Uridine 5 ' - Diphospho Research Articles

Formation of ethyl glucuronide by beta-glucuronidase

Substantial amounts of endogenous and xenobiotic compounds are transformed to ß-D-Glucuronides in the human body. The conjugation reaction, which requires uridine-5′-diphospho (UDP)-alpha-D-glucuronic acid as a cosubstrate is catalyzed by UDP-glucuronosyltransferases (UGTs). Beta-glucuronidase (GUSB), which is widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants, opposes this reaction: ß-D-glucuronides are hydrolyzed to ß-D-glucuronic acid and the corresponding aglycon in a configuration retaining manner. This study investigated GUSB activity in the presence of ethanol. Phenolphthalein-ß-D-glucuronide (PHPH-G), 4-nitrophenol-ß-D-glucuronide (4NP-G), morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide were used as substrates. Enzymatic reactions were carried out at 37 °C in buffer solution fortified with different amounts of ethanol (0–70% by volume) using GUSB from different sources ( E. coli , H. pomatia , bovine and human recombinant). The enzymatic reactions were stopped after 60 minutes and all samples were tested for ß-D-ethyl glucuronide (EtG) by a validated LC/MS/MS assay. In incubations of PHPH-G and 4NP-G, the release of aglyca was additionally measured by colorimetrical methods. It was found that EtG, which is a minor metabolite of ethanol but one of the most important direct biomarkers of alcohol consumption in man today, builts up from all investigated ß-D-glucuronides by GUSB activity in the presence of ethanol. The glucuronyl transfer reaction, which was neither observed in absence of ethanol nor in absence of GUSB, is detectable but minor at ethanol concentrations which are commonly observed in blood and tissues after consumption of alcoholic beverages. At higher ethanol concentrations (> 5% by volume), the transfer reaction is predominant and the majority of glucuronyl from ß-D-glucuronides is transferred to ethanol. Different types of donor substrates yielded different amounts of EtG in the presence of the same GUSB-activity and different types of GUSB yielded different amounts of EtG from the same donor substrate. The detection of EtG in several settings independent from human UGT-metabolism (e.g., EtG post collection synthesis in E.coli contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the observed mechanism. A contribution of GUSB activity to EtG kinetics in man is suspected and should be further investigated since high concentrations of ethanol (oral intake of alcoholic beverages) and high concentrations of ß-D-glucuronides (hepatobiliary secretion) are present in the gut, where the main GUSB activity is localized. Finally, the findings could be of interest in context of EtG hair analysis, since the prerequisites for in vitro formation of EtG in this study may also be found on head. There is an additional biochemical path and another source of EtG which deserves further attention in context of alcohol biomarker applications.

Beta-Glucuronidase Activity: Another Source of Ethyl Glucuronide.

Numerous classes of endogenous and xenobiotic compounds are conjugated to uridine-5'-diphospho (UDP)-alpha-D-glucuronic acid which is catalyzed by human UDP-Glucuronosyltransferases (UGTs). The resulting beta-D-glucuronides can be hydrolyzed to β-D-glucuronic acid and the corresponding aglycone in a configuration retaining manner by beta-glucuronidases (GUSBs), which are widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants. This study investigates GUSBs' activity in the presence of ethanol (0-70% by volume) using different β-D-glucuronides (phenolphthalein-β-D-glucuronide, 4-nitrophenol-β-D-glucuronide, morphine-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide and 1-/2-propyl-β-D-glucuronide) as substrates. It was found that β-D-ethyl glucuronide (EtG), which is a minor UGT-derived metabolite of ethanol in man and one of the most frequently used biomarkers of alcohol consumption today, builds up from all investigated β-D-glucuronides by means of GUSBs in the presence of ethanol. The glucuronyl transfer reaction, which was neither detected in the absence of ethanol nor in absence of GUSBs, is minor at ethanol concentrations which are commonly observed in blood and tiβues after consumption of alcoholic beverages, but predominant at higher concentrations of ethanol. In spite of in vitro characteristics, our observations point to an additional biochemical path and another source of EtG, which should be further evaluated in the context of alcohol biomarker applications. The detection of EtG in several settings independent from of human UGT-metabolism (e.g. EtG post post-collection synthesis in E.coli coli-contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the described mechanism.

