaejasmin A (INCA), a biflavonoid, is one of main active ingredients in the dried root of Stellera chamaejasme L., a widely used traditional Chinese medicine. In the present study, we identified the glucuronidation metabolite of INCA and characterized the UDP glucuronosyltransferases (UGTs) responsible for INCA glucuronidation. 7-O-glucuronide (M1) and 4'-O-glucuronide (M2) were identified by incubation of INCA with human liver microsomes (HLMs) in the presence of UDP glucuronic acid, and their structures were confirmed by high-resolution mass spectrometry and nuclear magnetic resonance analyses. Although INCA is a single enantiomer molecule, its M1 metabolite showed two equal-size peaks on a πNAP stationary phase but only one peak on a C(18) stationary phase, indicating that the 7-/7''- and 4'-/4'''-hydroxyl groups of INCA were in different spatial configurations relative to each other. Among the recombinant human UGT isoform test and correlation analysis, UGT1A1, UGT1A3, and UGT1A9 were found to mediate M1 formation, whereas only UGT1A3 mediated M2 formation. Kinetic studies showed obvious species differences between human, mouse, rat, dog, and pig liver microsomes. UGT1A1, HLMs, and human intestinal microsomes, but not human kidney microsomes, exhibited substrate inhibition for the formation of M1. UGT1A1-mediated formation of M1 showed a 6- and 11-fold higher V(max) than did UGT1A3- and UGT1A9-mediated formation of M1, respectively. The results of the relative activity factor assay showed that UGT1A1 contributed approximately 75% in the formation of M1. These findings collectively indicate that UGT1A1 is the major enzyme in the formation of M1, whereas UGT1A3 is the major enzyme in the formation of M2.

The Inhibition of the Components from Shengmai Injection towards UDP-Glucuronosyltransferase.

The mechanism of shengmai injection- (SMI-) related drug-drug interaction remains unclear. Evaluation of the inhibition potential of SMI's ingredients towards UDP-glucuronosyltransferases (UGTs) activity will provide a new insight to understand SMI-related drug-drug interaction. In vitro incubation system to model UGT reaction was used. Recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation reactions were employed to phenotype the inhibition profile of maidong's components towards the activity of UGT isoforms. Different inhibition potential of maidong's components towards various UGT isoforms was observed. Based on the inhibition kinetic investigation results, ophiopogonin D (OD) noncompetitively inhibited UGT1A6 and competitively inhibited UGT1A8, ophiopogonin D′ (OD′) noncompetitively inhibited UGT1A6 and UGT1A10, and ruscorectal (RU) exhibited competitive inhibition towards UGT1A4. The inhibition kinetic parameters were calculated to be 20.6, 40.1, 5.3, 9.0, and 0.02 μM, respectively. In combination with our previous results obtained for the inhibition of UGT isoforms by ginsenosides and wuweizi components, the important SMI ingredients exhibiting strong inhibition towards UGT isoforms were highlighted. All the results obtained in the present study provide a new insight to understand SMI-related drug-drug interaction.

Absolute Quantification of Cytochrome P450 and Uridine-Diphosphate Glucuronosyl Transferase Isoforms by Proteomics-based Approach

Abstract A strategy for the absolute quantitation of metabolic enzymes in rat liver microsomes was developed based on shotgun-based proteomics approach. Rat liver microsomes were digested with trypsin, and drug metabolizing enzymes were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) using standard curves based on custom synthesized specific peptides for each enzyme. The assays for cytochrome P450 (CYP450) and uridine diphosphoglucuronosyl transferase (UGT) showed good linearity ( r > 0.995) with lower limits of quantitation of 10 nM. A synthetic isotope labeled specific peptide was then used as internal standard to determine UGT1A1 by stable isotope dilution method. The labeled peptide behaved similarly to the unlabeled peptide in LC-MS/MS and the assay was linear in matrix solution. The UGT1A1 concentration was detected by the labeled peptide method to be 18.2 pmol mg −1 protein compared with 17.3 pmol mg −1 protein obtained by the standard curve method. Although the consistent results were achieved by the two methods, the stable isotope dilution method was more convenient and more suitable for high throughput determinations in complex systems.

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-β-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.

