RESUMO
The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor according to Fairbanks' protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (Ki = 160 nM against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human hepatocytes demonstrated that compound 5h is a anti-hyperglycaemic drug candidates performing slightly better than DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the potency of compound 5h since it lowered blood glucose levels by â¼36% at 30 mg kg-1 and â¼43% at 60 mg kg-1. The present study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such drug candidates.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Glicogênio Fosforilase/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Compostos de Espiro/farmacologia , Tiazóis/farmacologia , Animais , Glicemia/metabolismo , Ciclização , Teoria da Densidade Funcional , Glicogênio/metabolismo , Glicogênio Fosforilase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Concentração Inibidora 50 , Cinética , Lactonas/síntese química , Lactonas/química , Oxirredução , Ratos Zucker , Compostos de Espiro/síntese química , Compostos de Espiro/química , Estereoisomerismo , Temperatura , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Growth factors have key roles in liver physiology and pathology, particularly by promoting cell proliferation and growth. Recently, it has been shown that in mouse hepatocytes, epidermal growth factor receptor (EGFR) plays a crucial role in the activation of the xenosensor constitutive androstane receptor (CAR) by the antiepileptic drug phenobarbital. Due to the species selectivity of CAR signaling, here we investigated epidermal growth factor (EGF) role in CAR signaling in primary human hepatocytes. Primary human hepatocytes were incubated with CITCO, a human CAR agonist, or with phenobarbital, an indirect CAR activator, in the presence or absence of EGF. CAR-dependent gene expression modulation and PXR involvement in these responses were assessed upon siRNA-based silencing of the genes that encode CAR and PXR. EGF significantly reduced CAR expression and prevented gene induction by CITCO and, to a lower extent, by phenobarbital. In the absence of EGF, phenobarbital and CITCO modulated the expression of 144 and 111 genes, respectively, in primary human hepatocytes. Among these genes, only 15 were regulated by CITCO and one by phenobarbital in a CAR-dependent manner. Conversely, in the presence of EGF, CITCO and phenobarbital modulated gene expression only in a CAR-independent and PXR-dependent manner. Overall, our findings suggest that in primary human hepatocytes, EGF suppresses specifically CAR signaling mainly through transcriptional regulation and drives the xenobiotic response toward a pregnane X receptor (PXR)-mediated mechanism.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Hepatócitos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Recoverina/metabolismo , Adulto , Idoso , Células Cultivadas , Receptores ErbB/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Oximas/farmacologia , Fenobarbital/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway.
Assuntos
Hepacivirus/patogenicidade , Gotículas Lipídicas/metabolismo , Microtúbulos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Septinas/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Hepacivirus/fisiologia , Hepatite C/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Microtúbulos/virologia , Ácido Oleico/farmacologia , Septinas/genética , Replicação ViralRESUMO
Glycogen phosphorylase (GP) is a target for the treatment of hyperglycaemia in the context of type 2 diabetes. This enzyme is responsible for the depolymerization of glycogen into glucose thereby affecting the levels of glucose in the blood stream. Twelve new d-glucopyranosylidene-spiro-isoxazolines have been prepared from O-peracylated exo-D-glucals by regio- and stereoselective 1,3-dipolar cycloaddition of nitrile oxides generated in situ by treatment of the corresponding oximes with bleach. This mild and direct procedure appeared to be applicable to a broad range of substrates. The corresponding O-unprotected spiro-isoxazolines were evaluated as glycogen phosphorylase (GP) inhibitors and exhibited IC50 values ranging from 1 to 800 µM. Selected inhibitors were further evaluated in vitro using rat and human hepatocytes and exhibited significant inhibitory properties in the primary cell culture. Interestingly, when tested with human hepatocytes, the tetra-O-acetylated spiro-isoxazoline bearing a 2-naphthyl residue showed a much lower IC50 value (2.5 µM), compared to that of the O-unprotected analog (19.95 µM). The most promising compounds were investigated in Zucker fa/fa rat model in acute and sub-chronic assays and decreased hepatic glucose production, which is known to be elevated in type 2 diabetes. This indicates that glucose-based spiro-isoxazolines can be considered as anti-hyperglycemic agents in the context of type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Glucose/análogos & derivados , Glicogênio Fosforilase/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Animais , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucose/química , Glucose/farmacologia , Glicogênio Fosforilase/metabolismo , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Isoxazóis/síntese química , Isoxazóis/química , Isoxazóis/farmacologia , Estrutura Molecular , Ratos , Ratos Zucker , Compostos de Espiro/síntese química , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
Cytochrome P450 (CYP) expression and activity are not homogeneous in the liver lobules. Indeed, CYPs are mainly expressed and induced in centrilobular hepatocytes. The wingless-type MMTV integration site family (WNT)/ß-catenin pathway was identified as a major regulator of this zonal organization. We have recently demonstrated that in primary human hepatocytes (PHHs), the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor (AhR), but not of CYP3A4, is regulated by the WNT/ß-catenin pathway in response to WNT3a, its canonical activator. Here, we investigated whether glycogen synthase kinase 3ß (GSK3ß) inhibitors, which mimic the action of WNT molecules, could be used in PHHs to activate the ß-catenin pathway to study CYP expression. We assessed the activity of 6BIO (6-bromoindirubin-3'-oxime), CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile), and GSK3iXV (Pyridocarbazolo-cyclopentadienyl Ruthenium complex GSK3 inhibitor XV) that belong to structurally different families of GSK3ß inhibitors. Using small interfering RNAs, reporter gene assays, and molecular docking predictions, we demonstrated that GSK3ß inhibitors can activate the WNT/ß-catenin pathway in PHHs to regulate CYP2E1 expression. We also found that 6BIO and GSK3iXV are AhR full agonists that participate, through AhR signaling, to CYP1A2 induction. Conversely, CHIR99021 is an AhR partial agonist, and a pregnane X receptor ligand and partial agonist, thus regulating CYP1A2 and CYP3A4 gene expression in a ß-catenin-independent manner. In conclusion, GSK3ß inhibitors can activate the WNT/ß-catenin pathway in PHHs. Nevertheless, their role in CYP regulation should be analyzed with caution as these molecules can interact with xenosensors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Indutores das Enzimas do Citocromo P-450/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hepatócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Esteroides/agonistas , beta Catenina/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Indutores das Enzimas do Citocromo P-450/química , Indutores das Enzimas do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática/efeitos dos fármacos , Feminino , Genes Reporter/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Indóis/farmacologia , Masculino , Simulação de Acoplamento Molecular , Compostos Organometálicos/farmacologia , Oximas/farmacologia , Receptor de Pregnano X , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Criopreservação/métodos , Crioprotetores/farmacologia , Hepatócitos/transplante , Hepatopatias/terapia , Soluções para Preservação de Órgãos/farmacologia , Adulto , Idoso , Albuminas/biossíntese , Animais , Proliferação de Células , Sobrevivência Celular , Fator VII/biossíntese , Feminino , Ganciclovir/efeitos adversos , Hepatócitos/citologia , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Albumina Sérica/análise , Baço/citologia , Transplante Heterólogo , Ureia/metabolismoRESUMO
The wingless-type MMTV integration site family (WNT)/ß-catenin/adenomatous polyposis coli (CTNNB1/APC) pathway has been identified as a regulator of drug-metabolizing enzymes in the rodent liver. Conversely, little is known about the role of this pathway in drug metabolism regulation in human liver. Primary human hepatocytes (PHHs), which are the most physiologically relevant culture system to study drug metabolism in vitro, were used to investigate this issue. This study assessed the link between cytochrome P450 expression and WNT/ß-catenin pathway activity in PHHs by modulating its activity with recombinant mouse Wnt3a (the canonical activator), inhibitors of glycogen synthase kinase 3ß, and small-interfering RNA to invalidate CTNNB1 or its repressor APC, used separately or in combination. We found that the WNT/ß-catenin pathway can be activated in PHHs, as assessed by universal ß-catenin target gene expression, leucine-rich repeat containing G protein-coupled receptor 5. Moreover, WNT/ß-catenin pathway activation induces the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor, but not of CYP3A4, hepatocyte nuclear factor-4α, or pregnane X receptor (PXR) in PHHs. Specifically, we show for the first time that CYP2E1 is transcriptionally regulated by the WNT/ß-catenin pathway. Moreover, CYP2E1 induction was accompanied by an increase in its metabolic activity, as indicated by the increased production of 6-OH-chlorzoxazone and by glutathione depletion after incubation with high doses of acetaminophen. In conclusion, the WNT/ß-catenin pathway is functional in PHHs, and its induction in PHHs represents a powerful tool to evaluate the hepatotoxicity of drugs that are metabolized by CYP2E1.
Assuntos
Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2E1/genética , Regulação Enzimológica da Expressão Gênica , Hepatócitos/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/fisiologia , Adulto , Idoso , Linhagem Celular , Citocromo P-450 CYP3A/genética , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: In Huntington's disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice. RESULTS: To this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections. CONCLUSIONS: These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.
Assuntos
Corpo Estriado/efeitos dos fármacos , Doença de Huntington/tratamento farmacológico , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/uso terapêutico , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/uso terapêutico , Administração Bucal , Administração Retal , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Feminino , Células HeLa , Humanos , Proteína Huntingtina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/síntese química , Proteínas do Tecido Nervoso/farmacocinética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Teste de Desempenho do Rota-RodRESUMO
UNLABELLED: Hepatitis C virus (HCV) genotype 3a infection poses a serious health problem worldwide. A significant association has been reported between HCV genotype 3a infections and hepatic steatosis. Nevertheless, virological characterization of genotype 3a HCV is delayed due to the lack of appropriate virus cell culture systems. In the present study, we established the first infectious genotype 3a HCV system by introducing adaptive mutations into the S310 strain. HCV core proteins had different locations in JFH-1 and S310 virus-infected cells. Furthermore, the lipid content in S310 virus-infected cells was higher than Huh7.5.1 cells and JFH-1 virus-infected cells as determined by the lipid droplet staining area. CONCLUSION: This genotype 3a infectious cell culture system may be a useful experimental model for studying genotype 3a viral life cycles, molecular mechanisms of pathogenesis, and genotype 3a-specific antiviral drug development.
Assuntos
Técnicas de Cultura de Células , Hepacivirus/fisiologia , Antivirais , Linhagem Celular , Genótipo , Gotículas Lipídicas , Testes de Sensibilidade Microbiana , Mutação , Vírion , Replicação ViralRESUMO
One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrP(C) level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders.
Assuntos
Encéfalo/patologia , Regulação para Baixo , Técnicas de Transferência de Genes , Proteínas PrPC/metabolismo , Doenças Priônicas/patologia , RNA Interferente Pequeno/metabolismo , Animais , Astrócitos/metabolismo , Contagem de Células , Citocinas/biossíntese , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Análise de Sobrevida , Fatores de Tempo , Vacúolos/metabolismoRESUMO
OBJECTIVE: Adult primary human hepatocytes (PHHs) support the complete infection cycle of natural HCV from patients' sera. The molecular details underlying sera infectivity towards these cells remain largely unknown. Therefore, we sought to gain a deeper comprehension of these features in the most physiologically relevant culture system. DESIGN: Using kinetic experiments, we defined the optimal conditions to infect PHH and explored the link between cell organisation and permissivity. Based on their infectivity, about 120 sera were classified in three groups. Concentration of 52 analytes was measured in 79 selected sera using multiplexed immunobead-based analyte profiling. RESULTS: PHH permissivity towards HCV infection negatively correlated with cell polarisation and formation of functional bile canaliculi. PHH supported HCV replication for at least 2â weeks with de novo virus production. Depending on their reactivity, sera could be classified in three groups of high, intermediate or low infectivity toward PHH. Infectivity could not be predicted based on the donors' clinical characteristics, viral load or genotype. Interestingly, highly infectious sera displayed a specific cytokine profile with low levels of most of the 52 tested analytes. Among them, 24 cytokines/growth factors could impact hepatocyte biology and infection efficiency. CONCLUSIONS: We identified critical factors leading to efficient PHH infection by HCV sera in vitro. Overall, we showed that this cellular model provides a useful tool for studying the mechanism of HCV infection in its natural host cell, selecting highly infectious isolates, and determining the potency of drugs towards various HCV strains.
Assuntos
Hepacivirus/patogenicidade , Hepatócitos/virologia , Adulto , Biomarcadores/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Hepacivirus/metabolismo , Hepatócitos/fisiologia , Humanos , Cinética , Modelos Imunológicos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro/virologiaRESUMO
It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/-) γc(-/-) mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.
Assuntos
Adenoviridae/metabolismo , Hepatócitos/transplante , Fígado/patologia , Timidina Quinase/metabolismo , Transplante Heterólogo , Albuminas/metabolismo , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ganciclovir/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Hepatite B/patologia , Hepatite C/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante HomólogoRESUMO
Inflammation and infection have long been known to affect the activity and expression of enzymes involved in hepatic and extrahepatic drug clearance. Significant advances have been made to elucidate the molecular mechanisms underlying the complex cross-talk between inflammation and drug-metabolism alterations. The emergent role of ligand-activated transcriptional regulators, belonging to the nuclear receptor (NR) superfamily, is now well established. The NRs, pregnane X receptor, constitutive androstane receptor, retinoic X receptor, glucocorticoid receptor, and hepatocyte nuclear factor 4, and the basic helix-loop-helix/Per-ARNT-Sim family member, aryl hydrocarbon receptor, are the main regulators of the detoxification function. According to the panel of mediators secreted during inflammation, a cascade of numerous signaling pathways is activated, including nuclear factor kappa B, mitogen-activated protein kinase, and the Janus kinase/signal transducer and activator of transcription pathways. Complex cross-talk is established between these signaling pathways regulating either constitutive or induced gene expression. In most cases, a mutual antagonism between xenosensor and inflammation signaling occurs. This review focuses on the molecular and cellular mechanisms implicated in this cross-talk.
Assuntos
Inflamação/metabolismo , Receptor Cross-Talk , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Humanos , Inativação Metabólica , Inflamação/genética , Receptores Citoplasmáticos e Nucleares/genética , Transdução de SinaisRESUMO
Hepatitis C virus (HCV) genotype 3a is widespread worldwide, but no replication system exists for its study. We describe a subgenomic replicon system for HCV genotype 3a. We determined the consensus sequence of an HCV genome isolated from a patient, and constructed a subgenomic replicon using this clone. The replicon was transfected into HuH-7 cells and RNA replication was confirmed. We identified cell culture-adaptive mutations that increased colony formation multiple-fold. We have therefore established a genotype 3a replicon system that can be used to study this HCV genotype.
Assuntos
Hepacivirus/genética , Hepacivirus/fisiologia , RNA Viral/metabolismo , Replicon/genética , Replicação Viral/genética , Adaptação Fisiológica/genética , Linhagem Celular Tumoral , Ensaio de Unidades Formadoras de Colônias , Humanos , Mutação , FenótipoRESUMO
BACKGROUND: The metabolism of phenothiazine neuroleptics (promazine, perazine) in a primary culture of human hepatocytes after pretreatment of cells with those neuroleptics was studied. METHODS: The hepatocytes were pretreated with 25 µM promazine or perazine for 96 h. Then, the cells were incubated for 2, 4, 6, 8 and 24 h in the presence of neuroleptics. At the indicated time points, concentrations of phenothiazines and their metabolites (5-sulfoxides and N-desmethyl derivatives) were measured in the culture medium using HPLC with UV detection. RESULTS: Pretreatment of the primary culture of human hepatocytes with promazine or perazine resulted in accumulation of their metabolites in the culture medium. Such an effect was not observed in the case of control cultures (not pretreated with neuroleptics). CONCLUSION: The obtained results suggest that the tested phenothiazines may stimulate their own metabolism by inducing CYP1A2, CYP3A4 and CYP2C19 isoforms.
Assuntos
Antipsicóticos/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Hepatócitos/enzimologia , Perazina/metabolismo , Promazina/metabolismo , Idoso , Antipsicóticos/farmacologia , Biotransformação , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Remoção de Radical Alquila , Indução Enzimática , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Isoenzimas , Perazina/farmacologia , Cultura Primária de Células , Promazina/farmacologia , Espectrofotometria Ultravioleta , Sulfóxidos/metabolismo , Fatores de TempoRESUMO
Ochratoxin A (OTA) is a mycotoxin produced by fungi of two genera: Penicillium and Aspergillus. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic, and immunotoxic to several species of animals and to cause kidney and liver tumors in mice and rats. Biotransformation of OTA has not been entirely elucidated. Several metabolites have been characterized in vitro and/or in vivo, whereas other metabolites remain to be characterized. At present, data available regarding OTA metabolism and cytochrome inductions concern only rodents or in vitro systems. The aim of the present study was to explore the effect of OTA on mRNA expression of some cytochromes known to be regulated by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR), using primary cultures of human hepatocytes. Our results showed that OTA reduced hepatocyte viability in a dose-dependent manner. Using quantitative real-time reverse-transcription polymerase chain reaction, our study showed that treatment of primary cultured human hepatocytes with noncytotoxic increasing concentrations of OTA for 24 hours caused a significant upregulation of CYP3A4, CYP2B6, and, to a lesser extent, CYP3A5 and CYP2C9. PXR mRNA expression increased in only 1 treated liver, whereas CAR mRNA expression was not affected. OTA was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that OTA could activate PXR and AhR; however, further investigations are needed to confirm nuclear-receptor activation by OTA.
Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Ocratoxinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Ocratoxinas/administração & dosagem , Ocratoxinas/metabolismo , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Esteroides/efeitos dos fármacos , Receptores de Esteroides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aflatoxin B1 (AFB1), one of the most common mycotoxins found in human foods and animal feed, is principally hepatotoxic and hepatocarcinogenic. The aim of the present study was to explore the effect of AFB1 on messenger RNA (mRNA) expression of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) and some of their target cytochromes using primary cultures of human hepatocytes. Our results showed that AFB1, at noncytotoxic increasing concentrations, caused a significant upregulation of cytochrome P 2B6 (CYP2B6), CYP3A5, and to a lesser extent CYP3A4 and CYP2C9. Pregnane X receptor and CAR mRNA expression increased in the 3 treated livers. Aflatoxin B1 was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that AFB1 could activate PXR, CAR, and AhR; however, further investigations are needed to confirm nuclear receptor activation by AFB1.
Assuntos
Aflatoxina B1/toxicidade , Carcinógenos/toxicidade , Hepatócitos/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Receptor Constitutivo de Androstano , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genéticaRESUMO
The mycotoxin, patulin (PAT), which is frequently found in apples, grapes, oranges, pear, peaches, and in apple juices, has previously been shown to be cytotoxic, genotoxic, and mutagenic. In this study, we have investigated the effect of PAT on mRNA level of pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and their corresponding target cytochrome P450s. Using primary cultures of adult human hepatocytes, we evaluated PAT cytotoxicity on hepatocytes after 24 hours of treatment. Real time reverse-transcriptase polymerase chain reaction procedure was employed to determine the effect of PAT on receptors (PXR, CAR, and AhR) and cytochrome (CYP3A4, 2B6, 3A5, 2C9, 1A1, and 1A2) genes. Our results showed that PAT reduced hepatocyte viability. At a noncytotoxic range of PAT concentrations, PAT induced an upregulation of the PXR gene in the three treated hepatocytes cultures, whereas CAR was overexpressed in only 1 treated liver. PXR gene induction was accompanied by the enhancement of CYP2B6, 3A5, 2C9, and 3A4 expression. PAT was also found to induce an overexpression of AhR and CYP1A1 and CYP1A2 mRNA expression. These findings suggested that PAT may activate PXR and/or CAR and AhR. However, further investigations are needed to confirm nuclear receptor activation by PAT and to elucidate the molecular mechanism of PAT action.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Patulina/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Trifosfato de Adenosina/metabolismo , Análise de Variância , Sobrevivência Celular/efeitos dos fármacos , Receptor Constitutivo de Androstano , Humanos , Luciferases , Estrutura Molecular , Patulina/química , Receptor de Pregnano X , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The mycotoxin zearalenone (ZEN) is found worldwide as a contaminant in cereals and grains. ZEN subchronic and chronic toxicities are dominated by reproductive disorders in different mammalian species which have made ZEN established mammalian endocrine disrupter. Over the last 30 years of ZEN biotransformation study, the toxin was thought to undergo reductive metabolism only, with the generation in several species of α- and ß-isomers of zearalenol. However, recent investigations have noticed that the mycoestrogen is prone to oxidative metabolism leading to hydroxylation of ZEN though the involvement of different cytochromes P450 (CYPs) isoforms. The aim of the present study was to further explore the effect of ZEN on regulation of some CYPs using primary cultures of human hepatocytes. For this aim, using real time RT-PCR, we monitored in a first time, the effect of ZEN on mRNA levels of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR), nuclear receptors known to be involved in the regulation of some CYPs. In a second time, we looked for ZEN effect on expression of PXR, CAR and AhR corresponding phase I target genes (CYP3A4, CYP3A5, CYP2B6, CYP2C9, CYP1A1 and CYP1A2). Finally, we realised the luciferase assay in HepG2 treated with the toxin and transiently transfected with p-CYP3A4-Luc in the presence of a hPXR vector or transfected with p-CYPA1-Luc.Our results clearly showed that ZEN activated human PXR, CAR and AhR mRNA levels in addition to some of their phase I target genes mainly CYP3A4, CYP2B6 and CYP1A1 and at lesser extent CYP3A5 and CYP2C9 at ZEN concentrations as low as 0.1 µM.
Assuntos
Estrogênios não Esteroides/farmacologia , Hepatócitos/metabolismo , RNA Mensageiro/genética , Receptores de Hidrocarboneto Arílico/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores de Esteroides/agonistas , Zearalenona/farmacologia , Trifosfato de Adenosina/metabolismo , Idoso , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Receptor Constitutivo de Androstano , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Primers do DNA , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Luciferases/metabolismo , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In vitro production of human hepatocytes is of primary importance in basic research, pharmacotoxicology and biotherapy of liver diseases. We have developed a protocol of differentiation of human embryonic stem cells (ES) towards hepatocyte-like cells (ES-Hep). Using a set of human adult markers including CAAT/enhancer binding protein (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 (CYP7A1), CYP3A4 and constitutive androstane receptor (CAR), and fetal markers including alpha-fetoprotein, CYP3A7 and glutathione S-transferase P1, we analyzed the expression of a panel of 41 genes in ES-Hep comparatively with human adult primary hepatocytes, adult and fetal liver. The data revealed that after 21 days of differentiation, ES-Hep are representative of fetal hepatocytes at less than 20 weeks of gestation. The glucocorticoid receptor pathway was functional in ES-Hep. Extending protocols of differentiation to 4 weeks did not improve cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal epithelial (NPE) cells (NPE-Hep), ES-Hep expressed several adult and fetal liver makers at much greater levels (at least one order of magnitude), consistent with greater expression of liver-enriched transcription factors Forkhead box A2, C/EBPalpha, HNF4alpha and HNF6. It therefore seems that ES-Hep reach a better level of differentiation than NPE-Hep and that these cells use different lineage pathways towards the hepatic phenotype. Finally we showed that lentivirus-mediated expression of xenoreceptor CAR in ES-Hep induced the expression of several detoxification genes including CYP2B6, CYP2C9, CYP3A4, UDP-glycosyltransferase 1A1, solute carriers 21A6, as well as biotransformation of midazolam, a CYP3A4-specific substrate.