Metabolic conjugation reduces in vitro toxicity of the flavonoid nevadensin

The present study focuses on the association between metabolic capacity and toxicity of the natural occurring flavonoid nevadensin in vitro. Human colon (HT29), liver (HepG2) and bone marrow (KG1) carcinoma cells were used and strong cell line dependent differences in toxic effect strength were found. HepG2 and KG1 cells were more sensitive against nevadensin treatment in comparison to HT29 cells. High resolution mass spectrometry experiments showed that nevadensin is rapidly glucuronidated in HT29 cells, whereas KG1 cells do not metabolize nevadensin, thus glucuronidation was supposed to be a crucial metabolic pathway in vitro. To proof this suggestion, nevadensin glucuronides were isolated from pig liver microsomes und structurally elucidated via NMR spectroscopy. In HepG2 cells a cellular enrichment of nevadensin itself as well as nevadensin-7-O-glucuronide was determined by tandem mass spectrometry. A proteomic screening of uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT) in HT29 and HepG2 cells provided first hints that the isoforms UGT1A6 and UGT1A1 are responsible for nevadensin glucuronidation. Additionally, nevadensin was found to be a potent SULT inhibitor in HepG2 cells. In sum, the present study clearly illustrates the importance of obtaining detailed information about metabolic competence of cell lines which should be considered in the evaluation of toxic endpoints.

Natural alleles of a uridine 5'-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice.

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5'-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1YZN compared to EDR1YD1 , resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1YD1 and EDR1YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1YD1 and EDR1YZN varieties indicated that rice varieties harboring EDR1YZN and EDR1YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.

Potential of amentoflavone with antiviral properties in COVID-19 treatment.

Amentoflavone is one of the flavonoids that are known for their antiviral effects and many of them are predicted to have inhibitory effects against severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome Coronavirus (MERS-CoV) enzymes 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). Amentoflavone is a biflavonoid found in the herbal extracts of St. John's wort (Hypericum perforatum), Gingko biloba, Selaginella tamariscina, Torreya nucifera, and many other plants. Its pharmacological actions have been listed as antiviral, antibacterial, antioxidant, anti-inflammatory, antidiabetic, antidepressant, and neuroprotective. Molecular docking studies have found that amentoflavone binds strongly to the active site of the main protease (Mpro) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). As conventional antiviral medications are met with limited success against coronavirus disease-2019 (COVID-19) and vaccines are one of the only weapons against COVID-19 in the pharmaceutical armamentarium, traditional medicines are being considered for the forefront battle against COVID-19. Clinical studies with Hypericum and Gingko extract as additional or alternative drugs/supplements are registered. Here we review the potential of amentoflavone, an active agent in both Hypericum and Gingko extract as an adjunct therapy for COVID-19. Its anti-inflammatory, antioxidant, and sepsis preventive actions could provide protection against the "cytokine storm." Compared with the herbal extracts, which induce cytochrome P450 (CYP) and uridine 5'-diphospho (UDP)-glucuronosyltransferases (UGT) activity producing a negative herb-drug interaction, amentoflavone is a potent inhibitor of CYP3A4, CYP2C9, and UGT. Further studies into the therapeutic potential of amentoflavone against the coronavirus infection are warranted.

Effect of UGT2B7 Gene Polymorphism with Clinical Pediatric Epileptic patients on Sodium Valproate Monotherapy