Alteration of the Function of the UDP-Glucuronosyltransferase 1A Subfamily by Cytochrome P450 3A4: Different Susceptibility for UGT Isoforms and UGT1A1/7 Variants

Functional protein-protein interactions between UDP-glucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells that simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of serotonin, both Michaelis constant (Km) and maximal velocity (Vmax) were increased by CYP3A4. When CYP3A4 was coexpressed with either UGT1A1 or 1A7, the Vmax for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. S50 and Km both which are the substrate concentration giving 0.5 Vmax were little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7 and their effects on the interaction with CYP3A4. Although the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the coexpression of CYP3A4 restored the impaired function to a level comparable with the wild type. Similarly, simultaneous expression of CYP3A4 increased the Vmax of UGT1A7*1 (wild type) and *2 (N129K and R131K), whereas the same was not observed in UGT1A7*3 (N129K, R131K, and W208R). In the kinetics involving different concentrations of UDP-glucuronic acid (UDP-GlcUA), the Km for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The Km of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that (1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoform-specific manner and (2) nonsynonymous mutations in UGT1A7*3 reduce not only the ability of UGT to use UDP-GlcUA but also CYP3A4-mediated enhancement of catalytic activity, whereas CYP3A4 is able to restore the UGT1A1*6 function.

Interception of Benzo[a]pyrene-7,8-dione by UDP Glucuronosyltransferases (UGTs) in Human Lung Cells

Polycyclic aromatic hydrocarbons (PAHs) are environmental and tobacco carcinogens. Proximate carcinogenic PAH trans-dihydrodiols are activated by human aldo-keto reductases (AKRs) to yield electrophilic and redox-active o-quinones. Interconversion among benzo[a]pyrene (B[a]P)-7,8-dione, a representative PAH o-quinone, and its corresponding catechol generates a futile redox-cycle with the concomitant production of reactive oxygen species (ROS). We investigated whether glucuronidation of B[a]P-7,8-catechol by human UDP glucuronosyltransferases (UGTs) could intercept the catechol in three different human lung cells. RT-PCR showed that UGT1A1, 1A3, and 2B7 were only expressed in human lung adenocarcinoma A549 cells. The corresponding recombinant UGTs were examined for their kinetic constants and product profile using B[a]P-7,8-catechol as a substrate. B[a]P-7,8-dione was reduced to B[a]P-7,8-catechol by dithiothreitol under anaerobic conditions and then further glucuronidated by the UGTs in the presence of uridine-5′-diphosphoglucuronic acid as a glucuronic acid group donor. UGT1A1 catalyzed the glucuronidation of B[a]P-7,8-catechol and generated two isomeric O-monoglucuronsyl-B[a]P-7,8-catechol products that were identified by RP-HPLC and by LC-MS/MS. By contrast, UGT1A3 and 2B7 catalyzed the formation of only one monoglucuronide, which was identical to that formed in A549 cells. The kinetic profiles of three UGTs followed Michaelis–Menten kinetics. On the basis of the expression levels of UGT1A3 and UGT2B7 and the observation that a single monoglucuronide was produced in A549 cells, we suggest that the major UGT isoforms in A549 cells that can intercept B[a]P-7,8-catechol are UGT1A3 and 2B7.

Breast Cancer Resistance Protein-Mediated Efflux of Luteolin Glucuronides in HeLa Cells Overexpressing UDP-Glucuronosyltransferase 1A9

UDP-glucuronosyltransferases (UGTs) are responsible for the formation of glucuronides of polyphenolic flavonoids. This study investigated the UGT1A9-mediated glucuronidation of luteolin and the kinetics of luteolin glucuronide efflux. HeLa cells overexpressing UGT1A9 (HeLa-UGT1A9) were used to determine the kinetics of breast cancer resistance protein (BCRP)-mediated transport of luteolin glucuronides. Human UGT isoforms were used to determine glucuronidation rates. UGT1A9 was found to catalyze the production of four luteolin glucuronides, including three known monoglucuronides and a novel 3', 4'-diglucuronide. Ko143, a potent specific inhibitor of BCRP, significantly inhibited efflux of luteolin monoglucuronides from HeLa1A9 cells and increased their intracellular levels in a dose-dependent manner. The formation of luteolin diglucuronide was observed when intracellular concentration of total monoglucuronides went above 0.07nM. Intracellular accumulation of diglucuronide was detected at high monoglucuronide concentrations (>0.07nM). Diglucuronide production is speculated to be a compensatory pathway for luteolin disposition.