Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.389
Filtrar
1.
J Agric Food Chem ; 68(7): 2063-2070, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32009392

RESUMO

Luteolin is a typical flavonoid and broadly distributed in the plants. Oral bioavailability of luteolin is low owing to extensive metabolism. Regioselective glucuronidation by UDP-glucuronosyltransferases (UGTs) and liver uptake by organic anion transporting polypeptides (OATPs) of luteolin and consequent glucuronidation metabolites were studied. Luteolin-3'-O-glucuronide (L-3'-G) and luteolin-7-O-glucuronide (L-7-G) were the major metabolites in human liver microsomes. Further study demonstrated that UGT1A9 played a predominant role in the glucuronidation of luteolin. Transporter study showed that OATP1B1- and 1B3-transfected cells selectively uptake L-3'-G into cells but not luteolin or L-7-G. After intravenous administration of luteolin to mice, the area under the curve of L-3'-G in the plasma was the highest among luteolin, L-3'-G, and L-7-G. In the liver, the concentration of L-3'-G was significantly greater than L-7-G. In conclusion, OATP1B1 and OATP1B3 play an important role in the liver disposition of luteolin and its glucuronidation metabolites.


Assuntos
Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/metabolismo , Luteolina/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Transporte Biológico , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Luteolina/química , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética
2.
Anal Bioanal Chem ; 412(8): 1729-1740, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32030490

RESUMO

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Espectrometria de Massas/métodos , Microssomos Hepáticos/enzimologia , Adulto , Idoso , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/química , Feminino , Glucuronosiltransferase/química , Humanos , Masculino , Pessoa de Meia-Idade , Mapeamento de Peptídeos , Adulto Jovem
3.
Toxicol Lett ; 320: 46-51, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31812603

RESUMO

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.


Assuntos
Suplementos Nutricionais/efeitos adversos , Inibidores Enzimáticos/toxicidade , Interações Alimento-Droga , Glucuronosiltransferase/antagonistas & inibidores , Estilbenos/toxicidade , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Cinética , Taxa de Depuração Metabólica , Desintoxicação Metabólica Fase II , Modelos Biológicos , Medição de Risco , Estilbenos/farmacocinética
4.
Xenobiotica ; 50(1): 64-76, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31092094

RESUMO

The role that the phase-II reaction, glucuronidation, plays in the biotransformation of endo and xenobiotics is discussed with particular emphasis given to the UGT1A1 isoenzyme. This individual isoenzyme is responsible for both the mono and di-glucuronidation of bilirubin together with the glucuronidation of a number of xenobiotics of clinical interest (irinotecan, belinostat, atazanavir, pegvisomant).The review then discusses the roles that the various allelic variants of the UGT1A1 gene play in bilirubin metabolism and in particular how these allelic variants are involved in the clinical manifestation of the diseases of GS, CN1 and CN2.The review concludes with the roles that the UGT1A1*28 and UGT1A1*6 alleles play in adverse drug reactions (decreased glucuronidation of irinotecan, belinostat, atazanavir, pegvisomant) leading to increased exposure, reduced clearance and neutropenia (irinotecan, belinostat), increased risk for jaundice and hyperbilirubinaemia (atazanavir) and liver toxicity (pegvisomant) before discussing the future role of UGT1A1 in personalised medicine.


Assuntos
Glucuronosiltransferase/genética , Alelos , Bilirrubina , Genótipo , Glucuronosiltransferase/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Hiperbilirrubinemia/metabolismo , Irinotecano/metabolismo , Sulfonamidas/metabolismo
5.
Insect Sci ; 27(2): 276-291, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30136378

RESUMO

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are widely distributed within living organisms and share roles in biotransformation of various lipophilic endo- and xenobiotics with activated UDP sugars. In this study, it was found that the activity of UGTs in abamectin-resistant (AbR) strain was significantly higher (2.35-fold) than that in susceptible strain (SS) of Tetranychus cinnabarinus. Further analysis showed that 5-nitrouracil, the inhibitor of UGTs, could enhance the lethal effect of abamectin on mites. From the previous microarray results, we found an UGT gene (UGT201D3) overexpressed in AbR strain. Quantitative PCR analysis showed that UGT201D3 was highly expressed and more inducible with abamectin exposure in the AbR strain. After silencing the transcription of UGT201D3, the activity of UGTs was decreased and the susceptibility to abamectin was increased in AbR strain whereas it was not in SS. Furthermore, UGT201D3 gene was then successfully expressed in Escherichia coli. The recombinant UGT201D3 exhibited α-naphthol activity (2.81 ± 0.43 nmol/mg protein/min), and the enzyme activity could be inhibited by abamectin (inhibitory concentration at 50%: 57.50 ± 3.54 µmol/L). High-performance liquid chromatography analysis demonstrated that the recombinant UGT201D3 could effectively deplete abamectin (15.77% ± 3.72%) incubating with 150 µg protein for 6 h. These results provided direct evidence that UGT201D3 was involved in abamectin resistance in T. cinnabarinus.