Background: Pediatric epilepsy comprises of a chronic neurological disorders characterized by recurrent seizure attack. Sodium valproate is one of the common anti-epileptic drugs used in the treatment. Glucuronide conjugation is the major metabolic pathway of sodium valproate, carried out by the enzyme Uridine 5’-diphospho (UDP) glucuronosyl transferase (UGT) whose genetic polymorphisms may alter clinical outcome. Aim: To find the association between UGT2B7 genetic polymorphism and clinical outcome in terms of efficacy and tolerability in pediatric epileptic patients on sodium valproate monotherapy. Methods and Materials: In this cohort study, 75 pediatric epileptic patients aged 2-18 years receiving sodium valproate monotherapy for past one month were included from Justice K S Hegde Charitable Hospital, Mangalore, India after obtaining informed consent. Genetic polymorphism patterns of UGT2B7 (C161T, A268G, G211T) was evaluated by PCR-RFLP. Clinical outcome was measured in terms of responders and non-responders based on seizure control during the 6 month observation period. Tolerability was measured by estimating the hepatic, renal and other lab parameters. Clinical outcome in different UGT genotypes was compared by Chi square test. P value <0.05 was considered as significant. Results: Out of 75 patients, CC (41.3%), CT (38.7%), TT (20%) pattern was observed in UGT2B7 (C161T) gene, AA(14.7%), AG(42.7%), GG(42.7%) in (A268G) gene and GG(80%), GT(18.7%), TT(1.3%) in (G211T) gene. It was found that there was no statistical difference in clinical outcome with different UGT2B7 genetic polymorphism patterns. Conclusion: We conclude from our study that genetic polymorphism of UGT2B7 doesn’t have any role on the clinical outcome of epilepsy.

Intake of Molecular Hydrogen in Drinking Water Increases Membrane Transporters, p-Glycoprotein, and Multidrug Resistance-Associated Protein 2 without Affecting Xenobiotic-Metabolizing Enzymes in Rat Liver.

Molecular hydrogen (H2) has been shown to have antioxidant and anti-inflammatory activities that may reduce the development and progression of many diseases. In this study, hydrogen-rich water (HRW) was obtained by reacting hybrid magnesium–carbon hydrogen storage materials with water. Then, the effects of intake of HRW on the activities of xenobiotic-metabolizing enzymes, membrane transporters, and oxidative stress in rats were investigated. Rats were given HRW ad libitum for four weeks. The results showed that intake of HRW had no significant effect on the activities of various cytochrome P450 (CYP) enzymes (CYP1A1, 1A2, 2B, 2C, 2D, 2E1, 3A, and 4A), glutathione-S-transferase, and Uridine 5′-diphospho (UDP)-glucuronosyltransferase. Except for a mild lower plasma glucose concentration, intake of HRW had no effect on other plasma biochemical parameters in rats. p-Glycoprotein and multidrug resistance-associated protein (Mrp) 2 protein expressions in liver were elevated after intake of HRW. However, HRW had no significant effects on glutathione, glutathione peroxidase, or lipid peroxidation in liver. The results from this study suggest that consumption of HRW may not affect xenobiotic metabolism or oxidative stress in liver. However, intake of HRW may increase the efflux of xenobiotics or toxic substances from the liver into bile by enhancing p-glycoprotein and Mrp2 protein expressions.

Identification and interspecies characterization of UDP-glucuronosyltransferase isoforms catalyzing acacetin glucuronidation using recombinant UGT enzymes and microsomes

Abstract Objective To explore the glucuronic acid metabolism of acacetin in human liver and intestinal microsomes to better characterize human uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT) isoforms. In addition, interspecies comparisons were performed to identify the most appropriate experimental animal model for an in vivo study. Methods Liquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) were used to confirm the successful biosynthesis of acacetin-7-O-glucuronide. Human isoforms of UGT and isozyme-specific chemical inhibitors were used for recombinant assays. Acacetin glucuronidation kinetics were assessed by combining acacetin with recombinant human UGT isoforms or with microsomes from humans or experimental animals. Kinetic differences between species were assessed in vitro using the same approach. Results We identified multiple UGT isoforms that facilitated acacetin glucuronidation, and found that UGT1A1 was the major isoform that catalyzed this process. Acacetin-7-O-glucuronide formation exhibited clear substrate inhibition kinetics when combined with recombinant UGTs or with liver/intestinal microsomes derived from humans, monkeys, rats, mice, dogs, or pigs. Intrinsic metabolic clearance values of human intestinal microsomes were two-fold greater than those of human liver microsomes. Among the evaluated species, the Km value of dog microsomes (0.86 μM) was greatest in acacetin glucuronidation, while mice exhibited the highest CLint value, 5.05 mL/min/mg. The CLint values of microsomes derived from monkeys and minipigs were 1.99 mL/min/mg and 2.12 mL/min/mg, respectively, exhibiting similar intrinsic metabolic clearance activity to that observed in humans. Conclusion Monkey may represent a suitable model for experimental studies of acacetin pharmacokinetics owing to a high sequence homology of UGT1A1 and similar UGT1A1 glucuronidation activity to humans.

