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1.
Anal Bioanal Chem ; 412(2): 335-342, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31788715

RESUMO

Triclosan (TCS) is an antibacterial and antifungal compound found in many hygiene products, including toothpaste, soap, and detergents. However, this molecule can act as an endocrine disruptor and can induce harmful effects on human health and the environment. In this study, triclosan was biotransformed in vitro using human and rat liver fractions, to evaluate oxidative metabolism, the formation of reactive metabolites via the detection of GSH adducts, as well as glucuronide and sulfate conjugates using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). A deuterated analog of triclosan was also employed for better structural elucidation of specific metabolic sites. Several GSH adducts were found, either via oxidative metabolism of triclosan or its cleavage product, 2,4-dichlorophenol. We also detected glucuronide and sulfated conjugates of triclosan and its cleaved product. This study was aimed at understanding the routes of detoxification of this xenobiotic, as well as investigating any potential pathways related to additional toxicity via reactive metabolite formation. Graphical abstract.


Assuntos
Anti-Infecciosos Locais/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Triclosan/metabolismo , Animais , Glucuronídeos/metabolismo , Glutationa/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Sulfatos/metabolismo
2.
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
3.
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
4.
Chem Biol Interact ; 310: 108745, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31299240

RESUMO

Ursodeoxycholic acid (UDCA) is a major effective constituent of bear bile powder, which is widely used as function food in China and is documented in the Chinese pharmacopoeia as a traditional Chinese medicine. UDCA has been developed as the only accepted therapy by the US FDA for primary biliary cholangitis. Recently, the US FDA granted accelerated approval to obeticholic acid (OCA), a semisynthetic bile acid derivative from chenodeoxycholic acid, for primary biliary cholangitis. However, some perplexing toxicities of UDCA have been reported in the clinic. The present work aimed to investigate the difference between UDCA and OCA in regard to potential metabolic activation through acyl glucuronidation and hepatic accumulation of consequent acyl glucuronides. Our results demonstrated that the metabolic fates of UDCA and OCA were similar. Both UDCA and OCA were predominantly metabolically activated by conjugation to the acyl glucuronide in human liver microsomes. UGT1A3 played a predominant role in the carboxyl glucuronidation of both UDCA and OCA, while UGT2B7 played a major role in their hydroxyl glucuronidation. Further uptake studies revealed that OATP1B1- and 1B3-transfected cells could selectively uptake UDCA acyl glucuronide, but not UDCA, OCA, and OCA acyl glucuronide. In summary, the liver disposition of OCA is different from that of UDCA due to hepatic uptake, and liver accumulation of UDCA acyl glucuronide might be related to the perplexing toxicities of UDCA.


Assuntos
Ácido Quenodesoxicólico/análogos & derivados , Glucuronídeos/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Ácido Ursodesoxicólico/metabolismo , Animais , Transporte Biológico , Ácido Quenodesoxicólico/metabolismo , Humanos , Medicina Tradicional Chinesa , Ursidae , Ácido Ursodesoxicólico/análogos & derivados , Ácido Ursodesoxicólico/toxicidade
5.
J Pharm Biomed Anal ; 175: 112777, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31362246