Assuntos
Glucuronosiltransferase/metabolismo , Inseticidas , Ivermectina/análogos & derivados , Tetranychidae/metabolismo , Sequência de Aminoácidos , Animais , Escherichia coli , Feminino , Glucuronosiltransferase/genética , Resistência a Inseticidas , Interferência de RNA , Tetranychidae/genética , Uracila/análogos & derivados
6.
Chemosphere ; 238: 124645, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31472352

RESUMO

Bromophenols (BPs) are important organic compounds which have become dominant pollutants during these years. Our present study investigated the potential inhibition behaviour of BPs on the activity of one of the most important phase II drug-metabolizing enzymes (DMEs), UDP-glucuronosyltransferases (UGTs). Recombinant UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was utilized as the probe reaction. 100 µM of BPs was utilized as the inhibition screening concentrations, and the complete inhibition profile of UGT isoforms by BPs was obtained. UGT1A7 was the most vulnerable UGT isoform towards BPs. Some structure-activity relationship for the inhibition of UGTs by BPs was found, and this relationship can be furtherly explained by the hydrophobic contacts of BPs with the activity cavity of UGTs using in silico docking method. The inhibition kinetics determination showed that the inhibition kinetic parameter Ki value was calculated to be 2.85, 3.99 and 31.00 µM for the inhibition of UGT1A3, UGT1A7, and UGT2B7 by representative BPs, 2,4,6-TBP. Combined with in vivo exposure concentration of 2,4,6-TBP, in vitro-in vivo extrapolation (IVIVE) was employed to demonstrate the moderate possibility for the inhibition of UGT1A3 and UGT1A7 by 2,4,6-TBP. In conclusion, our study gave the full description towards the inhibition of BPs towards UGT isoforms, which will provide a new perspective for elucidating the toxicity mechanism of bromophenols (BPs).


Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Hidrocarbonetos Bromados/farmacologia , Fenóis/farmacologia , Catálise , Glucuronosiltransferase/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Acoplamento Molecular , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade
7.
J Pharm Biomed Anal ; 177: 112856, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31521020

RESUMO

Daphne genkwa Sieb. et Zucc., as a traditional oriental herb, has been widely distributed and employed in China. The major bioactive components in D. genkwa are flavonoid compounds, which showed pharmacological activities such as anti-inflammatory, analgesic, anti-tumor and immunomodulatory activities. In this study, we analyzed total flavonoids in D. genkwa and their metabolites in normal and adjuvant arthritis (AA) rat plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). A total of 4 metabolites in plasma, 9 metabolites in urine and 15 metabolites in feces were characterized respectively by LC-Q-TOF-MS technology in normal rat. And 9 of the metabolites were observed in the AA rat urine, while there was no prototype drug or its metabolites detected in plasma and fecal samples. The metabolic pathway mainly involves hydroxylation, methylation, glucuronide, sulfate conjugation, oxidation and reduction, during the phase I and phase II biotransformation pathway. All the information gained here will be greatly helpful in elucidating the potential biological and pharmacological mechanism of flavonoid in D. genkwa, thus providing new ideas for drug development.


Assuntos
Artrite Experimental/tratamento farmacológico , Daphne/química , Medicamentos de Ervas Chinesas/farmacocinética , Flavonoides/farmacocinética , Administração Oral , Animais , Artrite Experimental/sangue , Artrite Experimental/imunologia , Artrite Experimental/urina , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Fezes/química , Flavonoides/administração & dosagem , Flavonoides/química , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Glucuronosiltransferase/metabolismo , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Distribuição Tecidual
8.
Zhongguo Zhong Yao Za Zhi ; 44(18): 4043-4047, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31872743