Uridine 5'-diphospho-glucronosyltrasferase: Its role in pharmacogenomics and human disease.

Biotransformation is an enzyme-catalyzed process in which the body converts endogenous compounds, xenobiotics and toxic substances into harmless or easily excreted metabolites. The biotransformation reactions are classified as phase I and II reactions. Uridine 5'-diphospho (UDP)-glucuronosyltransferases (UGTs) are a superfamily of phase II enzymes which have roles in the conjugation of xenobiotics or endogenous compounds, including drugs and bilirubin, with glucuronic acid to make them easier to excrete. The method the human body uses to achieve glucuronidation may be affected by a large interindividual variation due to changes in the sequences of the genes encoding these enzymes. In the last five years, the study of the genetic variants of the UGTs at a molecular level has become important due to its association with several diseases and the ability to predict adverse events due to drug metabolism. In the present review, the structure and the prominent genetic variants of the UGT1A subfamily and their metabolic and clinical implications are described.

Adaptation in Caco-2 Human Intestinal Cell Differentiation and Phenolic Transport with Chronic Exposure to Blackberry (Rubus sp.) Extract.

As evidence mounts for a health-protective role of dietary phenolics, the importance of understanding factors influencing bioavailability increases. Recent evidence has suggested chronic exposure to phenolics may impact their absorption and metabolism. To explore alterations occurring from chronic dietary exposure to phenolics, Caco-2 cell monolayers were differentiated on Transwell inserts with 0-10 μM blackberry (Rubus sp.) total phenolics extracts rich in anthocyanins, flavonols, and phenolic acids. Following differentiation, apical to basolateral transport of phenolics was assessed from an acute treatment of 100 μM blackberry phenolics from 0 to 4 h. Additionally, differences in gene expression of transport and phase II metabolizing systems including ABC transporters, organic anion transporters (OATs), and uridine 5'-diphospho (UDP) glucuronosyltransferases (UGTs) were probed. After 4 h, 1 μM pretreated monolayers showed a significant (P < 0.05) decrease in the percentage of cumulative transport including less epicatechin (42.1 ± 0.53), kaempferol glucoside (23.5 ± 0.29), and dicaffeoylquinic acid (31.9 ± 0.20) compared to control. Finally, significant (P < 0.05) alterations in mRNA expression of key phase II metabolizing enzymes and transport proteins were observed with treatment. Therefore, adaptation to blackberry extract exposure may impact intestinal transport and metabolism of phenolics.

Effects of lemongrass oil and citral on hepatic drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in rats

The essential oil from a lemongrass variety of Cymbopogon flexuosus [lemongrass oil (LO)] is used in various food and aroma industry products and exhibits biological activities, such as anticancer and antimicrobial activities. To investigate the effects of 200 LO (200 mg/kg) and 400 LO (400 mg/kg) and its major component, citral (240 mg/kg), on drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in the liver, male Sprague-Dawley rats were fed a pelleted diet and administered LO or citral by gavage for 2 weeks. After 2 weeks of feeding, the effects of LO and citral on the metabolism and toxicity of acetaminophen were determined. The results showed that rats treated with 400 LO or citral had significantly reduced hepatic testosterone 6β-hydroxylation and ethoxyresorufin O-deethylation activities. In addition, NAD(P)H:quinone oxidoreductase 1 activity was significantly increased by citral, and Uridine 5′-diphospho (UDP) glucurosyltransferase activity was significantly increased by 400 LO in the rat liver. Treatment with 400 LO or citral reduced lipid peroxidation and reactive oxygen species levels in the liver. After acetaminophen treatment, however, LO and citral treatment resulted in little or no change in plasma alanine aminotransferase activity and acetaminophen-protein adducts content in the liver. Our results indicate that LO and citral may change the activities of drug-metabolizing enzymes and reduce oxidative stress in the liver. However, LO and citral may not affect the detoxification of acetaminophen.

Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians

The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5′-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities.

찔레꽃 및 찔레꽃 복합물 추출물이 고지방·고콜레스테롤 식이 흰쥐의 지질 함량 개선에 미치는 영향

본 연구에서는 고지방·고콜레스테롤 식이 흰쥐에서 찔레꽃과 찔레꽃 복합물이 혈청 및 간 조직의 지질대사에 미치는 영향을 관찰하였다. 실험군을 4군으로 나누어 정상 식이군(N군), 고지방·고콜레스테롤 식이군(HF군), 고지방·고콜레스테롤 식이에 찔레꽃을 5% 첨가한 군(HR군), 고지방·고콜레스테롤 식이에 찔레꽃 복합물을 첨가한 군(HRC군)으로 나누었다. 찔레꽃 복합물은 찔레꽃, 뱀딸기풀, 곰보배추, 조릿대로 구성되어 있다. 체중증가량과 식이효율은 HF군이 N군에 비해 유의적으로 증가하였고 HR군과 HRC군이 HF군에 비해 감소하였다. 혈청 중성지질은 HF군이 N군에 비해 유의적으로 증가하였고 HRC군은 유의적으로 크게 감소하였다. 혈청 HDL-cholesterol은 HR군과 HRC군이 HF군에 비해 증가하였다. 혈청 총콜레스테롤과 LDL-cholesterol은 HF군이 N군에 비해 유의적으로 증가하였고, 특히 동맥경화지수에서 HRC군은 HF군에 비해 유의적으로 감소하였다. 간 중성지질과 총콜레스테롤은 HF군이 N군에 비해 유의적으로 증가하였고 HR군과 HRC군은 HF군에 비해 유의적으로 감소하였다. 대변 중성지질과 총콜레스테롤은 HR군과 HRC군이 HF군에 비해 유의적으로 증가하였다. UDPglucuronyl transferase 활성은 HR군과 HRC군이 HF군에 비해 증가하였다. Total bile acid는 HR군과 HRC군이 HF군에 비해 증가하였고, 특히 HRC군은 HF군에 비해 유의적으로 증가하였다. 이러한 결과 찔레꽃과 찔레꽃 복합물은 UDP-glucuronyl transferase 활성, total bile acid 및 지질대사 조절에 효과적으로 활용될 수 있다고 사료된다.

Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

Glucuronidation mediated by uridine 5′-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

Elucidating butanol tolerance mediated by a response regulator Sll0039 in Synechocystis sp. PCC 6803 using a metabolomic approach.

Butanol is highly toxic to cyanobacterial cells, which could restrict the future application of this renewable system in producing carbon neutral biofuel butanol. To seek knowledge regarding butanol tolerance in cyanobacteria, a library of response regulator (RR) gene mutants of Synechocystis sp. PCC 6803 was screened. The results showed that the deletion mutant of an orphan RR-encoding genes ll0039 was more sensitive to butanol than the wild type, while complementation of the ∆slr1037 mutant with the sll0039 gene recovered its tolerance to the same level as the wild type, suggesting that the sll0039 gene was involved in the regulation of tolerance against butanol. Further analysis employing an integrated liquid chromatography-mass spectrometry (LC-MS)-based and gas chromatography-mass spectrometry (GC-MS)-based metabolomics was conducted to determine the possible regulatory network of butanol tolerance mediated by Sll0039. LC-MS analysis allowed the identification of several metabolites, such as adenosine 5'-diphosphate (ADP)-glucose, dihydroxyacetone phosphate (DHAP), D-ribose 5-phosphate (R5P), D-glucose 6-phosphate (G6P), D-fructose 6-phosphate (F6P), α-ketoglutaric acid (AKG), uridine 5'-diphospho (UDP)-glucose, and nicotinamide adenine dinucleotide phosphate (NADP), which were differentially regulated between the wild type and the ∆sll0039 mutant grown under butanol stress, while GC-MS analysis identified 1, 2, and 2 metabolic modules associated with the sll0039 gene deletion at 24, 48, and 72h, respectively, suggesting that they were under control directly or indirectly by Sll0039 RR. In addition, a metabolomic comparison of the metabolic responses to butanol stress was conducted in the ∆sll0039 mutant and the ∆slr1037 mutant previously found to be involved in butanol tolerance (Chen et al. Biotechnol Biofuels 7:89 2014a), and the results showed that the regulatory networks mediated by Sll0039 and Slr1037 could be functionally independent in Synechocystis. The results provided a metabolomic description of the butanol tolerance network regulated by Sll0039.