RESUMO

Dexmedetomidine (DMTD), an α2-adrenoceptor agonist, is commonly used for sedation and analgesia in intensive care unit (ICU) patients. The primary plasma metabolites of DMTD are its direct N-glucuronides, namely N3-glucuronide of dexmedetomidine (DG1) and N1-glucuronide of dexmedetomidine (DG2), accounting for 41% of DMTD metabolism in healthy volunteers. Since variations on the extent of N-glucuronidation could be one of the key factors contributing to the high interpatient differences of DMTD pharmacokinetics in ICU patients and its subsequent sedative effect. In order to fully evaluate the N-glucuronidation of DMTD in ICU patients, the current study aimed to develop a LC/MS/MS method to simultaneously quantify DMTD and its two major N-glucuronides, DG1 and DG2, in plasma samples and describe their pharmacokinetics in adult ICU patients. Solid-phase extraction cartridges were used to effectively extract DMTD, DG1 and DG2 from 0.4 mL plasma with the internal standard tolazoline. The method was applied in determining the pharmacokinetic profiles of DMTD, DG1, and DG2 in nine ICU patients (mean ±â€¯SD admission severity of illness APACHE II score 23 ±â€¯5) receiving dexmedetomidine infusion for 667 to 3518 min. Under the optimized LC/MS/MS conditions, no endogenous interference from blank plasma was observed. The linear range was 25-5000 pg/mL for DMTD, 50-5000 pg/mL for DG1, and 56-2800 pg/mL for DG2 with good linearity (r2 ranges: 0.997-0.999, 0.993-0.999, and 0.993-0.998 for DMTD, DG1 and DG2 respectively). The precision, accuracy and the stability of DMTD, DG1, and DG2 at their quality control concentrations complied with Food and Drug Administration bioanalytical criteria. The assay was applied in determining the pharmacokinetic profiles of DMTD, DG1, and DG2 in nine ICU patients. The range of AUCDG1/AUCDMTD (from 0.40 to 2.20) and AUCDG2/AUCDMTD (from 0.15 to 2.02) suggested large inter-patient differences in the glucuronidation of DMTD. The mean AUC0-∞ ratio between total glucuronides and DMTD in ICU patients receiving infusions (2.09, range 0.55-4.16) appeared lower than the reported value in healthy volunteers receiving bolus intravenous injection (2.86). This description of the pharmacokinetics of DMTD, DG1, and DG2 in ICU patients is novel and suggests that pathophysiological changes in critically ill patients may have potential to decrease the glucuronidation of DMTD.


Assuntos
Dexmedetomidina/farmacocinética , Hipnóticos e Sedativos/farmacocinética , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida/métodos , Estado Terminal , Feminino , Glucuronídeos/metabolismo , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem/métodos , Adulto Jovem
6.
J Mass Spectrom ; 54(8): 704-715, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31233253

RESUMO

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, N-glucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.


Assuntos
Flunitrazepam/urina , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Flunitrazepam/análogos & derivados , Flunitrazepam/metabolismo , Glucuronídeos/metabolismo , Humanos , Hidroxilação , Redes e Vias Metabólicas , Metaboloma , Oxirredução , Extração em Fase Sólida/métodos
7.
Biomed Chromatogr ; 33(10): e4602, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31116448

RESUMO

The present work describes novel methods using densitometry and indirect or off-line high performance thin-layer chromatography-mass spectrometry (HPTLC-MS) for the simultaneous detection and quantification of asenapine, propranolol and telmisartan and their phase II glucuronide metabolites. After chromatographic separation of the drugs and their metabolites the analytes were scraped, extracted in methanol and concentrated prior to mass spectrometric analysis. Different combinations of toluene and methanol-ethanol-n-butanol-iso-propanol were tested for analyte separation and the best results were obtained using toluene-methanol-ammonia (6.9:3.0:0.1, v/v/v) as the elution solvent. All of the drug-metabolite pairs were separated with a homologous retardation factor difference of ≥22. The conventional densitometric approach was also studied and the method performances were compared. Both of the approaches were validated following the International Conference on Harmonization guidelines, and applied to spiked human plasma samples. The major advantage of the TLC-MS approach is that it can provide much lower limits of detection (1.98-5.83 pg/band) and limit of quantitation (5.97-17.63 pg/band) with good precision (˂3.0% coefficient of variation) compared with TLC-densitometry. The proposed indirect HPTLC-MS method is simple yet effective and has tremendous potential in the separation and quantitation of drugs and their metabolites from biological samples, especially for clinical studies.