RESUMO

The purpose of this study was to investigate the effect of apigenin on UGT1 A1 enzyme activity and to predict the potential drug-drug interaction of apigenin in clinical use. First,on the basis of previous experiments,the binding targets and binding strength of apigenin to UGT1 A1 enzyme were predicted by computer molecular docking method. Then the inhibitory effect of apigenin on UGT1 A1 enzyme was evaluated by in vitro human liver microsomal incubation system. Molecular docking results showed that apigenin was docked into the active region of UGT1 A1 enzyme protein F,consistent with the active region of bilirubin docking,with moderate affinity. Apigenin flavone mother nucleus mainly interacted with amino acid residues ILE343 and VAL345 to form hydrophobic binding Pi-Alkyl. At the same time,the hydroxyl group on the mother nucleus and the amino acid residue LYS346 formed an additional hydrogen bond,which increased the binding of the molecule to the protein. These results suggested that the flavonoid mother nucleus structure had a special structure binding to the enzyme protein UGT1 A1,and the introduction of hydroxyl groups into the mother nucleus can increase the binding ability. In vitro inhibition experiments showed that apigenin had a moderate inhibitory effect on UGT1 A1 enzyme in a way of competitive inhibition,which was consistent with the results of molecular docking. The results of two experiments showed that apigenin was the substrate of UGT1 A1 enzyme,which could inhibit the activity of UGT1 A1 enzyme competitively,and there was a risk of drug interaction between apigenin and UGT1 A1 enzyme substrate in clinical use.


Assuntos
Apigenina/química , Bilirrubina/química , Interações de Medicamentos , Microssomos Hepáticos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Glucuronosiltransferase/metabolismo , Humanos , Ligações de Hidrogênio
9.
PLoS Negl Trop Dis ; 13(9): e0007687, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31513587

RESUMO

Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans.


Assuntos
Brugia Malayi/efeitos dos fármacos , Brugia Malayi/enzimologia , Glucuronosiltransferase/metabolismo , Albendazol/farmacologia , Animais , Antígenos de Helmintos/sangue , Brugia Malayi/imunologia , Brugia Malayi/metabolismo , Quimioterapia Combinada , Filariose Linfática/tratamento farmacológico , Filariose Linfática/prevenção & controle , Feminino , Filaricidas/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/genética , Humanos , Imunoglobulina E/sangue , Intestinos/enzimologia , Microfilárias/efeitos dos fármacos , Movimento , Probenecid/farmacologia , RNA Interferente Pequeno , Sulfimpirazona/farmacologia
10.
J Agric Food Chem ; 67(42): 11650-11656, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31554401

RESUMO

Occurring in hops (Humulus lupulus) and beer as a racemic mixture, (2R,2S)-8-prenylnaringenin (8-PN) is a potent phytoestrogen in hop dietary supplements used by women as alternatives to conventional hormone therapy. With a half-life exceeding 20 h, 8-PN is excreted primarily as 8-PN-7-O-glucuronide or 8-PN-4'-O-glucuronide. Human liver microsomes and 11 recombinant human UDP-glucuronosyltransferases (UGTs) were used to catalyze the formation of the two oxygen-linked glucuronides of purified (2R)-8-PN and (2S)-8-PN, which were subsequently identified using mass spectrometry and nuclear magnetic resonance spectroscopy. Formation of (2R)- and (2S)-8-PN-7-O-glucuronides predominated over the 8-PN-4'-O-glucuronides except for intestinal UGT1A10, which formed more (2S)-8-PN-4'-O-glucuronide. (2R)-8-PN was a better substrate for all 11 UGTs except for UGT1A1, which formed more of both (2S)-8-PN glucuronides than (2R)-8-PN glucuronides. Although several UGTs conjugated both enantiomers of 8-PN, some conjugated just one enantiomer, suggesting that human phenotypic variation might affect the routes of metabolism of this chiral estrogenic constituent of hops.


Assuntos
Flavanonas/química , Glucuronídeos/química , Glucuronosiltransferase/química , Extratos Vegetais/química , Biocatálise , Flavanonas/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Humulus/química , Humulus/metabolismo , Espectrometria de Massas , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Extratos Vegetais/metabolismo , Estereoisomerismo
11.
Appl Microbiol Biotechnol ; 103(19): 7953-7969, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31407037

