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1.
Toxicol Lett ; 338: 114-127, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33253781

RESUMO

In animal health risk assessment, hazard characterisation of feed additives has been often using the default uncertainty factor (UF) of 100 to translate a no-observed-adverse-effect level in test species (rat, mouse, dog, rabbit) to a 'safe' level of chronic exposure in farm and companion animal species. Historically, both 10-fold factors have been further divided to include chemical-specific data in both dimensions when available. For cats (Felis Sylvestris catus), an extra default UF of 5 is applied due to the species' deficiency in particularly glucuronidation and glycine conjugation. This paper aims to assess the scientific basis and validity of the UF for inter-species differences in kinetics (4.0) and the extra UF applied for cats through a comparison of kinetic parameters between rats and cats for 30 substrates of phase I and phase II metabolism. When the parent compound undergoes glucuronidation the default factor of 4.0 is exceeded, with exceptions for zidovudine and S-carprofen. Compounds that were mainly renally excreted did not exceed the 4.0-fold default. Mixed results were obtained for chemicals which are metabolised by CYP3A in rats. When chemicals were administered intravenously the 4.0-fold default was not exceeded with the exception of clomipramine, lidocaine and alfentanil. The differences seen after oral administration might be due to differences in first-pass metabolism and bioavailability. Further work is needed to further characterise phase I, phase II enzymes and transporters in cats to support the development of databases and in silico models to support hazard characterisation of chemicals particularly for feed additives.


Assuntos
Ração Animal/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Contaminação de Alimentos , Glucuronosiltransferase/metabolismo , Xenobióticos/farmacocinética , Animais , Gatos , Desentoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , Nível de Efeito Adverso não Observado , Ratos , Medição de Risco , Especificidade da Espécie , Especificidade por Substrato , Incerteza , Xenobióticos/administração & dosagem , Xenobióticos/toxicidade
2.
Toxicol Lett ; 338: 51-57, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33290829

RESUMO

Belinostat is a pan-histone deacetylase (HDAC) inhibitor which recently approved for the treatment of relapsed/refractory Peripheral T-cell lymphomas (PTCL). To assess drug-drug interactions (DDIs) potential of belinostat via inhibition of UDP-glucuronosyltransferases (UGTs), the effects of belinostat on UGTs activities were investigated using the non-selective probe substrate 4-methylumbelliferone (4-MU) and trifluoperazine (TFP) by UPLC-MS/MS. Belinostat exhibited a wide range of inhibition against UGTs activities, particularly a potent non-competitive inhibition against UGT1A3, and weak inhibition against UGT1A1, 1A7, 1A8, 2B4 and 2B7. Further, in vitro-in vivo extrapolation (IVIVE) approaches were used to predict the risk of DDI arising from inhibition of UGTs. Our data indicate that the intravenous infusion of belinostat at clinical available dose can contribute a significant increase to the AUC of co-administrated drugs primarily cleared by UGT1A3 or UGT1A1, which will result in potential DDIs. In contrast, oral administrated belinostat is unlikely to cause significant DDIs through inhibition of glucuronidation.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Sulfonamidas/farmacologia , Administração Oral , Antineoplásicos/administração & dosagem , Antineoplásicos/toxicidade , Interações Medicamentosas , Glucuronosiltransferase/metabolismo , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/toxicidade , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/toxicidade , Infusões Intravenosas , Cinética , Sulfonamidas/administração & dosagem , Sulfonamidas/toxicidade
3.
Xenobiotica ; 51(1): 115-121, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32811258

RESUMO

1. In the cynomolgus macaque, UDP-glucuronosyltransferases (UGTs) 1As have similar molecular and enzymatic characteristics to those of their human orthologs. However, genetic polymorphisms in major cynomolgus UGT1A1/6/9 have not been investigated. 2. We re-sequenced UGT1A1, UGT1A6, and UGT1A9 in 186 cynomolgus macaques (bred in Cambodia, China, or Indonesia) and 54 rhesus macaques and found 15, 13, and 26 non-synonymous variants, respectively. 3. Of these UGT1A1, UGT1A6, and UGT1A9 variants, respectively, 10, 9, and 12 were unique to cynomolgus macaques; 4, 1, and 2 were unique to rhesus macaques; and 1, 2, and 5 were found in both cynomolgus and rhesus macaques. The frequency of the UGT1A1 mutation G69R was 23%, 28%, and 63% in cynomolgus macaques bred in Cambodia, China, and Indonesia, respectively, and 97% in rhesus macaques. 4. The O-glucuronidation activities of liver microsomes from cynomolgus and rhesus macaques with respect to estradiol, serotonin, and propofol were measured. Among these activities, liver microsomes from cynomolgus macaques heterozygous for UGT1A1 G69R (n = 11) showed significantly reduced estradiol 3-O-glucuronidation activities compared with those from wild-type animals (n = 38). 5. These results suggest genetic variants such as UGT1A1 G69R could influence the UGT1A1-mediated glucuronidation of drugs in cynomolgus and rhesus macaques.


Assuntos
Glucuronosiltransferase/metabolismo , Sequência de Aminoácidos , Animais , Glucuronosiltransferase/genética , Macaca fascicularis/metabolismo , Macaca mulatta/metabolismo , Polimorfismo Genético
4.
Xenobiotica ; 51(1): 61-71, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32813611

RESUMO

UR-1102, a novel uricosuric agent for treating gout, has been confirmed to exhibit a pharmacological effect in patients. We clarified its metabolic pathway, estimated the contribution of each metabolic enzyme, and assessed the impact of genetic polymorphisms using human in vitro materials. Glucuronide, sulfate and oxidative metabolites of UR-1102 were detected in human hepatocytes. The intrinsic clearance by glucuronidation or oxidation in human liver microsomes was comparable, but sulfation in the cytosol was much lower, indicating that the rank order of contribution was glucuronidation ≥ oxidation > sulfation. Recombinant UGT1A1 and UGT1A3 showed high glucuronidation of UR-1102. We took advantage of a difference in the inhibitory sensitivity of atazanavir to the UGT isoforms and estimated the fraction metabolised (fm) with UGT1A1 to be 70%. Studies using recombinant CYPs and CYP isoform-specific inhibitors showed that oxidation was mediated exclusively by CYP2C9. The effect of UGT1A1 and CYP2C9 inhibitors on UR-1102 metabolism in hepatocytes did not differ markedly between the wild type and variants.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Glucuronosiltransferase/metabolismo , Gota/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Glucuronídeos/metabolismo , Gota/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxazinas/metabolismo , Piridinas/metabolismo
5.
Prostate ; 80(14): 1223-1232, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33258507

RESUMO

BACKGROUND: Antiandrogens are effective therapies that block androgen receptor (AR) transactivation and signaling in over 50% of castration-resistant prostate cancer (CRPC) patients. However, an estimated 30% of responders will develop resistance to these therapies within 2 years. JNJ-pan-AR is a broad-spectrum AR antagonist that inhibits wild-type AR as well as several mutated versions of AR that have emerged in patients on chronic antiandrogen treatment. In this work, we aimed to identify the potential underlying mechanisms of resistance that may result from chronic JNJ-pan-AR treatment. METHODS: The LNCaP JNJR prostate cancer subline was developed by chronically exposing LNCaP parental cells to JNJ-pan-AR. Transcriptomic and proteomic profiling was performed to identify potential drivers and/or biomarkers of the resistant phenotype. RESULTS: Several enzymes critical to intratumoral androgen biosynthesis, Aldo-keto reductase family 1 member C3 (AKR1C3), UGT2B15, and UGT2B17 were identified as potential upstream regulators of the JNJ-pan-AR resistant cells. While we confirmed the overexpression of all three enzymes in the resistant cells only AKR1C3 expression played a functional role in driving JNJ-pan-AR resistance. We also discovered that AKR1C3 regulates UGT2B15 and UGT2B17 expression in JNJ-pan-AR resistant cells. CONCLUSIONS: This study supports the rationale to further investigate the benefits of AKR1C3 inhibition in combination with antiandrogens to prevent CRPC disease progression.


Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/biossíntese , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Genômica , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Masculino , Antígenos de Histocompatibilidade Menor/biossíntese , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Proteômica , Receptores Androgênicos/metabolismo , Transcrição Genética
6.
Mol Pharmacol ; 98(6): 710-718, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33008919

RESUMO

Enzymes of the human UDP-glycosyltransferase (UGT) superfamily typically catalyze the covalent addition of the sugar moiety from a UDP-sugar cofactor to relatively low-molecular weight lipophilic compounds. Although UDP-glucuronic acid (UDP-GlcUA) is most commonly employed as the cofactor by UGT1 and UGT2 family enzymes, UGT2B7 and several other enzymes can use both UDP-GlcUA and UDP-glucose (UDP-Glc), leading to the formation of glucuronide and glucoside conjugates. An investigation of UGT2B7-catalyzed morphine glycosidation indicated that glucuronidation is the principal route of metabolism because the binding affinity of UDP-GlcUA is higher than that of UDP-Glc. Currently, it is unclear which residues in the UGT2B7 cofactor binding domain are responsible for the preferential binding of UDP-GlcUA. Here, molecular dynamics (MD) simulations were performed together with site-directed mutagenesis and enzyme kinetic studies to identify residues within the UGT2B7 binding site responsible for the selective cofactor binding. MD simulations demonstrated that Arg259, which is located within the N-terminal domain, specifically interacts with UDP-GlcUA, whereby the side chain of Arg259 H-bonds and forms a salt bridge with the carboxylate group of glucuronic acid. Consistent with the MD simulations, substitution of Arg259 with Leu resulted in the loss of morphine, 4-methylumbelliferone, and zidovudine glucuronidation activity, but morphine glucosidation was preserved. SIGNIFICANCE STATEMENT: Despite the importance of uridine diphosphate glycosyltransferase (UGT) enzymes in drug and chemical metabolism, cofactor binding interactions are incompletely understood, as is the molecular basis for preferential glucuronidation by UGT1 and UGT2 family enzymes. The study demonstrated that long timescale molecular dynamics (MD) simulations with a UGT2B7 homology model can be used to identify critical binding interactions of a UGT protein with UDP-sugar cofactors. Further, the data provide a basis for the application of MD simulations to the elucidation of UGT-aglycone interactions.


Assuntos
Arginina/genética , Glucuronosiltransferase/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo , Sítios de Ligação/genética , Coenzimas/metabolismo , Cristalografia por Raios X , Glucosiltransferases/genética , Glucosiltransferases/ultraestrutura , Glucuronídeos/metabolismo , Glucuronosiltransferase/genética , Glicosídeos/metabolismo , Células HEK293 , Humanos , Himecromona/metabolismo , Medicago truncatula , Simulação de Dinâmica Molecular , Morfina/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genética , Zidovudina/metabolismo
7.
Drug Metab Pharmacokinet ; 35(5): 466-474, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32883578

RESUMO

UDP-Glucuronosyltransferase (UGT) is a type I membrane protein localized to the endoplasmic reticulum (ER). UGT has a di-lysine motif (KKXX/KXKXX) in its cytoplasmic domain, which is defined as an ER retention signal. However, our previous study has revealed that UGT2B7, one of the major UGT isoform in human, localizes to the ER in a manner that is independent of this motif. In this study, we focused on another UGT isoform, UGT1A9, and investigated the role of the di-lysine motif in its ER localization, glucuronidation activity, and homo-oligomer formation. Immunofluorescence microscopy indicated that the cytoplasmic domain of UGT1A9 functioned as an ER retention signal in a chimeric protein with CD4, but UGT1A9 itself could localize to the ER in a di-lysine motif-independent manner. In addition, UGT1A9 formed homo-oligomers in the absence of the motif. However, deletion of the di-lysine motif or substitution of lysines in the motif for alanines, severely impaired glucuronidation activity of UGT1A9. This is the first study that re-defines the cytoplasmic di-lysine motif of UGT as an essential peptide for retaining glucuronidation capacity.


Assuntos
Biocatálise , Glucuronosiltransferase/metabolismo , Lisina/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Humanos
8.
PLoS One ; 15(9): e0237809, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915792

RESUMO

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.


Assuntos
Canalículos Biliares/efeitos dos fármacos , Desenvolvimento de Medicamentos/métodos , Hepatócitos/efeitos dos fármacos , Cultura Primária de Células/métodos , Quimeras de Transplante , Animais , Canalículos Biliares/citologia , Canalículos Biliares/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Ciclosporina/farmacologia , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Int J Nanomedicine ; 15: 5005-5016, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764932

RESUMO

Background and Aim: With the wide applications of chitosan and gold nanoparticles in drug delivery and many consumer products, there is limited available information about their effects on drug-metabolizing enzymes (DMEs). Changes in DMEs could result in serious drug interactions. Therefore, this study aimed to investigate the effects of exposure to chitosan or gold nanoparticles on hepatic Phase I and II DMEs, liver function and integrity, oxidative damage and liver architecture in male rats. Methods: Animals were divided into three equal groups: a control group, a group treated with chitosan nanoparticles (200 mg/kg, 50±5 nm) and a group treated with gold nanoparticles (4 mg/kg, 15±5 nm). Rats were orally administered their respective doses daily for 10 days. Results: Both chitosan and gold nanoparticles decreased the body weights by more than 10%. Gold nanoparticles reduced the activities of antioxidants (superoxide dismutase and catalase), and reduced glutathione level and elevated the malondialdehyde level in the liver. Gold nanoparticles caused significant reductions in CYP1A1, CYP2E1, quinone oxidoreductase1, and glutathione S-transferase and elevated CYP2D6 and N-acetyl transferase2. Chitosan elevated CYP2E1 and CYP2D6 and reduced UDP-glucuronosyltransferase 1A1. Both nanoparticles disturbed the architecture of the liver, but the deleterious effects after gold nanoparticles treatment were more prominent. Conclusion: Taken together, gold nanoparticles severely perturbed the DMEs and would result in serious interactions with many drugs, herbs, and foods.


Assuntos
Antioxidantes/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inativação Metabólica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Animais , Catalase/genética , Catalase/metabolismo , Quitosana/química , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Interações Medicamentosas , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Ouro/química , Ouro/farmacocinética , Fígado/metabolismo , Fígado/patologia , Masculino , Malondialdeído/metabolismo , Nanopartículas Metálicas/química , Ratos Wistar , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
10.
Drug Metab Pharmacokinet ; 35(4): 397-400, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32646660

RESUMO

UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. The cynomolgus macaque is a nonhuman primate species widely used in drug metabolism studies. The molecular characteristics of UGTs have been extensively investigated in humans, but they remain to be elucidated in cynomolgus macaques. In this study, cynomolgus macaque UGT3A1, UGT3A2, and UGT8A1 cDNAs were isolated and characterized. Amino acid sequences deduced from cynomolgus UGT3A1, UGT3A2, and UGT8A1 cDNAs were highly identical with their human orthologs (93, 96, and 99%, respectively) and were closely clustered in a phylogenetic tree. In the genome, cynomolgus UGT3A and UGT8A genes were located in the regions corresponding to those of their human orthologs. Among the 10 tissue types analyzed, expression of cynomolgus UGT3A1 and UGT3A2 mRNAs was detected in liver, kidney, and testis; the UGT3A1 and UGT3A2 mRNAs were most abundant in liver and testis, respectively. Cynomolgus UGT8A1 was most abundantly expressed in kidney, followed by brain, jejunum, and testis. These results suggest that cynomolgus UGT3As and UGT8A1 have molecular similarities to their human orthologs.


Assuntos
Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Animais , Glucuronosiltransferase/genética , Macaca fascicularis , Masculino , Filogenia
11.
Drug Metab Pharmacokinet ; 35(4): 401-404, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32651149

RESUMO

The cynomolgus macaque is a nonhuman primate species that is often used in drug metabolism studies during drug development. However, the localization of UDP-glucuronosyltransferases (UGTs), essential drug-metabolizing enzymes, has not been fully investigated in the liver and small intestine of cynomolgus macaques. In this study, UGT activities were analyzed in liver (five lobes) and small intestine (the duodenum and six sections from the proximal jejunum to the distal ileum) using typical probe substrates of human UGTs: 7-hydroxycoumarin, estradiol, serotonin, propofol, and zidovudine. In liver, UGT activities with respect to all substrates were detected, and the activity levels were similar in all liver lobes of the cynomolgus macaques tested. In contrast, in the small intestine, UGT activities toward all substrates were detected, but their levels generally decreased from jejunum to ileum in cynomolgus macaques. The localization of estradiol 3-O-glucuronosyltransferases and serotonin O-glucuronosyltransferases (which are mainly UGT1A enzymes) appear to be different in liver and small intestine. These results collectively suggest that, in cynomolgus macaques, UGT1As are differentially localized in the small intestine but are relatively homogeneously distributed in the liver.


Assuntos
Estradiol/metabolismo , Glucuronosiltransferase/metabolismo , Intestino Delgado/metabolismo , Fígado/metabolismo , Macaca fascicularis/metabolismo , Serotonina/metabolismo , Animais , Masculino
12.
J Biol Chem ; 295(31): 10709-10725, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32518167

RESUMO

Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Zymomonas mobilis, a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the order Sphingomonadales in the Z. mobilis genome. We first cloned the gene and expressed it in Escherichia coli, followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT in Sphingobium yanoikuyae and Bacteroides fragilis, which have been reported to produce glucuronosylceramide and α-galactosylceramide, respectively. We expressed the two homologs of the cer-glcat gene in E. coli and found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology.


Assuntos
Proteínas de Bactérias/metabolismo , Galactosilceramidas/biossíntese , Galactosiltransferases/metabolismo , Glucuronosiltransferase/metabolismo , Glicoesfingolipídeos/biossíntese , Zymomonas/enzimologia , Proteínas de Bactérias/genética , Bacteroides fragilis/enzimologia , Bacteroides fragilis/genética , Galactosilceramidas/genética , Galactosiltransferases/genética , Glucuronosiltransferase/genética , Glicoesfingolipídeos/genética , Zymomonas/genética
13.
Chem Biol Interact ; 325: 109115, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32380060

RESUMO

UDP-glucuronosyltransferases (UGTs) are a family of phase II drug metabolizing enzymes that catalyze glucuronidation of numerous endogenous and exogenous substrates. Carbon tetrachloride (CCl4) is widely used to develop liver injuries mimicking human liver diseases. However, effects of CCl4 on the expression and activities of UGTs and the mechanism have not been fully elucidated. The present study aims to elucidate the dysregulation patterns of major UGTs induced by CCl4. Biochemical and histopathological results showed that CCl4 exerted hepatotoxicity in rats. The mRNA levels of UGTs were all significantly reduced in acute liver injury rats. However, mRNA levels of UGT1A1, 1A6, 2B1 and 2B2 were up-regulated while the UGT2B3, 2B6 and 2B12 levels were reduced in chronic CCl4-induced liver fibrosis rats. The protein expression of UGT1A1, 1A6 and 2B were decreased in acute liver injury rats. UGT1A1 and 1A6 proteins were increased, whereas UGT2B protein was reduced in liver fibrosis rats. In addition, CCl4 inhibited the enzyme activities of UGTs in rats. Moreover, the dysregulation of UGTs was accompanied by the decreased mRNA expression of Nrf2, CAR, FXR, PXR, PPAR-α and their corresponding target genes, except for Nrf2, HO-1, AhR and CYP1A1 in liver fibrosis rats. These findings suggest that dysregulation of UGTs under CCl4 exposure is isoform-specific, which could have a complex impact on drug efficacy and endogenous metabolism. Different exposure durations of CCl4 (single vs multiple doses) could have differential effects on rat hepatic UGTs expression.


Assuntos
Tetracloreto de Carbono/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Glucuronosiltransferase/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fibrose , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismo
14.
Sci Rep ; 10(1): 8689, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457304

RESUMO

UDP-glucuronosyltransferases 1 A (UGT1A) enzymes are capable of detoxifying a broad range of endo- and xenobiotic compounds, which contributes to antioxidative effects, modulation of inflammation and cytoprotection. In the presence of low-function genetic UGT1A variants fibrosis development is increased in various diseases. This study aimed to examine the role of common UGT1A polymorphisms in NASH. Therefore, htgUGT1A-WT mice and htgUGT1A-SNP mice (carrying a common human haplotype present in 10% of the white population) were fed a high-fat Paigen diet for 24 weeks. Serum aminotransferase activities, hepatic triglycerides, fibrosis development and UGT1A expression were assessed. Microscopic examination revealed higher hepatic fat deposition and a significant induction of UGT1A gene expression in htgUGT1A-WT mice. In agreement with these observations, lower serum aminotransferase activities and lower expression levels of fibrosis-related genes were measured in htgUGT1A-SNP mice. This was accompanied by reduced PPARα protein levels in htgUGT1A-WT but not in SNP mice. Our data demonstrate a protective effect of a UGT1A SNP haplotype, leading to milder hepatic steatosis and NASH. Higher PPARα protein levels in animals with impaired UGT1A activity are the likely result of reduced glucuronidation of ligands involved in PPARα-mediated fatty acid oxidation and may lead to the observed protection in htgUGT1A-SNP mice.


Assuntos
Doença de Gilbert/genética , Glucuronosiltransferase/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Dieta Hiperlipídica , Fibrose , Doença de Gilbert/metabolismo , Doença de Gilbert/patologia , Glucuronosiltransferase/metabolismo , Haplótipos , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR alfa/genética , PPAR alfa/metabolismo , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Triglicerídeos/análise
15.
Toxicol Appl Pharmacol ; 398: 115032, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32387182

RESUMO

BACKGROUND AND PURPOSE: Irinotecan-induced diarrhea (IID) results from intestinal damages by its active metabolite SN-38. Alleviation of these damages has focused on lowering luminal SN-38 concentrations. However, it is unclear if the enteric bioavailability of SN-38 is mostly dependent on luminal SN-38 concentrations. EXPERIMENTAL APPROACH: Irinotecan (50 mg/kg, i.p. once daily for 6 days) was administered to female wildtype FVB, Mdr1a (-/-), Mrp2 (-/-) and Bcrp1 (-/-) mice for pharmacokinetic (PK), toxicokinetic (TK) and biodistribution studies. Plasma PK/TK profiles and tissues drug distribution were determined after first or sixth daily doses, along with activities of blood and gut esterases and intestinal Ugts. Caco-2 cells and bile-cannulate mice were used to further investigate intestinal and biliary disposition of irinotecan and its metabolites. KEY RESULTS: Significant differences in IID severity were observed with the susceptible rank of Bcrp1(-/-) > wildtype FVB > Mdr1a(-/-) > Mrp2(-/-). This rank order did not correlate with biliary excretion rates of SN-38/SN-38G. Rather, the severity was best correlated (R = 0.805) with the intestinal ratio of Css SN-38/SN-38G, a measure of gut Ugt activity. On the contrary, IID was poorly correlated with plasma AUC ratio of SN-38/SN-38G (R = 0.227). Increased intestinal esterase activities due to repeated dosing and gut efflux transporter functionality are the other key factors that determine SN-38 enteric exposures. CONCLUSION AND IMPLICATIONS: Intestinal SN-38 exposure is mainly affected by intestinal Ugt activities and blood esterase activities, and strongly correlated with severity of IID. Modulating intestinal SN-38 concentration and gut Ugt expression should be the focus of future studies to alleviate IID.


Assuntos
Diarreia/induzido quimicamente , Glucuronosiltransferase/metabolismo , Intestinos/efeitos dos fármacos , Irinotecano/farmacologia , Animais , Antineoplásicos Fitogênicos , Área Sob a Curva , Bile/metabolismo , Sistema Biliar/efeitos dos fármacos , Sistema Biliar/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Diarreia/metabolismo , Esterases/metabolismo , Feminino , Humanos , Camundongos , Distribuição Tecidual/efeitos dos fármacos
16.
Xenobiotica ; 50(10): 1170-1179, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32367776

RESUMO

Herbs are often administered in combination with therapeutic drugs, raising the possibility for herb-drug interactions (HDIs). Furoquinoline alkaloids are found in Rutaceae plants, which are structurally similar and have many medicinal properties. This study aims to investigate the inhibition of four furoquinoline alkaloids on the activity of UDP-glucuronosyltransferases (UGTs).The recombinant UGTs-catalyzed glucuronidation metabolism of 4-methylumbelliferone (4-MU) was utilized to investigate the inhibition potential. Inhibition type and parameters were determined, and in silico docking was employed to elucidate the inhibition difference of furoquinoline alkaloids towards UGTs.Dictamine, haplopine, γ-fagarine and skimmianine strongly inhibited UGT1A3, UGT1A7, UGT1A9 and UGT2B4, respectively. Among them, dictamnine inhibited more than 70% of the four UGTs. Inhibition kinetics determination showed that they all exerted competitive inhibition, and the inhibition kinetic constant (Ki) was determined to be 8.3, 7.2, 3.7 and 33.9 µM, respectively. In vitro-in vivo extrapolation (IVIVE) was employed to demonstrate the inhibition possibility for four alkaloids. Skimmianine was proved to be more suitable for clinical application. In silico docking study indicated that the hydrophobic interactions played a key role in the inhibition of furoquinoline alkaloids towards three of the four UGTs. In conclusion, monitoring the interactions between furoquinoline alkaloids and drugs mainly undergoing UGTs-catalyzed metabolism is necessary.


Assuntos
Inibidores Enzimáticos/metabolismo , Glucuronosiltransferase/metabolismo , Himecromona/metabolismo , Alcaloides , Simulação por Computador , Interações Ervas-Drogas , Humanos , Simulação de Acoplamento Molecular , Quinolinas
17.
Oncol Rep ; 43(4): 1067-1080, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32323779

RESUMO

The long pre­cancerous state of colorectal cancer (CRC) provides an opportunity to prevent the occurrence and development of CRC. The detoxification of CRC food­borne carcinogenic heterocyclic amines is highly dependent on UDP glucuronosyltransferase 1A (UGT1A)­mediated glucuronidation. Sulforaphane (SFN), a phytochemical, possesses antioxidant, anti­inflammatory and anticarcinogenic effects on the prevention of CRC. Previous studies revealed that SFN upregulates the expression of UGT1A. The aim of the present study was to investigate the regulatory mechanism of SFN­induced UGT1A upregulation and provide novel understanding on the basic research and chemoprevention of CRC. In the present study, the viability and proliferation of CRC cells (HT­29 and SW480) treated with SFN were assessed by MTT, colony formation and EdU assays. Flow cytometry was used to detect the cell cycle arrest and apoptosis of cells treated with different concentrations of SFN. The motility of cells was determined by wound healing and Transwell assays. Nuclear factor, erythroid 2 like 2 (Nrf2) short hairpin RNA (shRNA) and negative control shRNA lentiviruses were used for cell transfection. Reverse transcription­quantitative polymerase chain reaction and western blotting were employed to verify the role of Nrf2 in SFN­induced UGT1A. HT­29 and SW480 cells were divided into a control, an SFN and a PD98059 [an extracellular signal­regulated kinase (ERK) inhibitor] + SFN group. Western blotting detected the protein levels of Nrf2 and UGT1A. Intracellular levels of reactive oxygen species (ROS) were detected using a reactive oxygen assay kit. The results revealed that SFN inhibits cell proliferation and colony formation, promotes apoptosis, and reduces the migratory ability of CRC cells. The phosphorylation of ERK induced by SFN promoted Nrf2 accumulation. Furthermore, a significant increase in the levels of UGT1A was observed, which coincided with SFN­induced upregulation of Nrf2 levels in nuclear fractions. Pretreatment with PD58059 reversed the SFN­induced subcellular translocation of Nrf2 and the expression of UGT1A. In addition, SFN­induced high levels of ROS in CRC cells may be associated with the ERK signaling pathway. Collectively, these results indicated that SFN inhibited the proliferation of CRC cells and upregulated the expression of UGT1A in CRC cells via the ERK/Nrf2 signaling pathway.


Assuntos
Anticarcinógenos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Glucuronosiltransferase/metabolismo , Isotiocianatos/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Antioxidantes/farmacologia , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
18.
Ecotoxicol Environ Saf ; 197: 110611, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32294595

RESUMO

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


Assuntos
Hepatócitos/enzimologia , Fitoestrógenos/metabolismo , Salmo salar/metabolismo , Animais , Aquicultura , Biotransformação , Cromatografia Líquida , Genisteína/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Isoflavonas/metabolismo , Microssomos Hepáticos/enzimologia , Soja/química
19.
Zhongguo Zhong Yao Za Zhi ; 45(2): 412-417, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32237326

RESUMO

The bilirubin metabolism mediated by the phase Ⅱ metabolizing enzyme UGT1A1 in the liver was evaluated to study the potential hepatotoxicity risk based on investigation on the inhibitory effect of rhein and its metabolites on the UGT1A1 enzyme in Rhei Radix et Rhizoma. Firstly, in vitro liver microsomes incubation was used to initiate the phase Ⅱ metabolic reaction to investigate the inhibitory effect of rheinon UGT1A1 enzyme. Secondly, the phase Ⅰ and phase Ⅱ metabolic reactions were initiated to investigate the hepatotoxicity risk of rhein metabolites. It was found that the rhein and its phase Ⅱ metabolites had no significant inhibitory effect on UGT1A1 enzyme, but its phase Ⅰ metabolites significantly reduced UGT1A1 enzyme activity. Based on the metabolites analysis, it is speculated that the rhein phase Ⅰ metabolite rheinhydroxylate and its tautomers have certain hepatotoxicity risks, while the toxicity risk induced by the prototype and phase Ⅱ metabolites of rheinglucoside, rheinglucuronic acid and rhein sulfate is small.


Assuntos
Antraquinonas/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Medicamentos de Ervas Chinesas/toxicidade , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Glucuronosiltransferase/metabolismo , Humanos , Fígado/enzimologia , Rizoma
20.
Xenobiotica ; 50(8): 997-1008, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32116078

RESUMO

Corylifol A (CA), a phenolic compound from Psoralea corylifolia, possessed several biological properties but poor bioavailability. Here we aimed to investigate the roles of cytochromes P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and efflux transporters in metabolism and disposition of CA.Metabolism of CA was evaluated in HLM, expressed CYPs and UGTs. Chemical inhibitors and shRNA-mediated gene silencing of multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP) were performed to assess the roles of transporters in CA disposition.Three oxidated metabolites (M1-M3) and two glucuronides (M4-M5) were detected. The intrinsic clearances (CLint) values of M1 and M4 in HLM were 48.10 and 184.03 µL/min/mg, respectively. Additionally, CYP1A1, 2C8 and 2C19 were identified as main contributors with CLint values of 13.01-49.36 µL/min/mg, while UGT1A1, 1A7, 1A8 and 1A9 were with CLint values ranging from 85.01 to 284.07 µL/min/mg. Furthermore, activity correlation analysis proved CYP2C8, UGT1A1 and 1A9 were the main active hepatic isozymes. Besides, rats and monkeys were appropriate model animals. Moreover, dipyridamole and MK571 both could significantly inhibit M4 efflux. Gene silencing results also indicated MRP4 and BCRP were major contributors in HeLa1A1 cells.Taken together, CYPs, UGTs, MRP4 and BCRP were important determinants of CA pharmacokinetics.


Assuntos
Flavonas/metabolismo , Animais , Transporte Biológico , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Células HeLa , Humanos , Psoralea , Ratos
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