Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 19.063
Filtrar
1.
Chem Biol Interact ; 330: 109247, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866466

RESUMO

This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.


Assuntos
Fígado/metabolismo , Oxazinas/metabolismo , Frações Subcelulares/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Feminino , Herbicidas/química , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Ratos , Fatores Sexuais , Estereoisomerismo
2.
Pharm Biol ; 58(1): 247-252, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32223485

RESUMO

Context: Cepharanthine (CEP) extracted from the roots of Stephania cepharantha Hayata (Menispermaceae), has a range of therapeutic potential in clinical conditions. Whether it affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.Materials and methods: The effects of CEP (100 µM) on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs) with specific probe actions and probe substrates. In addition, the enzyme kinetic parameters were calculated.Results: The results showed that the activity of CYP3A4, CYP2E1 and CYP2C9 was inhibited by CEP, with IC50 values of 16.29, 25.62 and 24.57 µM, respectively, but other CYP isoforms were not affected. Enzyme kinetic studies showed that CEP was not only a non-competitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2E1 and CYP2C9, with Ki values of 8.12, 11.78 and 13.06 µM, respectively. Additionally, CEP is a time-dependent inhibitor for CYP3A4 with KI/Kinact value of 10.84/0.058 min/µM.Discussion and conclusions: The in vitro studies of CEP with CYP isoforms indicate that CEP has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2C9. Further clinical studies are needed to evaluate the significance of this interaction.


Assuntos
Benzilisoquinolinas/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Stephania/química , Benzilisoquinolinas/isolamento & purificação , Inibidores das Enzimas do Citocromo P-450/isolamento & purificação , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Raízes de Plantas/química
3.
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
4.
Arterioscler Thromb Vasc Biol ; 40(6): 1533-1542, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32268786

RESUMO

OBJECTIVE: Clopidogrel is a commonly used P2Y12 inhibitor to treat and prevent arterial thrombotic events. Clopidogrel is a prodrug that requires bioactivation by CYP (cytochrome P450) enzymes to exert antiplatelet activity. Diabetes mellitus is associated with an increased risk of ischemic events, and impaired ability to generate the active metabolite (AM) from clopidogrel. The objective of this study is to identify the mechanism of clopidogrel resistance in a murine model of diet-induced obesity (DIO). Approach and Results: C57BL/6J mice and IL-1R-/- mice were given high-fat diet for 10 weeks to generate a murine model of diet-induced obesity. Platelet aggregation and carotid arterial thrombosis were assessed in response to clopidogrel treatment. Wild-type DIO mice exhibited resistance to antiplatelet and antithrombotic effects of clopidogrel that was associated with reduced hepatic expression of CYP genes and reduced generation of the AM. IL (Interleukin)-1 receptor-deficient DIO (IL1R-/- DIO) mice showed no resistance to clopidogrel. Lack of resistance was accompanied by increased exposure of the clopidogrel AM. This resistance was also absent when wild-type DIO mice were treated with the conjugate of the clopidogrel AM, DT-678. CONCLUSIONS: These findings indicate that antiplatelet effects of clopidogrel may be impaired in the setting of diabetes mellitus due to reduced prodrug bioactivation related to IL-1 receptor signaling. Therapeutic targeting of P2Y12 in patients with diabetes mellitus using the conjugate of clopidogrel AM may lead to improved outcomes.


Assuntos
Clopidogrel/farmacocinética , Clopidogrel/uso terapêutico , Resistência a Medicamentos , Obesidade/complicações , Receptores de Interleucina-1/fisiologia , Animais , Trombose das Artérias Carótidas/prevenção & controle , Clopidogrel/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Diabetes Mellitus , Dieta Hiperlipídica , Modelos Animais de Doenças , Fibrinolíticos , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/enzimologia , Obesidade/etiologia , Obesidade/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas , Pró-Fármacos/farmacocinética , Pró-Fármacos/uso terapêutico , Receptores de Interleucina-1/deficiência
5.
Pharmacol Rep ; 72(1): 156-165, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32016858

RESUMO

BACKGROUND: Lycopene as a naturally occurring carotenoid is a common part of the human diet. Several beneficial properties of lycopene have been identified, with the most studied being anti-cancer and antioxidant activity. However, no evidence of possible drug-drug or drug-food supplement interactions has been found. METHODS: We studied the in vivo effect of lycopene on the selected rat liver cytochromes P450 (CYPs): CYP1A2, CYP2B, CYP2C11, CYP2C6, CYP2D, and CYP3A. Lycopene was administered to rats intragastrically at doses of 4, 20, and 100 mg/kg/day for 10 consecutive days. Total protein content, P450 Content, and metabolic activity of selected CYPs were evaluated in the rat liver microsomal fraction. RESULTS: Increased CYP2B, CYP2D, and CYP3A metabolic activities were observed in animals treated with the lycopene dose of 100 mg/kg/day. The content of CYP3A1 protein was increased by the dose of 100 mg/kg/day and CYP3A2 protein was increased by all administered doses of lycopene. CONCLUSION: The results of our study indicate that lycopene increased the metabolic activity of enzymes that are orthologues to the most clinically important human enzymes involved in xenobiotic metabolism. The risk of pharmacokinetic interactions between lycopene dietary supplements and co-administered drugs should be evaluated.


Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Licopeno/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Licopeno/administração & dosagem , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Ratos Wistar
6.
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
7.
Molecules ; 25(4)2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32093091

RESUMO

Inhibition of cytochrome P450 (CYP) alters the pharmacokinetic parameters of the drug and causes drug-drug interactions. Salicylic acid been used for the treatment of colorectal cancer (CRC) and chemoprevention in recent decades. Thus, the aim of this study was to examine the in vitro inhibitory effect of salicylic acid on CYP2E1 activity in rat liver microsomes (RLMs) using high-performance liquid chromatography (HPLC). High-performance liquid chromatography analysis of a CYP2E1 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 282 nm using 60% H2O, 25% acetonitrile, and 15% methanol as mobile phase. The CYP2E1 assay showed a good linearity (R2 > 0.999), good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80-120%), and low detection (4.972 µM and 1.997 µM) and quantitation limit values (15.068 µM and 6.052 µM), for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Salicylic acid acts as a mixed inhibitor (competitive and non-competitive inhibition), with Ki (inhibition constant) = 83.56 ± 2.730 µM and concentration of inhibitor causing 50% inhibition of original enzyme activity (IC50) exceeding 100 µM (IC50 = 167.12 ± 5.460 µM) for CYP2E1 enzyme activity. Salicylic acid in rats would have both low and high potential to cause toxicity and drug interactions with other drugs that are substrates for CYP2E1.


Assuntos
Inibidores do Citocromo P-450 CYP2E1/farmacologia , Citocromo P-450 CYP2E1/metabolismo , Microssomos Hepáticos/enzimologia , Ácido Salicílico/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Ratos Sprague-Dawley
8.
Am J Physiol Endocrinol Metab ; 318(2): E262-E275, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31821038

RESUMO

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase (FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog (PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.


Assuntos
Proteínas de Transporte/biossíntese , Lipoproteínas VLDL/biossíntese , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , Microssomos Hepáticos/enzimologia , Apolipoproteína B-100/biossíntese , Apolipoproteína B-100/genética , Linhagem Celular , Células Cultivadas , Inibidores da Síntese de Ácidos Graxos/farmacologia , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
9.
J Pharmacol Exp Ther ; 372(3): 320-330, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31882454

RESUMO

Unraveling the molecular mechanisms by which genetic variants of cytochrome P450 2A6 lead to different metabolic phenotypes remains a long-standing but important challenge. CYP2A6 is an enzyme involved in the metabolism of several clinical drugs as well as the metabolic activation of carcinogenic nitrosamines. Herein, CYP2A6 genotypes and phenotypes, as indicated by protein content [by liquid chromatography-mass spectrometry (MS)/MS] and metabolic activities [Vmax, clearance (CL)], were determined for 90 human liver samples. We determined the median, range, and interindividual and intraindividual variation of CYP2A6 content and activity at the microsomal, liver tissue, and whole liver level and predicted hepatic in vivo clearance by in vitro-in vivo extrapolation based on CYP2A6-mediated coumarin metabolism by each CYP2A6 genotype. These results reveal how different CYP2A6 genotypes yield different phenotypic traits in protein content and enzyme activity. For relative Vmax, CL, and protein content, the intraindividual percentage coefficients of variation (ICVs) were 41.0% (18.8%-125.1%), 28.5% (2.39%-133.5%), and 27.8% (2.68%-88.0%), respectively. The high ICVs implied large intraindividual variation at different levels, sometimes in a genotype-dependent manner. Intergenotype analysis revealed that the CYP2A6*4 allele demonstrated the most obvious effect on phenotypic outcomes, both in protein content and in metabolic activity. Indeed, decreased CYP2A6 protein content with the CYP2A6*4 genotype might explain the decreased metabolic activity from the molecular to the organismal level. These findings may allow useful predictions for CYP2A6-mediated drug metabolism on an individual patient basis in accord with the goal of achieving personalized medicine. SIGNIFICANCE STATEMENT: We provide the median, range, and interindividual and intraindividual variation in CYP2A6 content at the microsomal, liver tissue, and whole liver level by liquid chromatography-mass spectrometry (MS)/MS as well as activities at the protein, microsomal, liver tissue, and whole liver level both in vitro and at the organismal level based on CYP2A6-mediated coumarin metabolism with each CYP2A6 genotype, thereby allowing us to elucidate how different CYP2A6 genotypes yield differing phenotypic traits (protein content and enzyme activity), facilitating the development of personalized medicine.


Assuntos
Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismo , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Modelos Biológicos , Polimorfismo Genético , Grupo com Ancestrais do Continente Asiático/genética , Frequência do Gene , Genótipo , Humanos , Técnicas In Vitro , Taxa de Depuração Metabólica , Fenótipo , Valor Preditivo dos Testes
10.
Pharm Biol ; 58(1): 60-63, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31868554

RESUMO

Context: 23-Hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis (Bunge) Regel (Ranunculaceae), exhibits potential antitumor activity. Its metabolism, however, has not yet been studied.Objective: This study focuses on the metabolism of 23-HBA in vitro by human liver microsomes.Materials and methods: The metabolic kinetics of 23-HBA (0.5-100 µM) and the effects of selective CYP450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) inhibitors on metabolism of 23-HBA were evaluated in human liver microsomes incubation system and then determined by LC-MS method. The Michaelis-Menten parameters Km and Vmax were initially estimated by analysing Lineweaver-Burk plot. The clearance (CLint) was also calculated.Results: The Vmax, Km, and CLint of 23-HBA were 256.41 ± 11.20 pmol/min/mg, 11.10 ± 1.07 µM, and 23.10 ± 1.32 µL/min/mg, respectively. The metabolism of 23-HBA was significantly inhibited by furafylline (0.05 µM, p < 0.01) and ketoconazole (0.02 µM, p < 0.05). Ticlopidine (1.3 µM, p < 0.05) could inhibit the metabolism of 23-HBA, while the other inhibitors (sulfaphenazole and quinidine) showed nonsignificant inhibition on the metabolism of 23-HBA.Discussion and conclusions: This is the first investigation of the metabolism of 23-HBA in human liver microsomes. The in vitro study indicates that CYP1A2 and CYP3A4 are mainly involved in the metabolism of 23-HBA. Special attention should be given to the pharmacokinetic and clinical outcomes when 23-HBA was co-administrated with other compounds mainly undergoing CYP1A2/CYP3A4-mediated metabolism. Further studies are needed to evaluate the significance of this interaction and strengthen the understanding of traditional Chinese medicine.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Triterpenos/metabolismo , Cromatografia Líquida , Humanos , Técnicas In Vitro , Isoenzimas , Espectrometria de Massas , Pulsatilla/química
11.
Physiol Res ; 68(Suppl 1): S51-S58, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31755290

RESUMO

Evaluation of possible interactions with enzymes of drug metabolism is an important part of studies on safety and, in general, on the properties of any drug or biologically active compound. Here, focus is given on interactions of three sesquiterpenes (beta-caryophyllene oxide (CAO), trans-nerolidol (tNER) and farnesol (FAR)) with CYP3A4. To determine the CYP3A4 activity, specific substrates testosterone (TES) and midazolam (MDZ) were used. In human liver microsomes, the CAO inhibited the MDZ 1´-hydroxylation by mixed type inhibition and K(i) 46.6 microM; TES 6beta-hydroxylation was inhibited more strongly by tNER by the same mechanism and with K(i) of 32.5 microM. Results indicated a possibility of different mode of interaction of both compounds within the active site of CYP3A4 and this was why the molecular docking study was done. The docking experiments showed that the studied sesquiterpenes (CAO and tNER) bound to the CYP3A4 active site cause a significant decrease of binding affinity of substrates tested which corresponded well to the inhibition studies. The inhibition observed, however, most probably does not pose a real harm to microsomal drug metabolism as the levels of sesquiterpenes in plasma (assuming the use of these compounds as spices or flavoring additives) does not usually exceed micromolar range. Hence, the interaction of drugs metabolized by CYP3A4 with sesquiterpenes is less probable.


Assuntos
Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Sesquiterpenos Policíclicos/farmacologia , Sesquiterpenos/farmacologia , Domínio Catalítico , Citocromo P-450 CYP3A/química , Farneseno Álcool/química , Farneseno Álcool/farmacologia , Humanos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Sesquiterpenos Policíclicos/química , Sesquiterpenos/química
12.
Pharmacology ; 104(5-6): 296-302, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31587003

RESUMO

INTRODUCTION: Cynaroside is a biological component isolated from Lonicera japonica Thunb, and it possesses numerous pharmacological activities. However, whether cynaroside affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. The purpose of this study was to investigate the effects of cynaroside on 8 major CYP isoforms in human liver microsomes (HLMs). METHODS: In this study, the inhibitory effects of cynaroside on the 8 human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using HLMs. RESULTS: The results showed that cynaroside inhibited the activity of CYP1A2, 3A4, and 2C9, with IC50 values of 21.74, 15.88, and 16.58 µmol/L, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that cynaroside was not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP1A2 and 2C9, with Ki values of 7.33, 11.60, and 8.09 µmol/L, respectively. In addition, cynaroside is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.049/11.62 µmol/L-1min-1. CONCLUSION: The in vitro studies of cynaroside with CYP isoforms indicate that cynaroside has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, 3A4, and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Glucosídeos/farmacologia , Luteolina/farmacologia , Interações Medicamentosas , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia
13.
Mutat Res ; 846: 403072, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31585629

RESUMO

A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59-) or mutant reticulocyte frequencies (RETCD59-) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.


Assuntos
Benzofenoneídio/toxicidade , Carcinógenos/toxicidade , Ensaio Cometa , Proteínas de Membrana/genética , Testes para Micronúcleos , Ativação Metabólica , Animais , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/ultraestrutura , Masculino , Microssomos Hepáticos/enzimologia , Testes de Mutagenicidade , Mutação , Estudo de Prova de Conceito , Ratos , Ratos Sprague-Dawley , Reticulócitos/efeitos dos fármacos , Reticulócitos/ultraestrutura
14.
Mutat Res ; 846: 403057, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31585630

RESUMO

Canola (or rapeseed) oil and waste vegetable oil (WVO) are used commonly to make biodiesel fuels composed completely from these oils (B100) or as blends with petroleum diesel (B0). However, no studies have reported the mutagenic potencies of the particulate matter with diameter ≤2.5 µm (PM2.5) or the mutagenicity emission factors, such as revertants/MJthermal (rev/MJth) for these biodiesel emissions. Using strains TA98 and TA100 with the Salmonella (Ames) mutagenicity assay, we determined these metrics for organic extracts of PM2.5 of emissions from biodiesel containing 5% soy oil (soy B5); 5, 20, 50, and 100% canola (canola B5, B20, B50, B100), and 100% waste vegetable oil (WVO B100). The mutagenic potencies (rev/mg PM2.5) of the canola B100 and WVO B100 emissions were generally greater than those of B0, whereas the mutagenicity emission factors (rev/MJth, rev/kg fuel, and rev/m3) were less, reflecting the lower PM emissions of the biodiesels relative to B0. Nearly all the rev/mg PM2.5 and rev/MJth values were greater in TA98 with S9 than without S9, indicating a relatively greater role for polycyclic aromatic hydrocarbons, which require S9, than nitroarenes, which do not. In TA100 -S9, the rev/mg PM2.5 and rev/MJth for the biodiesels were generally ≥ to those of B0, indicating that most of these biodiesels produced more direct-acting, base-substitution mutagenic activity than did B0. For B100 biodiesels and petroleum diesel, the rev/MJth in TA98 + S9 ranked: petroleum diesel > canola > WVO > soy. The diesel emissions generally had rev/MJth values orders of magnitude higher than those of large utility-scale combustors (natural gas, coal, oil, or wood) but orders of magnitude lower than those of inefficient open burning (e.g., residential wood fireplaces). These comparative data of the potential health effects of a variety of biodiesel fuels will help inform the life-cycle assessment and use of biodiesel fuels.


Assuntos
Poluentes Atmosféricos/toxicidade , Biocombustíveis/toxicidade , Resíduos Industriais , Óleos Vegetais/toxicidade , Óleo de Brassica napus/toxicidade , Salmonella/efeitos dos fármacos , Óleo de Soja/toxicidade , Emissões de Veículos/toxicidade , Ativação Metabólica , Animais , Microssomos Hepáticos/enzimologia , Testes de Mutagenicidade , Tamanho da Partícula , Material Particulado/toxicidade , Ratos , Salmonella/genética
15.
Mutat Res ; 846: 503094, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31585631

RESUMO

In contributing to this Special Issue of Mutation Research dedicated to Professor Bruce N. Ames in recognition of his 90th birthday in December 2018, we intend to portray the importance not only of the Ames Salmonella/mammalian-microsome mutagenicity assay in some of our studies over the years, but also the importance of the insight that Bruce Ames brought to the field of genetic toxicology.


Assuntos
Testes de Mutagenicidade , Salmonella/efeitos dos fármacos , Ativação Metabólica , Poluentes Ocupacionais do Ar/toxicidade , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Flavonoides/química , Flavonoides/toxicidade , Engenharia Genética , Humanos , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Nevirapina/química , Nevirapina/toxicidade , Exposição Ocupacional , Quercetina/toxicidade , Ratos , Salmonella/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Especificidade da Espécie , Toxicologia/métodos , Urina/química
16.
Mutat Res ; 846: 403070, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31585632

RESUMO

Bruce Ames has had an enormous impact on human health by developing facile methods for the identification of mutagens. This research also provided important insights into the relationship between mutagenesis and carcinogenesis. Bruce is a highly innovative and creative individual who has followed his interests across disciplines into diverse fields of inquiry. The present author had the pleasure of spending a sabbatical in the Ames lab and utilized the Ames test in multiple aspects of his research. He describes both in this honorific to Bruce on the occasion of his 90th birthday.


Assuntos
Bioquímica/história , Genética/história , Testes de Mutagenicidade/história , Ativação Metabólica , Animais , California , História do Século XX , História do Século XXI , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Mutagênese , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Ratos , Ratos Sprague-Dawley , Salmonella/efeitos dos fármacos , Salmonella/genética
17.
Pharm Biol ; 57(1): 571-576, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31456483

RESUMO

Context: Kaempferitrinis (KF) is a bioactive flavonoid and possesses numerous pharmacological activities. However, whether KF affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. Objective: This study investigates the effects of KF on eight major CYP isoforms in human liver microsomes (HLMs). Materials and methods: In vitro, HLMs were used to investigate the inhibitory effects of KF (100 µM) on the eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8), and corresponding probe substrates were used. Enzyme kinetic studies (0-50 µM of KF) were conducted to determine the inhibition mode of KF on CYP enzymes. Results: The results showed that KF inhibited the activity of CYP1A2, 3A4, and 2C9, with IC50 values of 20.56, 13.87, and 14.62 µM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that KF was not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP1A2 and 2C9, with Ki values of 7.11, 10.24, and 7.58 µM, respectively. In addition, KF is a time-dependent inhibitor for CYP3A4 with KI/Kinact value of 10.85/0.036 min/µM. Discussion: The in vitro studies of KF with CYP isoforms indicate that KF has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, 3A4, and 2C9. Conclusion: It is recommended that KF should not be used with other drugs metabolized by CYP1A2, 3A4, and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Quempferóis/farmacologia , Fígado/efeitos dos fármacos , Humanos , Técnicas In Vitro , Isoenzimas , Cinética , Fígado/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia
18.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366067