RESUMO

Two sustainable and cost-effective cascade enzymatic systems were developed to regenerate uridine diphosphate (UDP)-α-D-glucose and UDP-ß-L-rhamnose from sucrose. The systems were coupled with the UDP generating glycosylation reactions of UDP sugar-dependent glycosyltransferase (UGT) enzymes mediated reactions. As a result, the UDP generated as a by-product of the GT-mediated reactions was recycled. In the first system, YjiC, a UGT from Bacillus licheniformis DSM 13, was used for transferring glucose from UDP-α-D-glucose to naringenin, in which AtSUS1 from Arabidopsis thaliana was used to synthesize UDP-α-D-glucose and fructose as a by-product from sucrose. In the second system, flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from A. thaliana was used to transfer rhamnose from UDP-ß-L-rhamnose to quercetin, in which AtSUS1 along with UDP-ß-L-rhamnose synthase (AtRHM1), also from A. thaliana, were used to produce UDP-ß-L-rhamnose from the same starter sucrose. The established UDP recycling system for the production of naringenin glucosides was engineered and optimized for several reaction parameters that included temperature, metal ions, NDPs, pH, substrate ratio, and enzymes ratio, to develop a highly feasible system for large-scale production of different derivatives of naringenin and other natural products glucosides, using inexpensive starting materials. The developed system showed the conversion of about 37 mM of naringenin into three different glucosides, namely naringenin, 7-O-ß-D-glucoside, naringenin, 4'-O-ß-D-glucoside, and naringenin, 4',7-O-ß-D-diglucoside. The UDP recycling (RCmax) was 20.10 for naringenin glucosides. Similarly, the conversion of quercetin to quercetin 7-O-α-L-rhamnoside reached a RCmax value of 10.0.


Assuntos
Flavanonas/metabolismo , Glucosídeos/metabolismo , Glucuronosiltransferase/metabolismo , Hexosiltransferases/metabolismo , Quercetina/metabolismo , Sacarose/metabolismo , Arabidopsis/enzimologia , Bacillus licheniformis/enzimologia , Biocatálise , Glucuronosiltransferase/isolamento & purificação , Hexosiltransferases/isolamento & purificação
12.
Life Sci ; 234: 116770, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421085

RESUMO

Aim Licoricidin has multiple pharmacological activities. The present study was designed to investigate the permeability and pharmacokinetic behavior of licoricidin using in vitro models. MATERIAL AND METHODS: First, human liver microsomes and recombinant human supersomes were used to investigate the interactions between metabolic enzymes and licoricidin. In addition, rat, minipig, rabbit, dog, monkey, and human liver microsomes were used to determine metabolic differences among species. The parallel artificial membrane permeability assay (PAMPA) was used to explore licoricidin permeability behavior. KEY FINDINGS: At 100 µM, licoricidin strongly inhibited CYP2C9, CYP2C19, CYP3A4, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17. Licoricidin metabolism exhibited considerable differences among species; dog, pig, and rat liver microsomes showed higher metabolic capacity than the other species. Seven licoricidin metabolites were identified by liquid chromatography-tandem mass spectrometry, and hydration and hydroxylation were the major metabolic pathways. The PAMPA permeability of licoricidin was moderate at both pH 4.0 and 7.4. SIGNIFICANCE: The present study will support further pharmacological or toxicological research on licoricidin.


Assuntos
Benzopiranos/metabolismo , Benzopiranos/farmacocinética , Animais , Inibidores das Enzimas do Citocromo P-450/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/metabolismo , Haplorrinos , Humanos , Redes e Vias Metabólicas , Microssomos Hepáticos/metabolismo , Permeabilidade , Coelhos , Ratos , Especificidade da Espécie , Suínos , Porco Miniatura
13.
Drug Metab Pharmacokinet ; 34(4): 280-286, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31262603

RESUMO

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.


Assuntos
Anticorpos/imunologia , Glucuronosiltransferase/imunologia , Reações Antígeno-Anticorpo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Microssomos/imunologia , Microssomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas
14.
J Toxicol Sci ; 44(7): 459-469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31270302

RESUMO

Phenobarbital (PB) and Di (2-ethylhexyl) phthalate (DEHP), an anti-epileptic drug and a plasticizer used in flexible polyvinylchloride formulations, respectively, are well-known typical hepatotoxicants. This study investigated the effects of PB (100 mg/kg/day) or DEHP (500 mg/kg/day) on the endocrine system in intact juvenile/peripubertal male rats exposed for 31 days beginning on postnatal day 23. Slight hormone level changes, histopathological changes in thyroid gland or induction of UDP-glucuronosyltransferase in liver were observed in both the PB and DEHP groups. One of the assumed mechanisms inducing thyroid effects is predictable to be secondary changes based on the enhancement in thyroid hormone metabolism via the induction of hepatic microsomal enzymes. No reproductive system-related changes in organ weights, histopathology, and sexual maturation were observed in both groups. Lower testosterone level was observed in the PB group. CYP2B and CYP3A, which are involved in testosterone metabolism, were induced in liver of the PB group. There was no change of 17ß-hydroxysteroid dehydrogenase activity in testis of both groups. Lower testosterone level in the PB-treated male rats was attributed to an indirect, hepatotoxicity-associated effect on the reproductive system and not to direct effects on testis such as the antiandrogenic activity and the inhibition of steroidogenesis. These results did not indicate that PB or DEHP exposure affects the endocrine system directly.