Prevalence of UGT1A6 polymorphisms in children with epilepsy on valproate monotherapy.

Valproate is a commonly used anticonvulsant drug. Uridine 5΄-diphospho (UDP)-glucuronosyltransferase (UGT) contributes to around 50% of valproate metabolism and its polymorphisms may be important for explaining the considerable variation in valproate levels in patients with epilepsy. This study was aimed to analyze the genetic polymorphisms of UGT1A6 in Indian children with epilepsy and their potential influence on the pharmacokinetics of valproate. This cross-sectional study was carried out in the Department of Pediatrics, All India Institutes of Medical Sciences (AIIMS), New Delhi, between March 2011 and July 2012. Children aged 3-12 years diagnosed with epilepsy on valproate monotherapy for at least 1 month were enrolled. They underwent a detailed clinical examination. The UGT1A6 polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Random samples were checked by genetic sequencing. The steady-state plasma concentrations of valproate were measured by High Performance Liquid Chromatography (HPLC) and associated with UGT1A6 polymorphisms. A total of 80 children were studied. The prevalence of UGT1A6 T19G was as follows: TT (45%), TG (38.8%), and GG (16.3%); that of UGT1A6 A541G was: AA (48.8%), AG (38.8%), and GG (12.5%); and that of UGT1A6 A552C was: AA (43.8%), AC (40%), and CC (16.3%). The association between valproate doses or standardized serum valproate concentration and the various UGT1A6 genotypes could not be studied reliably in this small study population. The frequencies of UGT1A6 geneotypes and alleles were reported in the study population.

Effects of co-treatment with sulforaphane and autophagy modulators on uridine 5'-diphospho-glucuronosyltransferase 1A isoforms and cytochrome P450 3A4 expression in Caco-2 human colon cancer cells.

Sulforaphane (SFN), which is highly enriched in cruciferous vegetables, has been investigated for its cancer chemopreventive properties and ability to induce autophagy. Uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT)1A induction is one of the mechanisms that is responsible for the cancer chemopreventive activity of SFN. The current study demonstrates that rapamycin may enhance the chemopreventive effects of SFN on Caco-2 cells; this may be partially attributed to nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2)- and human pregnane X receptor (hPXR)-mediated UGT1A1, UGT1A8 and UGT1A10 induction. These results indicate that targeting autophagy modulation may be a promising strategy for increasing the chemopreventive effects of SFN in cases of colon cancer.

Alterations in Hepatic mRNA Expression of Phase II Enzymes and Xenobiotic Transporters after Targeted Disruption of Hepatocyte Nuclear Factor 4 Alpha

Hepatocyte nuclear factor 4 alpha (HNF4a) is a liver-enriched master regulator of liver function. HNF4a is important in regulating hepatic expression of certain cytochrome P450s. The purpose of this study was to use mice lacking HNF4a expression in liver (HNF4a-HNull) to elucidate the role of HNF4a in regulating hepatic expression of phase II enzymes and transporters in mice. Compared with male wild-type mice, HNF4a-HNull male mouse livers had (1) markedly lower messenger RNAs (mRNAs) encoding the uptake transporters sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide (Oatp) 1a1, Oatp2b1, organic anion transporter 2, sodium phosphate cotransporter type 1, sulfate anion transporter 1, sodium-dependent vitamin C transporter 1, the phase II enzymes Uridine 5'-diphospho (UDP)-glucuronosyltransferase (Ugt) 2a3, Ugt2b1, Ugt3a1, Ugt3a2, sulfotransferase (Sult) 1a1, Sult1b1, Sult5a1, the efflux transporters multidrug resistance-associated protein (Mrp) 6, and multidrug and toxin extrusion 1; (2) moderately lower mRNAs encoding Oatp1b2, organic cation transporter (Oct) 1, Ugt1a5, Ugt1a9, glutathione S-transferase (Gst) m4, Gstm6, and breast cancer resistance protein; but (3) higher mRNAs encoding Oatp1a4, Octn2, Ugt1a1, Sult1e1, Sult2a2, Gsta4, Gstm1-m3, multidrug resistance protein (Mdr) 1a, Mrp3, and Mrp4. Hepatic signaling of nuclear factor E2-related factor 2 and pregnane X receptor appear to be activated in HNF4a-HNull mice. In conclusion, HNF4a deficiency markedly alters hepatic mRNA expression of a large number of phase II enzymes and transporters, probably because of the loss of HNF4a, which is a transactivator and a determinant of gender-specific expression and/or adaptive activation of signaling pathways important in hepatic regulation of these phase II enzymes and transporters.