RESUMO

NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Desmetilação/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Isoquinolinas/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Biotransformação/efeitos dos fármacos , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Cães , Corantes Fluorescentes/química , Humanos , Isoquinolinas/química , Cinética , Macaca fascicularis , Camundongos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Suínos , Porco Miniatura , Teofilina/análogos & derivados , Teofilina/farmacologia
19.
Toxicol Lett ; 313: 188-195, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31284022

RESUMO

Brucine is one of the main bioactive and toxic constituents of the herb drug Semen Strychni. Here we aimed to determine dosing time-dependent hepatotoxicity of brucine, and to investigate the role of metabolism in generation of brucine chronotoxicity. Brucine was administered to wild-type or Npas2-/- (a clock disrupted model) mice at different circadian time points for toxicity and pharmacokinetic characterization. The hepatotoxicity was evaluated by plasma alanine aminotransferase and aspartate aminotransferase measurements and histopathological analysis. The role of Cyp3a11 in brucine metabolism was determined by chemical inhibition assays and Cyp3a11-overexpressing HEK293 cells. Hepatic circadian Cyp3a11 mRNA and protein levels were determined by qPCR and Western blotting, respectively. The toxicity of brucine was more severe in the light phase [Zeitgeber time (ZT) 2 and ZT8] than in the dark phase (ZT14 and ZT20). Chemical inhibition and substrate metabolism assays suggested Cyp3a11 as a significant contributor to brucine metabolism. The Cyp3a11 mRNA, protein and activity in the livers of wild-type mice displayed significant circadian fluctuations. Npas2 ablation markedly down-regulated Cyp3a11 mRNA, protein and activity, and abrogated their circadian rhythms. The circadian time differences in brucine pharmacokinetics and liver distribution were lost in Npas2-/- mice, so were the time differences in brucine hepatotoxicity. In conclusion, chronotoxicity of brucine was determined by circadian variations in Cyp3a11 metabolism. The findings have implications in improving brucine (and possibly Semen Strychni) efficacy via dosing time optimization.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Ritmo Circadiano , Citocromo P-450 CYP3A/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Fotoperíodo , Estricnina/análogos & derivados , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ritmo Circadiano/genética , Cronoterapia Farmacológica , Células HEK293 , Humanos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Estricnina/administração & dosagem , Estricnina/metabolismo , Estricnina/farmacocinética , Estricnina/toxicidade
20.
Toxicol Lett ; 313: 196-204, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31278966

RESUMO

Fipronil is a chiral insecticide employed worldwide in crops, control of public hygiene and control of veterinary pests. Humans can be exposed to fipronil through occupational, food, and environmental contamination. Therefore, the risk assessment of fipronil in humans is important to protect human health. Fipronil sulfone is the major metabolite formed during fipronil metabolism by humans. Since the CYP450 enzymes are the main ones involved in drug metabolism, the evaluation of their inhibition by fipronil and its main metabolite is important to predict drug-pesticide interactions. The aim of this work was to investigate the inhibition effects of rac-fipronil, S-fipronil, R-fipronil and fipronil sulfone on the main human CYP450 isoforms. The results showed that CYP2D6 is the only CYP450 isoform inhibited by these xenobiotics. In addition, no enantioselective differences were observed in the inhibition of CYP450 isoforms by fipronil and its individuals' enantiomers. Rac-fipronil, S-fipronil and R-fipronil are moderate CYP2D6 inhibitors showing a competitive inhibition profile. On the other hand, the metabolite fipronil sulfone showed to be a strong inhibitor of CYP2D6 also by competitive inhibition. These results highlight the importance of metabolite evaluation on pesticide safety since the metabolism of fipronil into fipronil sulfone increases the risk of pesticide-drug interactions for drugs metabolized by CYP2D6.


Assuntos
Inibidores do Citocromo P-450 CYP2D6/toxicidade , Citocromo P-450 CYP2D6/metabolismo , Praguicidas/toxicidade , Pirazóis/toxicidade , Citocromo P-450 CYP2D6/química , Inibidores do Citocromo P-450 CYP2D6/química , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Biológicos , Praguicidas/química , Conformação Proteica , Pirazóis/química , Medição de Risco , Relação Estrutura-Atividade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA