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1.
Heliyon ; 5(9): e02466, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31538121

RESUMO

In this study, we used reporter gene assays in COS-1 cells to examine the activation of rat pregnane X receptor (PXR), rat constitutive androstane receptor (CAR) and rat peroxisome-proliferator activated receptor (PPAR)α by pyrethroid pesticides, and to understand the effects of metabolic modification on their activities. All eight pyrethroids tested in this study showed rat PXR agonistic activity; deltamethrin was the most potent, followed by cis-permethrin and cypermethrin. However, when the pyrethroids were incubated with rat liver microsomes, their rat PXR activities were decreased to various extents. Cis- and trans-permethrin showed weak rat CAR agonistic activity, while the other pyrethroids were inactive. However, fenvalerate showed dose-dependent inverse agonistic activity toward rat CAR, and this activity was reduced after metabolism. None of the pyrethroids showed rat PPARα agonistic activity, but a metabolite of cis-/trans-permethrin and phenothrin, 3-phenoxybenzoic acid, activated rat PPARα. Since PXR, CAR and PPARα regulate various xenobiotic/endobiotic-metabolizing enzymes, activation of these receptors by pyrethroids may result in endocrine disruption due to changes of hormone-metabolizing activities.

2.
Food Chem Toxicol ; 133: 110792, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31472229

RESUMO

Parabens are widely used as preservatives in personal care products, medicines and foods, resulting in substantial human exposures, even though some harmful effects, such as endocrine-disrupting activity, have been reported. Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are members of the nuclear receptor superfamily, regulate the metabolism of endogenous substrates including hormones. Therefore, we hypothesized that parabens may alter hormone-metabolizing activities by acting on these receptors, and such changes could contribute to the endocrine-disrupting activity. To test this idea, we systematically examined the effects of 17 parabens on these receptors using reporter gene assays. Nine parabens significantly activated human and rat PXR. Parabens with C2-C5 (linear and branched) side chains were most active. Butylparaben and isobutylparaben also significantly activated rat CAR. We found that long-side-chain (C7-C12) parabens showed up to 2-fold activation of PPARα at 10 µM. Furthermore, pentylparaben and hexylparaben showed rat PXR antagonistic activity and rat CAR inverse agonistic activity. The activity of butylparaben towards PXR and CAR was lost after carboxylesterase-mediated metabolism. These findings confirm that parabens influence the activities of PXR, CAR and PPARα, and thus have the potential to contribute to endocrine disruption by altering hormone metabolism.


Assuntos
PPAR alfa/metabolismo , Parabenos/farmacologia , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Agonismo Inverso de Drogas , Humanos , Masculino , Microssomos Hepáticos/metabolismo , PPAR alfa/agonistas , PPAR alfa/genética , Parabenos/metabolismo , Receptor de Pregnano X/antagonistas & inibidores , Receptor de Pregnano X/genética , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética
3.
Biol Pharm Bull ; 42(8): 1366-1375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31366871

RESUMO

Drug-induced liver injury (DILI) is a common side effect of several medications and is considered a major factor responsible for the discontinuation of drugs during their development. Cholestasis is a DILI that results from impairment of bile acid transporters, such as the bile salt export pump (BSEP), leading to accumulation of bile acids. Both in vitro and in vivo studies are required to predict the risk of drug-induced cholestasis. In the present study, we used chimeric mice with humanized liver as a model to study drug-induced cholestasis. Administration of a single dose of ketoconazole or rifampicin, known to potentially cause cholestasis by inhibiting BSEP, did not result in elevated levels of alkaline phosphatase (ALP), which are known hepatic biomarkers. The concentration of taurodeoxycholic acid increased in the liver after ketoconazole administration, whereas rifampicin resulted in increased tauromuricholic acid and taurocholic acid (TCA) levels in the liver and plasma. Furthermore, rifampicin resulted in an increase in the uniform distribution of a compound with m/z 514.3, presumed as TCA through imaging mass spectrometry. The mRNA levels of bile acid-related genes were also altered after treatment with ketoconazole or rifampicin. We believe these observations to be a part of a feedback mechanism to decrease bile acid concentrations. The changes in bile acid concentrations results may reflect the initial responses of the human body to cholestasis. Furthermore, these findings may contribute to the screening of drug candidates, thereby avoiding drug-induced cholestasis during clinical trials and drug development.