Assuntos
Anticonvulsivantes/toxicidade , Dietilexilftalato/toxicidade , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/patologia , Fenobarbital/toxicidade , Plastificantes/toxicidade , Animais , Animais Recém-Nascidos , Anticonvulsivantes/administração & dosagem , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Fígado/enzimologia , Masculino , Fenobarbital/administração & dosagem , Ratos Sprague-Dawley , Testosterona/metabolismo , Glândula Tireoide/diagnóstico por imagem , Glândula Tireoide/patologia , Fatores de Tempo
15.
Zhongguo Zhong Yao Za Zhi ; 44(11): 2367-2372, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31359665

RESUMO

To evaluate the hepatotoxicity risks of physcion on the basis of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1A1(UGT1A1 enzyme). The monomers were added into the rat liver microsomes to test the hepatotoxicity by using bilirubin as UGT1A1 enzyme substrate, with apparent inhibition constant K_i as the evaluation index. Liver microsome incubation in vitro was adopted to initiate phase Ⅱ metabolic reaction and investigate the inhibitory effect of physcion. Then the phase Ⅰ and Ⅱ metabolic reactions were initiated to investigate the comprehensive inhibition of metabolites and prototype components. The results showed that when only the phase Ⅱ reaction was initiated, physcion directly acted on the UGT1A1 enzyme in a prototype form, exhibited weak inhibition and the inhibition type was mixed inhibition; When the phase Ⅰ and Ⅱ reactions were initiated simultaneously, the inhibitory effects of physcion on UGT1A1 enzyme became strong and the inhibition type was mixed inhibition, suggesting that physcion had phase Ⅰ and Ⅱ metabolic processes, and the metabolites had strong inhibitory effect on UGT1A1 enzyme. This experiment preliminarily proved that the metabolites of physcion may be the main components to induce hepatotoxicity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Emodina/análogos & derivados , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Animais , Emodina/toxicidade , Cinética , Ratos
16.
Int J Mol Sci ; 20(15)2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31349586

RESUMO

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II detoxification enzymes involved in glycosylation of lipophilic endobiotics and xenobiotics, including phytoalexins. Nicotine, one of the most abundant secondary plant metabolites in tobacco, is highly toxic to herbivorous insects. Plant-herbivore competition is the major impetus for the evolution of large superfamilies of UGTs and other detoxification enzymes. However, UGT functions in green peach aphid (Myzus persicae) adaptation are unknown. In this study, we show that UGT inhibitors (sulfinpyrazone and 5-nitrouracil) significantly increased nicotine toxicity in M. persicae nicotianae, suggesting that UGTs may be involved in nicotine tolerance. In total, 101 UGT transcripts identified in the M. persicae genome/transcriptome were renamed according to the UGT Nomenclature Committee guidelines and grouped into 11 families, UGT329, UGT330, UGT339, UGT341-UGT345, and UGT348-UGT350, with UGT344 containing the most (57). Ten UGTs (UGT330A3, UGT339A2, UGT341A6, UGT342B3, UGT343C3, UGT344D5, UGT344D8, UGT348A3, UGT349A3, and UGT350A3) were highly expressed in M. persicae nicotianae compared to M. persicae sensu stricto. Knockdown of four UGTs (UGT330A3, UGT344D5, UGT348A3, and UGT349A3) significantly increased M. persicae nicotianae sensitivity to nicotine, suggesting that UGT expression in this subspecies may be associated with nicotine tolerance and thus host adaptation. This study reveals possible UGTs relevant to nicotine adaptation in tobacco-consuming M. persicae nicotianae, and the findings will facilitate further validation of the roles of these UGTs in nicotine tolerance.


Assuntos
Adaptação Biológica , Afídeos/fisiologia , Glucuronosiltransferase/metabolismo , Nicotina/metabolismo , Sequência de Aminoácidos , Animais , Afídeos/classificação , Afídeos/efeitos dos fármacos , Sequência Conservada , Resistência a Medicamentos/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/química , Glucuronosiltransferase/genética , Família Multigênica , Nicotina/farmacologia , Filogenia , Domínios Proteicos
17.
Microb Cell Fact ; 18(1): 118, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262296

RESUMO

BACKGROUND: Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5'-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. RESULTS: To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L-1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. CONCLUSIONS: This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA.


Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Uridina Difosfato Ácido Glucurônico/biossíntese , Vias Biossintéticas , Escherichia coli/genética , Glucuronatos/biossíntese , Glucuronosiltransferase/metabolismo
18.
Food Chem Toxicol ; 131: 110542, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31163218

RESUMO

S-equol, an active metabolite of the soy isoflavone daidzein, is mainly metabolized into glucuronide(s) by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, S-equol glucuronidation was examined in the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice using a kinetic analysis. CLint values for 7- and 4'-glucuronidation by liver microsomes were higher than those by intestinal microsomes in all species. CLint values for total glucuronidation (sum of 7- and 4'-glucuronidation) were rats (7.6) > monkeys (5.8) > mice (4.9) > dogs (2.8) > humans (1.0) for liver microsomes, and rats (9.6) > mice (2.8) > dogs (1.3) ≥ monkeys (1.2) > humans (1.0) for intestinal microsomes, respectively. Regarding regioselective glucuronidation by liver and intestinal microsomes, CLint values were 7-glucuronidation > 4'-glucuronidation for humans, monkeys, dogs, and mice, and 4'-glucuronidation > 7-glucuronidation for rats. These results suggest that the metabolic abilities of UGT enzymes toward S-equol in the liver and intestines markedly differ among humans, monkeys, dogs, rats, and mice.


Assuntos
Equol/metabolismo , Glucuronídeos/biossíntese , Microssomos Hepáticos/metabolismo , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Cães , Equol/química , Glucuronosiltransferase/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Cinética , Macaca fascicularis , Camundongos , Pessoa de Meia-Idade , Ratos Sprague-Dawley , Estereoisomerismo , Adulto Jovem
19.
Gene ; 706: 115-123, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31082503

RESUMO

BACKGROUND: UGT2B7 was recently acknowledged as a new critical enzyme involved in biotransformation of a variety of carcinogens, whose function was reported to be significantly associated with its encoding gene (UGT2B7) polymorphisms. However, results regarding the associations between single nucleotide polymorphisms (SNPs) of UGT2B7 and cancer risk still remained controversial. Therefore, a meta-analysis was conducted to further elucidate the role of UGT2B7 SNPs on cancer susceptibilities. METHODS: PubMed, EMBASE, Cochrane library, Chinese National Knowledge Infrastructure (CNKI), Technology of Chongqing (VIP) and Wan Fang Database were searched for eligible studies until March 2019. All analysis was carried out using the Review Manager 5.3 software. Subgroup analyses were performed by cancer types, ethnicity or source of controls. RESULTS: 13 studies with a total of 7688 cancer cases and 11,281 controls were included in this meta-analysis. The results showed that UGT2B7 rs7439366 increased the colorectal cancer risk in dominant model (OR = 0.76, 95% CI = 0.61-0.95, P = 0.02). However, as for the rs7435335 and rs12233719, we did not find their associations with cancer risk in all genetic models. In addition, the rs7441774 was found to be associated with breast cancer risk and significantly reduced papillary thyroid cancer risk in rs3924194 was also observed. Nevertheless, these findings remained to be further proven in future studies since these 2 SNPs were only respectively involved in 1 study. CONCLUSION: This meta-analysis confirmed the association of UGT2B7 rs7439366 with colorectal cancer risk, which may be a potential promising biomarker for prediction of colorectal cancer risk.


Assuntos
Neoplasias Colorretais/genética , Glucuronosiltransferase/genética , Neoplasias/genética , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença/genética , Genótipo , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/fisiologia , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
20.
Orphanet J Rare Dis ; 14(1): 114, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122244

RESUMO

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.


Assuntos
Fragilidade Osmótica/fisiologia , Esferócitos/metabolismo , Esferocitose Hereditária/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/genética , Anquirinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Lactente , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mutação/genética , Fragilidade Osmótica/genética , Patologia Molecular , República da Coreia , Espectrina/genética , Espectrina/metabolismo , Esferocitose Hereditária/genética , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA