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1.
FASEB J ; 2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32421247

RESUMO

Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.

2.
Food Chem Toxicol ; 125: 182-189, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30610934

RESUMO

The present study aimed to determine the effects of cigarette smoke on the regulation of hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes in male BALB/c mice exposed to nose-only cigarette smoke for 4 days. There were no significant increases in serum liver injury markers (alanine aminotransferase and aspartate aminotransferase) or oxidative stress (total antioxidant capacity, malondialdehyde, and glutathione disulfide/reduced glutathione) following cigarette smoke exposure, but malondialdehyde was elevated in the bronchoalveolar lavage fluid of smoke-exposed mice. Additionally, the hepatic microsomal protein levels of Cyp1a and Cyp2b, and the activities of ethoxyresorufin O-deethylase, pentoxyresorufin O-depenylase, and chlorzoxazone 6-hydrxylase, were elevated in smoke-exposed mice. Interestingly, the hepatic activities of GST toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrynic acid, but not cumene hydroperoxide were enhanced by cigarette smoke exposure, which was consistent with the increased expression levels of mu- and pi-class GSTs, but not alpha-class GSTs, observed in immunoblot analyses. These findings indicate that the short-term inhalation of cigarette smoke induces drug-metabolizing enzymes such as CYP1A, CYP2B, and mu/pi-class GSTs in the absence of hepatic injury and oxidative stress. Furthermore, smoking may alter hepatic drug metabolism, as well as the disposition and toxicity of xenobiotics, including some therapeutic drugs and cigarette smoke constituents.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/metabolismo , Fígado/enzimologia , Fumaça , Tabaco , Animais , Camundongos , Nariz
3.
Biomol Ther (Seoul) ; 26(2): 167-174, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28605831

RESUMO

Alterations in sulfur amino acid metabolism are associated with an increased risk of a number of common late-life diseases, which raises the possibility that metabolism of sulfur amino acids may change with age. The present study was conducted to understand the age-related changes in hepatic metabolism of sulfur amino acids in 2-, 6-, 18- and 30-month-old male C57BL/6 mice. For this purpose, metabolite profiling of sulfur amino acids from methionine to taurine or glutathione (GSH) was performed. The levels of sulfur amino acids and their metabolites were not significantly different among 2-, 6- and 18-month-old mice, except for plasma GSH and hepatic homocysteine. Plasma total GSH and hepatic total homocysteine levels were significantly higher in 2-month-old mice than those in the other age groups. In contrast, 30-month-old mice exhibited increased hepatic methionine and cysteine, compared with all other groups, but decreased hepatic S-adenosylmethionine (SAM), S-adenosylhomocysteine and homocysteine, relative to 2-month-old mice. No differences in hepatic reduced GSH, GSH disulfide, or taurine were observed. The hepatic changes in homocysteine and cysteine may be attributed to upregulation of cystathionine ß-synthase and down-regulation of γ-glutamylcysteine ligase in the aged mice. The elevation of hepatic cysteine levels may be involved in the maintenance of hepatic GSH levels. The opposite changes of methionine and SAM suggest that the regulatory role of SAM in hepatic sulfur amino acid metabolism may be impaired in 30-month-old mice.

4.
Oncotarget ; 8(41): 71054-71069, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-29050342

RESUMO

Epoxyeicosatrienoic acid (EET) production via cytochrome P450 (CYP) epoxygenases closely correlates with the progression of breast cancer. However, its role in the development of chemoresistant breast cancers has yet to be elucidated. Here, we found that CYP3A4 expression and its epoxy-product, 11,12-epoxyeicosatrienoic acid (11,12-EET) was enhanced in tamoxifen (TAM)-resistant MCF-7 (TAMR-MCF-7) breast cancer cells compared to control MCF-7 cells. Treatment of TAMR-MCF-7 cells with ketoconazole and azamulin (selective CYP3A4 inhibitors) or 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET antagonist) inhibited cellular proliferation and recovered the sensitivity to 4-hydroxytamoxifen. Chick chorioallantoic membrane and trans-well migration analyses revealed that the enhanced angiogenic, tumorigenic, and migration intensities of TAMR-MCF-7 cells were also significantly suppressed by ketoconazole and 14,15-EEZE. We previously reported that Pin1, a peptidyl prolyl isomerase, is a crucial regulator for higher angiogenesis and epithelial-mesenchymal transition characteristics of TAMR-MCF-7 cells. EET inhibition suppressed E2F1-dependent Pin1 gene transcription, and Pin1 silencing also blocked cell proliferation, angiogenesis, and migration of TAMR-MCF-7 cells. Our findings suggest that the CYP3A4-mediated EET pathway represents a potential therapeutic target for the treatment of tamoxifen-resistant breast cancer.

5.
J Pharm Biomed Anal ; 131: 309-315, 2016 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-27619177

RESUMO

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.


Assuntos
Anticorpos Monoclonais/sangue , Autoanticorpos/sangue , Química Farmacêutica/normas , Animais , Anticorpos Monoclonais/toxicidade , Autoanticorpos/efeitos dos fármacos , Química Farmacêutica/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Macaca fascicularis , Masculino , Reprodutibilidade dos Testes
6.
Food Chem Toxicol ; 96: 244-53, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27523289

RESUMO

In this study, the hepatic expression of cytochrome P450 (CYP) enzymes, including CYP1A1/2, 2B1, 2C11, 2E1, 3A1/2, and 4A, was investigated in 5-week-old (insulinresistant state) and 11-week-old (diabetic) Zucker diabetic fatty (ZDF) rats. Serum glucose and glycated hemoglobin levels were increased in 11-week-old ZDF rats, but not in 5-weekold ZDF rats. Hyperinsulinemia was observed in both age groups. The microsomal protein, total CYP, CYP reductase, CYP1A1/2, and CYP3A1 levels did not differ between 5- and 11-week-old ZDF rats and their respective control rats, while CYP4A was up-regulated in both groups. Hepatic levels of cytochrome b5, CYP2B1, CYP2C11, CYP2E1, and CYP3A2 were decreased in 5-week-old ZDF rats, but not in 11-week-old ZDF rats. Similarly, pentoxyresorufin O-depentylase, testosterone 2α- and 16α-hydroxylase, chlorzoxazone 6- hydroxylase, and midazolam 1'- and 4-hydroxylase activities were decreased only in 5-weekold ZDF rats. Based on these results, the 5-week-old ZDF rats exhibited down-regulation of the major CYP enzymes. These results suggest that hepatic expression of CYP enzymes may be dysregulated during development in ZDF rats. With the exception of CYP2B1 and CYP4A, the hepatic levels and activities of CYP were comparable between 11-week-old ZDF and control rats, suggesting that xenobiotic metabolism is normally regulated in the early diabetic state.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Fatores Etários , Animais , Immunoblotting , Masculino , Ratos , Ratos Zucker
7.
Eur J Med Chem ; 117: 113-24, 2016 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-27092411

RESUMO

Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) in the metabolic pathway of arachidonic acid and has been considered as an important therapeutic target for chronic diseases such as hypertension, diabetes and inflammation. Although many urea derivatives are known as sEH inhibitors, the enantioselectivity of the inhibitors is not highlighted in spite of the stereoselective hydrolysis of EETs by sEH. In an effort to explore the importance of enantioselectivity in the urea scaffold, a series of enantiomers with the stereocenter adjacent to the urea nitrogen atom were prepared. The selectivity of enantiomers of 1-(α-alkyl-α-phenylmethyl)-3-(3-phenylpropyl)ureas showed wide range differences up to 125 fold with the low IC50 value up to 13 nM. The S-configuration with planar phenyl and small alkyl groups at α-position is crucial for the activity and selectivity. However, restriction of the free rotation of two α-groups with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity between the enantiomers, despite the increase in activity up to 13 nM. The hydrophilic group like sulfonamido group at para position of 3-phenylpropyl motif of 1-(α-alkyl-α-phenylmethyl-3-(3-phenylpropyl)urea improves the activity as well as enantiomeric selectivity. All these ureas are proved to be specific inhibitor of sEH without inhibition against mEH.


Assuntos
Inibidores Enzimáticos/síntese química , Epóxido Hidrolases/antagonistas & inibidores , Ureia/análogos & derivados , Doença Crônica/tratamento farmacológico , Descoberta de Drogas , Humanos , Isomerismo , Solubilidade , Estereoisomerismo
8.
Chem Res Toxicol ; 28(5): 872-85, 2015 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-25860621

RESUMO

Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for the prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for the bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferase-dependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.


Assuntos
Antineoplásicos Alquilantes/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Ciclofosfamida/toxicidade , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Antineoplásicos Alquilantes/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ciclofosfamida/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Microssomos Hepáticos/metabolismo , NADP/metabolismo
9.
Food Chem Toxicol ; 76: 94-102, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25533794

RESUMO

meso-Dihydroguaiaretic acid (MDGA) is a major component of Myristica fragrans and Machilus thunbergii that is traditionally used as a spice and for medicinal purposes. Despite reports of various biological activities exerted by MDGA, there is no information regarding its metabolic properties. The purpose of this study was to determine the metabolic stability and cytochrome P450 (CYP) inhibitory potential of MDGA, using pooled human liver microsomes (HLMs) to characterize its metabolic properties. In addition, pharmacokinetic analysis was performed in mice treated intravenously (5 mg/kg) or orally (20 mg/kg) with MDGA for comparison with our in vitro results. The half-life of MDGA in HLMs and mouse liver microsomes incubated with NADPH, UDPGA or NADPH plus UDPGA was 25.41 and 22.74, 0.39 and 0.20 or 0.28 and 0.22 min, respectively. In our pharmacokinetic study, MDGA rapidly declined in plasma and had low bioavailability, which was attributable to extensive metabolism by UDP-glucuronosyltransferases and CYPs. Among CYP isoforms, CYP2E1 activity was selectively inhibited by MDGA through a competitive inhibitory mode, with an inhibitory constant (Ki) value of 13.1 µM. These results suggest that MDGA can be used as a selective CYP2E1 inhibitor in vitro, which warrants evaluation of the pharmacological significance of MDGA-induced CYP2E1 inhibition.


Assuntos
Guaiacol/análogos & derivados , Lignanas/farmacocinética , Microssomos Hepáticos/efeitos dos fármacos , Animais , Inibidores do Citocromo P-450 CYP2E1/sangue , Inibidores do Citocromo P-450 CYP2E1/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Guaiacol/sangue , Guaiacol/farmacocinética , Meia-Vida , Humanos , Lauraceae/química , Lignanas/sangue , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microssomos Hepáticos/metabolismo
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