Assuntos
Cromatografia em Camada Delgada/métodos , Densitometria/métodos , Glucuronídeos , Preparações Farmacêuticas , Espectrometria de Massas por Ionização por Electrospray/métodos , Administração Oral , Cromatografia Líquida de Alta Pressão , Glucuronídeos/sangue , Glucuronídeos/isolamento & purificação , Glucuronídeos/metabolismo , Glucuronídeos/farmacocinética , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , Limite de Detecção , Modelos Lineares , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/isolamento & purificação , Preparações Farmacêuticas/metabolismo , Reprodutibilidade dos Testes
8.
BMC Microbiol ; 19(1): 99, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096909

RESUMO

BACKGROUND: Consuming red and processed meat has been associated with an increased risk of colorectal cancer (CRC), which is partly attributed to exposure to carcinogens such as heterocyclic amines (HCA) formed during cooking and preservation processes. The interaction of gut microbes and HCA can result in altered bioactivities and it has been shown previously that human gut microbiota can transform mutagenic HCA to a glycerol conjugate with reduced mutagenic potential. However, the major form of HCA in the colon are glucuronides (HCA-G) and it is not known whether these metabolites, via stepwise microbial hydrolysis and acrolein conjugation, are viable precursors for glycerol conjugated metabolites. We hypothesized that such a process could be concurrently catalyzed by bacterial beta-glucuronidase (B-GUS) and glycerol/diol dehydratase (GDH) activity. We therefore investigated how the HCA-G PhIP-N2-ß-D-glucuronide (PhIP-G), a representative liver metabolite of PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), which is the most abundant carcinogenic HCA in well-cooked meat, is transformed by enzymatic activity of human gut microbial representatives of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. RESULTS: We employed a combination of growth and enzymatic assays, and a bioanalysis approach combined with metagenomics. B-GUS of Faecalibacterium prausnitzii converted PhIP-G to PhIP and GDH of Flavonifractor plautii, Blautia obeum, Eubacterium hallii, and Lactobacillus reuteri converted PhIP to PhIP-M1 in the presence of glycerol. In addition, B-GUS- and GDH-positive bacteria cooperatively converted PhIP-G to PhIP-M1. A screen of genes encoding B-GUS and GDH was performed for fecal microbiome data from healthy individuals (n = 103) and from CRC patients (n = 53), which revealed a decrease in abundance of taxa with confirmed GDH and HCA transformation activity in CRC patients. CONCLUSIONS: This study for the first time demonstrates that gut microbes mediate the stepwise transformation of PhIP-G to PhIP-M1 via the intermediate production of PhIP. Findings from this study suggest that targeted manipulation with gut microbes bearing specific functions, or dietary glycerol supplementation might modify gut microbial activity to reduce HCA-induced CRC risk.


Assuntos
Bactérias/enzimologia , Dieta , Microbioma Gastrointestinal , Glucuronidase/metabolismo , Glucuronídeos/metabolismo , Propanodiol Desidratase/metabolismo , Bactérias/genética , Bacteroidetes/enzimologia , Bacteroidetes/genética , Biotransformação , Carcinógenos/metabolismo , Neoplasias Colorretais , Fezes/química , Fezes/microbiologia , Firmicutes/enzimologia , Firmicutes/genética , Glicerol/química , Humanos , Imidazóis/metabolismo , Carne/análise , Metagenômica , Proteobactérias/enzimologia , Proteobactérias/genética
9.
J Steroid Biochem Mol Biol ; 191: 105350, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30959153

RESUMO

Testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronides can be deconjugated to active androgen in gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC-MS/MS proteomics. The fractional contribution of individual transporters (ft) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5'-nucleotidase assay). The glucuronides of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.


Assuntos
Glucuronídeos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Testosterona/metabolismo , Transporte Biológico , Humanos , Mucosa Intestinal/metabolismo , Rim/metabolismo , Fígado/metabolismo
10.
Eur J Drug Metab Pharmacokinet ; 44(5): 629-638, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30879263

RESUMO

BACKGROUND AND OBJECTIVES: Dichotomization of pharmacokinetic exposure measures in exposure-response relationship studies provides results that are interpretable in clinical care. Several methods exist in the literature on how to define the cut-off values needed for the dichotomization process. Commonly, the sample median is utilized to define the dichotomizing value; however, statistical methods based on the exposure metric and its association with the outcome are argued to result in a more proper definition of the optimal cut-point. The Youden index is a recommended statistical method to define the cut-off value. The current analysis objective is to compare the dichotomization results based on the Youden index versus median methods. METHODS: Utilizing mycophenolic acid (MPA) exposure data and its related acute rejection and leukopenia outcome variables, the current study compared the MPA exposure-response relationship outcomes when MPA exposure is dichotomized via the Youden index versus median methods. Univariate logistic models were utilized to quantify the relationships between MPA exposure, including total MPA, unbound MPA, and the acyl-glucuronide metabolite of MPA, and the probabilities of acute rejection and leukopenia. RESULTS: The overall trend of the results of the logistic models demonstrated a general similarity in the inferred exposure-response associations when considering either the Youden index-based or the median-based dichotomization methods. CONCLUSION: The results demonstrated in this analysis suggest that both the Youden index and the median methods provide similar conclusions when dichotomization of a continuous variable is considered. However, confirmation of these conclusions comes from future powered studies that include a larger number of subjects.


Assuntos
Glucuronídeos/metabolismo , Ácido Micofenólico/metabolismo , Adolescente , Feminino , Glucuronídeos/efeitos adversos , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/metabolismo , Transplante de Rim/métodos , Leucopenia/induzido quimicamente , Modelos Logísticos , Masculino , Ácido Micofenólico/efeitos adversos
11.
Drug Metab Pharmacokinet ; 34(3): 194-200, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30880146

RESUMO

Isosteviol is a lead compound whose cardioprotective property has been partly explained by its regulation of ion channels and interference with signalling pathways in the metabolism of some fatty acids. This study determined the metabolic stability of isosteviol in human liver microsomes and H9c2 cell line, and the identity of its metabolites in human and rat liver fractions. Isosteviol was largely unmetabolized in H9c2 cells and in NADPH-only supplemented human liver fractions, suggesting a very limited contribution of phase I biotransformation to its hepatic clearance. The in vitro half-life of isosteviol in UDPGA-only supplemented medium was observed to be 24.9 min with an estimated intrinsic clearance of 0.349 mL/min/kg in man. Analysis by LC-MS/MS and Q-tof showed that isosteviol is mainly metabolised to its acyl-ß-D-glucuronide in humans and rats. Mono-hydroxy-isosteviol and dihydroisosteviol were also identified. Rat liver fraction, however, generated dihydroxy-isosteviol in addition to two mono-hydroxy derivatives. Further studies confirmed that dihydroisosteviol is subsequently biotransformed to its acyl-ß-D-glucuronide in man and rat. These findings suggest that future studies of the efficacy and toxicity of isosteviol might have to consider xenobiotics that alter the glucuronidation pathways significantly in man.


Assuntos
Cardiotônicos/metabolismo , Diterpenos de Caurano/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Biotransformação , Técnicas de Cultura de Células , Linhagem Celular , Estabilidade de Medicamentos , Glucuronídeos/metabolismo , Humanos , NADP/metabolismo , Ratos , Especificidade da Espécie
12.
Toxins (Basel) ; 11(4)2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925814

RESUMO

Applying post-harvest control measures such as adding mycotoxin detoxifying agents is a frequently-used mitigation strategy for mycotoxins. EFSA states that the efficacy of these detoxifiers needs to be tested using specific biomarkers for exposure. However, the proposed biomarkers for exposure are not further optimized for specific target species. Hence, the goal of this study was a) to evaluate the most suitable biomarkers for deoxynivalenol (DON) and zearalenone (ZEN) in porcine plasma, urine and feces; and DON, aflatoxin B1 (AFB1) and ochratoxin A (OTA) in plasma and excreta of broiler chickens and b) to determine the efficacy of a candidate detoxifier, as a proof-of-concept study. Therefore, a mixture of mycotoxins was administered as a single oral bolus with or without detoxifying agent. In accordance with literature AFB1, OTA, and DON-sulphate (DON-S) proved optimal biomarkers in broilers plasma and excreta whereas, in pigs DON-glucuronide (DON-GlcA) and ZEN-glucuronide (ZEN-GlcA) proved the optimal biomarkers in plasma, DON and ZEN-GlcA in urine and, ZEN in feces. A statistically significant reduction was seen between control and treatment group for both AFB1 and DON in broiler plasma, under administration of the mycotoxin blend and detoxifier dose studied suggesting thus, beneficial bioactivity.


Assuntos
Micotoxinas/toxicidade , Animais , Biomarcadores/metabolismo , Galinhas , Fezes/química , Glucuronídeos/metabolismo , Masculino , Micotoxinas/sangue , Micotoxinas/farmacocinética , Micotoxinas/urina , Sulfatos/metabolismo , Suínos
13.
Eur J Drug Metab Pharmacokinet ; 44(4): 549-556, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30820844

RESUMO

BACKGROUND: D-luciferin is one of the most commonly used substrates in bioluminescence imaging for real-time monitoring of sophisticated biological processes in models of human biology or disease in vitro and in vivo. D-luciferin is rapidly cleared from blood circulation after being exogenously delivered in vivo and the presence of phenolic groups indicates that glucuronide conjugation is a possible metabolic pathway for the compound. OBJECTIVES: This study aimed to characterize the glucuronidation pathway of D-luciferin in human liver microsomes (HLM) and human intestine microsomes (HIM). METHODS: HLM and HIM were employed to catalyze the glucuronidation of D-luciferin in vitro. The activity of recombinant uridine-diphospho-glucuronosyltransferase (UGT) isoforms towards D-luciferin glucuronidation was screened. Chemical inhibition assay and kinetic analysis was combined to determine the UGT isoforms responsible for the formation of D-luciferin glucuronide in HLM and HIM. RESULTS: D-luciferin could be catalyzed to form one mono-glucuronide which was characterized as 6'-O-glucuronide in HLM and HIM. UGT1A1, 1A3, 1A6, 1A8, 1A9 and 1A10 participated in the formation of D-luciferin glucuronide, with UGT1A1 exhibiting the highest catalytic activity. Both chemical inhibition assays and kinetic analysis showed that UGT1A1 and UGT1A3 played important roles in D-luciferin-6'-O-glucuronidation in HLM and HIM, with UGT1A6 also giving a non-negligible contribution to this biotransformation in HLM. CONCLUSIONS: UGT1A1, UGT1A3 and UGT1A6 were responsible for 6'-O-glucuronidation of D-luciferin in HLM, while UGT1A1 and UGT1A3 were the major contributors to this biotransformation in HIM.


Assuntos
Benzotiazóis/metabolismo , Glucuronídeos/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Biotransformação/fisiologia , Cães , Glucuronosiltransferase/metabolismo , Cobaias , Humanos , Mucosa Intestinal/metabolismo , Cinética , Fígado , Macaca fascicularis , Masculino , Redes e Vias Metabólicas/fisiologia , Camundongos , Microssomos Hepáticos/metabolismo , Coelhos , Ratos , Suínos , Porco Miniatura
14.
Biomed Chromatogr ; 33(7): e4538, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30912169

RESUMO

A liquid chromatography/electrospray ionization-tandem mass spectrometry-based method was developed for the identification of the conjugation positions of the monoglucuronides of 25-hydroxyvitamin D3 [25(OH)D3 ] and 24,25-dihydroxyvitamin D3 [24,25(OH)2 D3 ] in human urine. The method employed derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D3 -3- and -25-glucuronides, or 24,25(OH)2 D3 -3-, -24- and -25-glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D3 and 24,25(OH)2 D3 , respectively. The 3-glucuronide was not present for 24,25(OH)2 D3 , unlike 25(OH)D3 , thus we found that selective glucuronidation occurs at the C24-hydroxy group for 24,25(OH)2 D3 .


Assuntos
Colecalciferol/urina , Cromatografia Líquida/métodos , Glucuronídeos/urina , Espectrometria de Massas em Tandem/métodos , Colecalciferol/química , Colecalciferol/metabolismo , Glucuronídeos/química , Glucuronídeos/metabolismo , Humanos , Espectrometria de Massas por Ionização por Electrospray/métodos
15.
Drug Test Anal ; 11(7): 1094-1108, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30845374

RESUMO

In the search for improved laboratory methods for the diagnosis of ethylene glycol poisoning, the in vivo formation of a glucuronide metabolite of ethylene glycol was hypothesized. Chemically pure standards of the ß-O-glucuronide of ethylene glycol (EG-GLUC) and a deuterated analog (d4 -EG-GLUC) were synthesized. A high-performance liquid chromatography and tandem mass spectrometry method for determination of EG-GLUC in serum after ultrafiltration was validated. Inter-assay precision (%RSD) was 3.9% to 15.1% and inter-assay %bias was -2.8% to 12.2%. The measuring range was 2-100 µmol/L (0.48-24 mg/L). Specificity testing showed no endogenous amounts in routine clinical samples (n = 40). The method was used to analyze authentic, clinical serum samples (n = 31) from patients intoxicated with ethylene glycol. EG-GLUC was quantified in 15 of these samples, with a mean concentration of 6.5 µmol/L (1.6 mg/L), ranging from 2.3 to 15.6 µmol/L (0.55 to 3.7 mg/L). In five samples, EG-GLUC was detected below the limit of quantification (2 µmol/L) and it was below the limit of detection in 11 samples (1 µmol/L). Compared to the millimolar concentrations of ethylene glycol present in blood after intoxications and potentially available for conjugation, the concentrations of EG-GLUC found in clinical serum samples are very low, but comparable to concentrations of ethyl glucuronide after medium dose ethanol intake. In theory, EG-GLUC has a potential value as a biomarker for ethylene glycol intake, but the pharmacokinetic properties, in vivo/vitro stability and the biosynthetic pathways of EG-GLUC must be further studied in a larger number of patients and other biological matrices.


Assuntos
Etilenoglicol/metabolismo , Etilenoglicol/envenenamento , Glucuronídeos/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Etilenoglicol/sangue , Glucuronídeos/sangue , Humanos , Limite de Detecção , Espectrometria de Massas em Tandem/métodos
16.
J Pharmacol Exp Ther ; 369(2): 234-243, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30850392

RESUMO

UDP-glucuronosyltransferase (UGT) 2A1 is an important enzyme in the detoxification of polycyclic aromatic hydrocarbons found in cigarette smoke. This enzyme is expressed in aerodigestive tract tissues including lung as both its wild-type and exon 4-deleted splice variant isoforms, with the latter acting as a negative regulator of wild-type UGT2A1 activity. UGT2A1 regulation may also be mediated by microRNA (miRNA). To identify miRNA important in the regulation of UGT2A1, expression analysis in tandem with in silico analysis suggested miR-196a-5p and miR-196b-5p as potential top candidates. Significant reductions in firefly luciferase activity were observed in human embryonic kidney cell line 293 cells cotransfected with the wild-type UGT2A1 3'-untranslated region (UTR)-containing luciferase plasmid and either miR-196a-5p (62%, P = 0.00080) or miR-196b-5p (60%, P = 0.00030) mimics. In pull-down assays, there was a 3.4- and 5.2-fold increase in miR-196a-5p (P = 0.054) and miR-196b-5p (P = 0.035), respectively, using the UGT2A1 3'-UTR biotinylated mRNA probe as compared with the ß-actin coding region control mRNA probe. UGT2A1 mRNA was reduced by 25% (P = 0.058) and 35% (P = 0.023) in H146 and H1944 cells, respectively, after overexpression of the miR196a-5p mimic. A similar 32% (P = 0.030) and 41% (P = 0.016) reduction was observed after over-expression of the miR-196b-5p mimic. In H146 cells transfected with miRNA mimic together with a small interfering RNA (siRNA) specific for the UGT2A1 splice variant, a significant reduction in 3-hydroxy-benzo[a]pyrene-glucuronide formation was observed. The miR-196a-5p- and miR-196b-55p-treated cells exhibited reductions of 35% (P = 0.047) and 44% (P = 0.0063), respectively. These data suggest that miR-196a-5p and miR-196b-5p play an important role in UGT2A1 regulation within the lung and potentially other aerodigestive tract tissues.


Assuntos
Glucuronosiltransferase/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/genética , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos
17.
Biol Pharm Bull ; 42(2): 187-193, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30713251

RESUMO

The objective of this study was to characterize pharmacokinetics and metabolism of (±)-cremastranone (CMT) in mouse. Plasma concentrations of CMT following a single oral dose (10 mg/kg) were all below quantitation limit throughout 24-h time course, indicating poor oral bioavailability. Its plasma levels declined rapidly, with a half-life (t1/2) of 1.5 ± 0.3 min following a single intravenous dose (5 mg/kg). They were below the quantitation limit after 15 min post-dosing. CMT showed a high plasma clearance (CLp) of 7.73 ± 3.09 L/h/kg. Consistently, CMT was metabolized rapidly, with a t1/2 < 1 min when it was incubated with liver or intestine S9 fractions of mouse and human in the presence of cofactors for CYP450, uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT), and sulfotransferase (ST). Further studies showed that CMT was metabolized by CYP450, UGT, and ST in vitro in liver S9 fractions of mouse and human, with UGT being the major enzyme responsible for its rapid metabolism. CMT was metabolized by UGT and ST in intestine S9 fractions of mouse and human. Mono-demethylated (M1), mono-glucuronide (M2), and mono-sulfate (M3 and M4) metabolites were tentatively identified in vitro. In conclusion, the pharmacokinetics of CMT is suboptimal as a systemic agent, especially as an oral therapy, due to its extensive metabolism. This report provides possible structural modifications to design CMT derivatives with better pharmacokinetic properties.


Assuntos
Isoflavonas/metabolismo , Isoflavonas/farmacocinética , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Injeções Intravenosas , Isoflavonas/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microssomos Hepáticos/metabolismo , Sulfotransferases/metabolismo , Distribuição Tecidual , Difosfato de Uridina/metabolismo
18.
Drug Metab Dispos ; 47(4): 436-443, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709838

RESUMO

Clopidogrel acyl-ß-d-glucuronide is a mechanism-based inhibitor of cytochrome P450 2C8 in human liver microsomes (HLMs). However, time-dependent inactivation (TDI) of CYP2C8 could not be detected in an earlier study in human recombinant CYP2C8 (Supersomes). Here, we investigate whether different enzyme sources exhibit differences in detection of CYP2C8 TDI under identical experimental conditions. Inactivation of CYP2C8 by amiodarone (100 µM), clopidogrel acyl-ß-d-glucuronide (100 µM), gemfibrozil 1-O-ß-glucuronide (100 µM), and phenelzine (100 µM) was investigated in HLMs and three recombinant human CYP2C8 preparations (Supersomes, Bactosomes, and EasyCYP Bactosomes) using amodiaquine N-deethylation as the marker reaction. Furthermore, the inactivation kinetics of CYP2C8 by clopidogrel glucuronide (5-250 µM) was determined in Supersomes and Bactosomes. Amiodarone caused weak TDI in all enzyme preparations tested, while the extent of inactivation by clopidogrel glucuronide, gemfibrozil glucuronide, and phenelzine varied markedly between preparations, and even different Supersome lots. Both glucuronides caused strong inactivation of CYP2C8 in HLMs, Bactosomes and in one Supersome lot (>50% inhibition), but significant inactivation could not be reliably detected in other Supersome lots or EasyCYP Bactosomes. In Bactosomes, the concentration producing half of kinact (KI) and maximal inactivation rate (kinact) of clopidogrel glucuronide (14 µM and 0.054 minute-1) were similar to those determined previously in HLMs. Phenelzine caused strong inactivation of CYP2C8 in one Supersome lot (91% inhibition) but not in HLMs or other recombinant CYP2C8 preparations. In conclusion, different enzyme sources and different lots of the same recombinant enzyme preparation are not equally sensitive to detect inactivation of CYP2C8, suggesting that recombinant CYPs should be avoided when identifying mechanism-based inhibitors.


Assuntos
Citocromo P-450 CYP2C8/metabolismo , Amiodarona/metabolismo , Clopidogrel/metabolismo , Genfibrozila/metabolismo , Glucuronídeos/metabolismo , Humanos , Cinética , Microssomos Hepáticos/metabolismo , Fenelzina/metabolismo , Sensibilidade e Especificidade
19.
Toxicol Lett ; 306: 43-52, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30769082

RESUMO

Plant-derived mycotoxin conjugates like deoxynivalenol-3-glucoside can be partly hydrolyzed to their aglycones in vivo, albeit to different extent depending on the mycotoxin conjugate and on the animal species. The aim of this work was to investigate the metabolization of the trichothecene mycotoxin nivalenol (NIV) and the fate of its modified form NIV-3-glucoside (NIV3G) in rats. To that end, 350 µg/kg body weight of NIV and the equimolar dose of NIV3G were administered to six rats by gavage in a 5 × 6 design and excreta were collected for 2 days after each treatment. For further analysis of NIV and NIV3G metabolites in rat urine and feces, seven novel NIV- and NIV3G metabolites including NIV sulfonates (NIVS) 1, 2 and 3, deepoxy-NIV (DNIV), DNIV sulfonate 2, NIV3G sulfonate (NIV3GS) 2 and NIV-3-glucuronide were produced, isolated and characterized. Subsequently, LC-MS/MS based methods for determination of NIV, NIV3G and their metabolites in excreta samples were developed, validated and applied. The biological recoveries of administered toxins in the form of their fecal and urinary metabolites were 57 ± 21% for NIV and 94 ± 36% for NIV3G. The majority of NIV and NIV3G metabolites was excreted into feces, with DNIV and NIVS 2 as major NIV metabolites and NIV3GS 2 and DNIV as major metabolites of NIV3G. Only 1.5% of the administered NIV3G was recovered in urine, with NIV3G itself as major urinary metabolite. The biological recovery of free NIV in urine was approximately 30 times lower after treatment with NIV3G than after administration of NIV, indicating that exposure of rats to NIV3G results in lower toxicity than exposure to NIV.


Assuntos
Micotoxinas/metabolismo , Tricotecenos/metabolismo , Animais , Biotransformação , Fezes/química , Glucosídeos/metabolismo , Glucosídeos/toxicidade , Glucuronídeos/metabolismo , Contagem de Leucócitos , Masculino , Micotoxinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Tricotecenos/farmacocinética , Tricotecenos/toxicidade
20.
Biopharm Drug Dispos ; 40(2): 94-98, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30636046

RESUMO

Licoricidin is a major prenylated isoflavone of Glycyrrhiza uralensis Fisch. (Leguminosae), and its pharmacological effects have been reported frequently. Typically, flavonoids having multiple hydroxyl groups are unambiguous substrates for glucuronyl conjugation by UDP-glucuronosyltransferases (UGTs). The pharmacological effects of flavonoids are derived from the conjugation of glucuronide to yield the bioactive metabolite. Here, the metabolism of licoricidin in pooled human liver microsomes (HLMs) was investigated using high-resolution quadrupole-orbitrap mass spectrometry. One metabolite (M1) was identified in HLMs after incubation with licoricidin in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA) and NADPH. The structure of M1 was determined as a monoglucuronyl licoricidin, which was selectively produced by UGT1A9. Licoricidin showed a higher metabolic ratio and rapid metabolism with the recombinant human UGT1A9 than mycophenolic acid, a well-known UGT1A9 substrate. In conclusion, the selective formation of 7-glucuronyl licoricidin by UGT1A9 in HLMs could serve as a new selective substrate to determine the activity of UGT1A9 in vitro.


Assuntos
Benzopiranos/metabolismo , Benzopiranos/farmacologia , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Benzopiranos/isolamento & purificação , Glycyrrhiza uralensis/química , Humanos , Técnicas In Vitro , Microssomos Hepáticos/metabolismo , Raízes de Plantas/química
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