Assuntos
Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colestase/metabolismo , Cetoconazol/efeitos adversos , Fígado/efeitos dos fármacos , Rifampina/efeitos adversos , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Ácidos e Sais Biliares/sangue , Doença Hepática Induzida por Substâncias e Drogas/sangue , Colestase/sangue , Colestase/induzido quimicamente , Humanos , Cetoconazol/sangue , Cetoconazol/farmacocinética , Fígado/metabolismo , Masculino , Camundongos , Rifampina/sangue , Rifampina/farmacocinética
4.
Biol Pharm Bull ; 42(3): 327-336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828063

RESUMO

Predicting human pharmacokinetics (PK) such as clearance (CL) and volume of distribution (Vd) is a critical component of drug discovery. These predictions are mainly performed by in vitro-in vivo extrapolation (IVIVE) using human biological samples, such as hepatic microsomes and hepatocytes. However, some issues with this process have arisen, such as inconsistencies between in vitro and in vivo findings; the integration of predicted CYP, non-CYP and transporter-mediated human PK; and the difficulty of evaluating very metabolically stable compounds. Various approaches to solving these issues have been reported. Allometric scaling using experimental animals has also often been used. However, this method has also shown many problems due to interspecies differences, albeit that various correction methods have been proposed. Another approach involves the production of chimeric mice with humanized liver via the transplantation of human hepatocytes into mice. The livers of these mice are repopulated mostly with human hepatocytes and express human drug-metabolizing enzymes and drug transporters, suggesting that these mice are useful for solving the issues of IVIVE and allometric scaling, and more reliably predicting human PK. In this review, we summarize human PK prediction methods using IVIVE, allometric scaling and chimeric mice with humanized liver, and discuss the utility of predicting human PK in drug discovery by comparing these chimeric mice with IVIVE and allometric scaling.


Assuntos
Descoberta de Drogas/métodos , Fígado/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos , Preparações Farmacêuticas/sangue , Farmacocinética
5.
Toxicol Appl Pharmacol ; 370: 133-144, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30880217

RESUMO

Liver resection is performed to remove tumors in patients with liver cancer, but the procedure's suitability depends on the regenerative ability of the liver. It is important to consider the effects of exogenous factors, such as diets, on liver regeneration for the recovery of function. The evaluation of drug metabolism during liver regeneration is also necessary because liver dysfunction is generally observed after the operation. Here, we investigated the influence of a purified diet (AIN-93G) on liver regeneration and changes in the mRNA expression of several cytochrome P450 (CYP) isoforms in the liver and small intestine using a two-thirds partial hepatectomy (PH) mouse model fed with a standard diet (MF) and a purified diet. Liver regeneration was significantly delayed in the purified diet group relative to that in the standard diet group. The liver Cyp2c55 and Cyp3a11 expression was increased at 3 day after PH especially in the purified diet group. Bile acid may partly cause the differences in liver regeneration and CYP expression between two types of diets. On the other hand, Cyp3a13 expression in the small intestine was transiently increased at day 1 after PH in both diet groups. The findings suggest that compensatory induction of the CYP expression occurred in the small intestine after attenuation of drug metabolism potential in the liver. The present results highlight the importance of the relationship between liver regeneration, drug metabolism, and exogenous factors for the effective treatment, including surgery and medication, in patients after liver resection or transplantation.

6.
Biochem Biophys Res Commun ; 509(1): 287-293, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30587336

RESUMO

Cytochrome P450 (CYP) 3A4 plays an important role in drug metabolism. Although transcriptional regulation of CYP3A expression by chemicals has been comprehensively studied, its post-translational regulation is not fully understood. We previously reported that acetaminophen (APAP) caused accumulation of functional CYP3A protein via inhibition of CYP3A protein degradation through reduction of glycoprotein 78 (gp78), an E3 ligase of the ubiquitin proteasome system. Furthermore, N-acetyl-m-aminophenol, a regioisomer of APAP causes CYP3A protein accumulation, whereas p-acetamidobezoic acid, in which a hydroxy group of APAP was substituted for a carboxy group, did not lead to the same effects. However, the mechanism underlying the reduction of gp78 protein expression by APAP has not yet been elucidated. In this study, we selected 32 compounds including a phenolic hydroxyl group such as APAP and explored the compounds that increased CYP3A enzyme activity to analyze their common mechanism. Four compounds, including salicylate, increased CYP3A enzyme activity and led to the accumulation of functional CYP3A protein similarly to APAP. APAP and salicylate activate p38 mitogen-activated protein kinase (p38 MAPK). gp78 is known to be phosphorylated by p38 MAPK; so, we investigated the relationship between p38 MAPK and CYP3A. APAP activated p38 MAPK, decreased gp78 protein expression, and subsequently induced CYP3A protein expression in a time-dependent manner. When SB203580, a p38 MAPK inhibitor, was co-administered with APAP, the inhibitory effects of APAP on CYP3A protein degradation were suppressed. In this study, we demonstrated the involvement of the p38 MAPK-gp78 pathway in suppressing CYP3A protein degradation by APAP. Salicylate derivatives may also suppress the CYP3A protein degradation.


Assuntos
Acetaminofen/farmacologia , Analgésicos não Entorpecentes/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Citocromo P-450 CYP3A/metabolismo , Salicilatos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Proteólise/efeitos dos fármacos , Ratos Sprague-Dawley
7.
Nihon Yakurigaku Zasshi ; 151(5): 213-220, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29760366

RESUMO

To develop new drugs with high efficacy and safety, it is important to predict the pharmacological, toxicological, and pharmacokinetic profiles of drug candidates in humans. Chimeric mice with a humanized liver are mice in which human hepatocytes have been transplanted, such that mouse liver cells are replaced with human hepatocytes; these mice have been used as prediction models. Studies performed thus far indicate that chimeric mice with a humanized liver can be used for the prediction of human-specific metabolite formation and plasma concentration-time curves for several drugs. Furthermore, studies advocate the utility of chimeric mice with a humanized liver for modelling drug-induced hepatotoxicity and disease such as hepatitis virus infection in safety and pharmacological evaluations respectively. Taken together, these findings indicate that chimeric mice with a humanized liver can be used to evaluate the relationship between pharmacokinetics, toxicity, and efficacy; the contribution by active metabolites may also be assessed. In recent years, new and improved animal models have been developed to overcome the disadvantages of chimeric mice with a humanized liver. It is expected that their usefulness for optimization of drug candidates and translational research in drug discovery and development will further increase.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Fígado/efeitos dos fármacos , Animais , Quimera , Descoberta de Drogas , Humanos , Modelos Animais
8.
Biochem Pharmacol ; 154: 28-38, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29678521

RESUMO

As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O6-benzylguanine as substrates. 17ß-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC50 values. We also evaluated the potential DDI between an AOX substrate (O6-benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-24) of O6-benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context.


Assuntos
Ativação Metabólica/efeitos dos fármacos , Aldeído Oxidase/antagonistas & inibidores , Aldeído Oxirredutases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ativação Metabólica/fisiologia , Aldeído Oxidase/metabolismo , Aldeído Oxirredutases/metabolismo , Animais , Quimera , Relação Dose-Resposta a Droga , Interações de Medicamentos/fisiologia , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos SCID , Preparações Farmacêuticas/metabolismo , Ftalazinas/metabolismo , Ftalazinas/farmacologia
9.
Biol Pharm Bull ; 41(5): 722-732, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29445054

RESUMO

Differentiated HepaRG cells maintain liver-specific functions such as drug-metabolizing enzymes. In this study, the feasibility of HepaRG cells as a human hepatocyte model for in vitro toxicity assessment was examined using selected hepatotoxic compounds. First, basal drug-metabolizing enzyme activities (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, uridine 5'-diphospho-glucuronosyltransferase [UGT], and sulfotransferases [SULT]) were measured in HepaRG, human hepatocytes, and HepG2 cells. Enzyme activities in differentiated HepaRG cells were comparable to those in human hepatocytes and much higher than those in HepG2 cells, except for SULT activity. Second, we examined the cytotoxicity of hepatotoxic compounds, acetaminophen (APAP), aflatoxin B1 (AFB1), cyclophosphamide (CPA), tamoxifen (TAM), and troglitazone (TGZ) in HepaRG cells and human hepatocytes. AFB1- and CPA-induced cytotoxicities against HepaRG cells were comparable to those against human hepatocytes. Furthermore, the cytotoxicities of these compounds were inhibited by 1-aminobenzotriazole (ABT), a broad CYP inhibitor, in both cells and were likely mediated by metabolic activation by CYP. Finally, toxicogenomics analysis of HepG2 and HepaRG cells after exposure to AFB1 and CPA revealed that numerous p53-related genes were upregulated- and the expression of these genes was greater in HepaRG than in HepG2 cells. These results suggest that gene expression profiles of HepaRG cells were affected more considerably by the toxic mechanisms of AFB1 and CPA than the profiles of HepG2 cells were. Therefore, our investigation shows that HepaRG cells could be useful human hepatic cellular models for toxicity studies.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/metabolismo , Testes de Toxicidade/métodos , Linhagem Celular , Células Cultivadas , Sistema Enzimático do Citocromo P-450/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Perfilação da Expressão Gênica , Glucuronosiltransferase/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Preparações Farmacêuticas/metabolismo , Sulfotransferases/metabolismo , Proteína Supressora de Tumor p53/genética
10.
Metallomics ; 10(2): 337-345, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29345269

RESUMO

Tributyltin (TBT), a common organotin environmental pollutant, has been widely used as a component of marine antifouling paints. We previously reported that exposure to TBT inhibits the expression and DNA binding of nuclear respiratory factor-1 (NRF-1) and causes neurotoxicity. In the present study, we focused on the epigenetic effects of TBT and investigated whether TBT decreases NRF-1 expression via epigenetic modifications in SH-SY5Y human neuroblastoma cells. First, we found that exposure to 300 nM TBT decreases NRF-1 expression. We examined epigenetic changes induced by TBT, and showed that TBT causes hypermethylation of the NRF-1 promoter region, increases the amount of methyl-CpG-binding protein 2 (MeCP2) bound to the NRF-1 promoter, and alters the expression of DNA methyltransferases and ten-eleven translocation (TET) demethylation enzymes. These results suggest that epigenetic changes play an important role in regulation of NRF-1 expression. Next, we investigated effect of NRF-1 expression decrease on cells, and TBT reduces mitochondrial membrane potential and overexpression of NRF-1 rescued this reduction in membrane potential. Thus, we suggested that NRF-1 is important for maintaining mitochondrial membrane potential. Our study indicates that TBT causes epigenetic changes such as hypermethylation, which increases recruitment of MeCP2 to the NRF-1 promoter and probably lead to decreased of NRF-1 expression and mitochondrial membrane potential. Therefore, this research provides new evidence of the epigenetic action caused by organotin.


Assuntos
Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Potencial da Membrana Mitocondrial , Neuroblastoma/genética , Fator 1 Nuclear Respiratório/genética , Compostos de Trialquitina/farmacologia , Sobrevivência Celular , Genoma Humano , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Fator 1 Nuclear Respiratório/metabolismo , Regiões Promotoras Genéticas , Sulfitos , Células Tumorais Cultivadas
11.
Arch Toxicol ; 92(1): 401-409, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28725974

RESUMO

Glutamate receptor 2 (GluA2/GluR2) is one of the four subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR); an increase in GluA2-lacking AMPARs contributes to neuronal vulnerability to excitotoxicity because of the receptor's high Ca2+ permeability. Carbofuran is a carbamate pesticide used in agricultural areas to increase crop productivity. Due to its broad-spectrum action, carbofuran has also been used as an insecticide, nematicide, and acaricide. In this study, we investigated the effect of carbofuran on GluA2 protein expression. The 9-day treatment of rat primary cortical neurons with 1 µM and 10 µM carbofuran decreased GluA2 protein expression, but not that of GluA1, GluA3, or GluA4 (i.e., other AMPAR subunits). Decreased GluA2 protein expression was also observed on the cell surface membrane of 10 µM carbofuran-treated neurons, and these neurons showed an increase in 25 µM glutamate-triggered Ca2+ influx. Treatment with 50 µM glutamate, which did not affect the viability of control neurons, significantly decreased the viability of 10 µM carbofuran-treated neurons, and this effect was abolished by pre-treatment with 300 µM 1-naphthylacetylspermine, an antagonist of GluA2-lacking AMPAR. At a concentration of 100 µM, but not 1 or 10 µM, carbofuran significantly decreased acetylcholine esterase activity, a well-known target of this chemical. These results suggest that carbofuran decreases GluA2 protein expression and increases neuronal vulnerability to glutamate toxicity at concentrations that do not affect acetylcholine esterase activity.


Assuntos
Carbofurano/toxicidade , Córtex Cerebral/citologia , Ácido Glutâmico/toxicidade , Neurônios/efeitos dos fármacos , Receptores de AMPA/metabolismo , Acetilcolinesterase/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/embriologia , Inibidores da Colinesterase/toxicidade , Feminino , Proteínas Ligadas por GPI/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Gravidez , Ratos Wistar , Espermina/análogos & derivados , Espermina/farmacologia
12.
Drug Metab Pharmacokinet ; 33(1): 31-39, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29183653

RESUMO

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery.


Assuntos
Quimera/metabolismo , Descoberta de Drogas/tendências , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Quimera/genética , Descoberta de Drogas/métodos , Humanos , Fígado/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Camundongos , Camundongos Transgênicos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química
13.
J Toxicol Sci ; 42(5): 589-596, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28904294

RESUMO

It is important to consider susceptibility to drug-induced toxicity between animals and humans. Chimeric mice with a humanized liver are expected to predict hepatotoxicity in humans. Drug-induced phospholipidosis (DIPL), in which phospholipids accumulate, is a known entity. In this study, we examined whether chimeric mice can reveal species differences in DIPL. Changes in various phosphatidylcholine (PhC) molecules were investigated in the liver of chimeric mice after administering amiodarone, which induces phospholipidosis. Liquid chromatography-tandem mass spectrometry revealed that levels of PhCs tended to increase in the liver after administration of amiodarone. The liver of chimeric mice consists of human hepatocytes and residual mouse hepatocytes. We used imaging mass spectrometry (IMS) to evaluate the increase of PhCs in human and mouse hepatocytes after administration of amiodarone. IMS visualizes localization of endogenous and exogenous molecules in tissues. The IMS analysis suggested that the localized levels of several PhCs tended to be higher in the human hepatocytes than those in mouse hepatocytes, and PhC levels changed in response to amiodarone. Chimeric mice with a humanized liver will be useful to evaluate species differences in DIPL between mice and humans.


Assuntos
Amiodarona/toxicidade , Hepatócitos/metabolismo , Hepatócitos/transplante , Lipidoses/induzido quimicamente , Lipidoses/metabolismo , Fígado/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Quimeras de Transplante , Animais , Cromatografia Líquida , Humanos , Camundongos , Especificidade da Espécie , Espectrometria de Massas em Tandem
14.
J Toxicol Sci ; 42(5): 605-613, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28904296

RESUMO

A large number of chemicals are routinely detected in aquatic environments, and these chemicals may adversely affect aquatic organisms. Accurate risk assessment requires understanding drug-metabolizing systems in aquatic organisms because metabolism of these chemicals is a critical determinant of chemical bioaccumulation and related toxicity. In this study, we evaluated mRNA expression levels of nuclear receptors and drug-metabolizing enzymes as well as cytochrome P450 (CYP) activities in pro-metamorphic tadpoles, froglets, and adult frogs to determine how drug-metabolizing systems are altered at different life stages. We found that drug-metabolizing systems in tadpoles were entirely immature, and therefore, tadpoles appeared to be more susceptible to chemicals compared with metamorphosed frogs. On the other hand, cyp1a mRNA expression and CYP1A-like activity were higher in tadpoles. We found that thyroid hormone (TH), which increases during metamorphosis, induced CYP1A-like activity. Because endogenous TH concentration is significantly increased during metamorphosis, endogenous TH would induce CYP1A-like activity in tadpoles.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica/genética , Metamorfose Biológica/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/fisiologia , Xenopus/genética , Xenopus/fisiologia , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
15.
Int J Mol Sci ; 18(8)2017 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-28800112

RESUMO

Tributyltin (TBT), which has been widely used as an antifouling agent in paints, is a common environmental pollutant. Although the toxicity of high-dose TBT has been extensively reported, the effects of low concentrations of TBT are relatively less well studied. We have previously reported that low-concentration TBT decreases α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor subunit 2 (GluR2) expression in cortical neurons and enhances neuronal vulnerability to glutamate. However, the mechanism of this TBT-induced GluR2 decrease remains unknown. Therefore, we examined the effects of TBT on the activity of transcription factors that control GluR2 expression. Exposure of primary cortical neurons to 20 nM TBT for 3 h to 9 days resulted in a decrease in GluR2 mRNA expression. Moreover, TBT inhibited the DNA binding activity of nuclear respiratory factor-1 (NRF-1), a transcription factor that positively regulates the GluR2. This result indicates that TBT inhibits the activity of NRF-1 and subsequently decreases GluR2 expression. In addition, 20 nM TBT decreased the expression of genes such as cytochrome c, cytochrome c oxidase (COX) 4, and COX 6c, which are downstream of NRF-1. Our results suggest that NRF-1 inhibition is an important molecular action of the neurotoxicity induced by low-concentration TBT.


Assuntos
Poluentes Ambientais/toxicidade , Fator 1 Relacionado a NF-E2/metabolismo , Neurônios/efeitos dos fármacos , Receptores de AMPA/metabolismo , Compostos de Trialquitina/toxicidade , Animais , Células Cultivadas , Poluentes Ambientais/farmacologia , Células HEK293 , Humanos , Fator 1 Relacionado a NF-E2/genética , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de AMPA/genética , Compostos de Trialquitina/farmacologia
16.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1061-1062: 110-116, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28728084

RESUMO

A depth filter is widely used in biopharmaceutical manufacturing process. In this study, we found that buffer exchange to reduce conductivity dramatically improved the removal of impurities in depth filtration. The host cell protein clearance was comparable to that of protein A affinity chromatography, which is generally used as a first capture step in monoclonal antibody purification. In addition, a combination of different depth filters showed enhanced purification. Additional flow-through purification is possible without any sample preparation, and a biopharmaceutical-quality purity level (<100ppm for host cell protein and <5% for high molecular weight species) can be attained. These results represent the possibility of an antibody manufacturing process using an entirely flow-through mode.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus
17.
Biol Pharm Bull ; 40(7): 1121-1124, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28674256

RESUMO

Tributyltin (TBT), a common environmental contaminant, is widely used as an antifouling agent in paint. We previously reported that exposure of primary cortical neurons to TBT in vitro decreased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit glutamate receptor 2 (GluR2) expression and subsequently increased neuronal vulnerability to glutamate. Therefore, to identify whether GluR2 expression also decreases after TBT exposure in vivo, we evaluated the changes in GluR2 expression in the mouse brain after prenatal or postnatal exposure to 10 and 25 ppm TBT through pellet diets. Although the mean feed intake and body weight did not decrease in TBT-exposed mice compared with that in control mice, GluR2 expression in the cerebral cortex and hippocampus decreased after TBT exposure during the prenatal period. These results indicate that a decrease in neuronal GluR2 may be involved in TBT-induced neurotoxicity, especially during the fetal period.


Assuntos
Encéfalo/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Receptores de AMPA/metabolismo , Compostos de Trialquitina/toxicidade , Animais , Peso Corporal , Encéfalo/metabolismo , Comportamento Alimentar , Feminino , Camundongos , Gravidez
18.
Sci Rep ; 7: 46668, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28443637

RESUMO

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, mainly characterised by the progressive loss of dopaminergic neurons. MPP+ has been widely used as a PD-related neurotoxin, and their reports suggested the several hypotheses for neuronal cell death. However, most of these hypotheses come from the studies about the acute MPP+ exposure. We previously revealed that mild MPP+ exposure (10 and 200 µM), which induces gradual cell death, impairs autophagosome degradation at 48 h. In the present study, we further investigated the specific events of mild MPP+ exposure and revealed that mild MPP+ exposure causes the cell death through glucose starvation, but not acute toxic model (2.5 and 5 mM). At 36 h after mild MPP+ exposure, autophagosome synthesis was enhanced owing to glucose starvation and continued to enhance until 48 h, despite impaired autophagosome degradation. Inhibition of autophagosome synthesis reduced mild MPP+-induced cell death. In conclusion, we clarified that glucose starvation-enhanced autophagosome synthesis occurs at an earlier stage than impaired autophagosome degradation and is important in mild MPP+ toxicity.


Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Glucose/metabolismo , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Herbicidas/farmacologia , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Interferência de RNA
19.
J Appl Toxicol ; 37(9): 1030-1035, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28299817

RESUMO

Many concerns have been expressed regarding the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. The disruption of thyroid hormones in the neonatal period may lead to permanent effects on thyroid hormone homeostasis as well as related developmental disorders, as thyroid hormones are essential for regulating the growth and differentiation of many tissues. To understand the long-term alteration in gene expressions by neonatal administration of thyroid hormone-like chemicals in general, we identified genes whose expression was altered in the liver, an important component of the thyroid hormone axis, by neonatal exposure to triiodothyronine (T3). T3 was administered to male F344 rats on postnatal days 1, 3, and 5 (week 0). At 8 weeks of age, cDNA microarray analysis was used to identify hepatic genes whose expression was altered by neonatal exposure to T3. Among the up-regulated genes that were identified, the expression of Olr59, Ethe1, and Slc10a2 increased specifically in rats neonatally exposed to T3. Interestingly, altered hepatic expression of these genes indeed increased when a hydroxylated polybrominated diphenyl ether (PBDE), OH-BDE42, which is capable of binding to the TR, was given neonatally. Our data demonstrated that neonatal exposure to thyroid hormones could affect the long-term expression of the genes, which could be useful markers for neonatal effects by thyroid hormone-disrupting chemicals. Copyright © 2017 John Wiley & Sons, Ltd.


Assuntos
Dioxigenases/metabolismo , Proteínas Mitocondriais/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores Odorantes/metabolismo , Simportadores/metabolismo , Tri-Iodotironina/sangue , Tri-Iodotironina/farmacologia , Animais , Animais Recém-Nascidos , Dioxigenases/genética , Disruptores Endócrinos/toxicidade , Regulação da Expressão Gênica , Éteres Difenil Halogenados/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Proteínas Mitocondriais/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Receptores Odorantes/genética , Simportadores/genética , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo
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