Human UDP-glucuronosyltransferase UGT2A2: cDNA construction, expression, and functional characterization in comparison with UGT2A1 and UGT2A3

Characterize the expression and glucuronidation activities of the human uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT) 2A2. UGT2A1 was cloned from nasal mucosa mRNA. Synthetic cDNA for UGT2A2 was constructed assuming exon sharing between UGT2A1 and UGT2A2 (Mackenzie et al., Pharmacogenetics and Genomics 2005, 15:677-685). Exon 1 of UGT2A2 was amplified from genomic DNA and combined with exons 2-6 of UGT2A1. UGT2A3 was cloned from liver mRNA. Quantitative reverse-transcribed-PCR (RT-PCR) was used to evaluate the expression of all the three UGTs of subfamily 2A in different tissues. Recombinant UGT2A1, UGT2A2 and UGT2A3 were expressed in baculovirus-infected insect cells and analyzed for glucuronidation activity towards different substrates. DNA sequencing of RT-PCR products from human nasal mucosa mRNA, confirmed exon sharing between UGT2A1 and UGT2A2. In addition, it indicated that the N-terminal signal peptide sequence of UGT2A2 is the longest among the human UGTs. Quantitative RT-PCR revealed that both UGT2A1 and UGT2A2 are mainly expressed in the nasal mucosa, and that their expression level in fetal samples was much higher than in adults. Activity assays with recombinant UGTs 2A1-2A3 showed broad substrate selectivity for UGT2A1 and UGT2A2. Although glucuronidation rates and substrate affinities were mostly higher in UGT2A1, the Km values for UDP-glucuronic acid were similar in both UGTs. In addition, there were regioselectivity differences between the two UGTs and, with a few substrates, particularly ethinylestradiol, the activity of UGT2A2 was higher. UGT2A2 is mainly expressed in the nasal mucosa and it has glucuronidation activity towards several different endobiotic and xenobiotic substrates.

Stereoselectivity in Drug Metabolism: Molecular Mechanisms and Analytical Methods

About 50% of therapeutic drugs are currently administered as a racemate, a mixture of equal proportions of two enantiomers. In an achiral environment, the enantiomers of a chiral drug show identical chemical and physical properties. However, they can present different chemical and pharmacological behavior in a chiral environment such as in the body. The interaction of two enantiomers with a chiral macromolecule, such as an enzyme or receptor, is three dimensional in nature, forming diastereomeric complexes resulting in a chiral recognition process. Moreover, when administered as a racemate, two enantiomers can display the pharmacokinetic processes (absorption, distribution, metabolism and excretion) in a stereoselective manner. Among these processes, stereoselectivity plays a central role in the metabolism due to the involvement of the enzymatic system. Thus, the purpose of the current review is to present important aspects related to stereochemistry of drug metabolism, with emphasis on molecular mechanisms involved in enzyme mediated reactions, such as those catalyzed by cytochrome P450, uridine 5'-diphospho (UDP)-glucuronosyltransferases and sulfotransferases. Additionally, recent advances regarding the analysis of chiral drugs and their metabolites employing different analytical techniques (high-performance liquid chromatography-HPLC and capillary electrophoresis-CE) will also be outlined.