RESUMO
Cholestasis is a pathophysiological process caused by the damage of hepatocytes or obstruction of bile flow, which often leads to emotional disorder in central nervous system. Alpha-naphthylisothiocyanate (ANIT) is the most widely used chemical to induce cholestatic models; however, the neurobehavior of ANIT-induced cholestatic model has not been investigated. The present study was designed to evaluate the anxiety-like behavior of cholestatic mice induced by a single (i.p.) injection of ANIT and its potential mechanism. For validating the model, the alanine aminotransferase, glutamic oxaloacetic transaminase, alkaline phosphatase, and total bile acid in the serum of mice were detected, and the pathological sections of hepatic lobes were also observed. After that, a series of behavioral tests were used to detect the anxiety-like behavioral changes of the ANIT-induced cholestatic mice, and then the level of 5-hydroxytryptamine and 5-hydroxyindole acetic acid in serum and prefrontal cortex were detected. Our data showed that ANIT-induced cholestatic mice exhibited increased anxiety-like behaviors in the open-field test and elevated plus maze test. Moreover, the concentration of 5-hydroxyindole acetic acid significantly decreased in the serum and the prefrontal cortex of ANIT-induced cholestatic mice compared with the control group. In addition, the expression of 5-hydroxytryptamine 1A, 5-hydroxytryptamine 2C, 5-hydroxytryptamine 3A, and 5-hydroxytryptamine 7 receptors increased in the prefrontal cortex of the model mice compared to their controls. Our results suggest that ANIT-induced cholestatic mice can display anxiety-like behavior closely related with enhanced serotoninergic signaling transduction in central nervous system.
Assuntos
1-Naftilisotiocianato/administração & dosagem , Ansiedade/induzido quimicamente , Ansiedade/metabolismo , Colestase/induzido quimicamente , Colestase/metabolismo , Serotonina/metabolismo , 1-Naftilisotiocianato/sangue , Animais , Ansiedade/complicações , Comportamento Animal/efeitos dos fármacos , Colestase/complicações , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Serotonina/sangue , Transdução de Sinais/efeitos dos fármacosRESUMO
Chronic glucocorticoid overexposure predisposes to the development of atherosclerotic cardiovascular disease in humans. Cholestatic liver disease is associated with increased plasma glucocorticoid levels. Here, we determined - in a preclinical setting - whether the chronic presence of cholestatic liver disease also induces a concomitant negative impact on atherosclerosis susceptibility. Hereto, regular chow diet-fed atherosclerosis-susceptible hypercholesterolemic apolipoprotein E (APOE)-knockout mice were treated with the bile duct toxicant alpha-naphthylisothiocyanate (ANIT) for 8 weeks. ANIT exposure induced the development of fibrotic cholestatic liver disease as evident from collagen deposits and compensatory bile duct hyperproliferation within the liver and the rise in plasma levels of bilirubin (+60%; P < 0.01) and bile acids (10-fold higher; P < 0.01). Adrenal weights (+22%; P < 0.01) and plasma corticosterone levels (+72%; P < 0.01) were increased in ANIT-treated mice. In contrast, atherosclerosis susceptibility was not increased in response to ANIT feeding, despite the concomitant increase in plasma free cholesterol (+30%; P < 0.01) and cholesteryl ester (+42%; P < 0.001) levels. The ANIT-induced hypercorticosteronemia coincided with marked immunosuppression as judged from the 50% reduction (P < 0.001) in circulating lymphocyte numbers. However, hepatic glucocorticoid signaling was not enhanced after ANIT treatment. It thus appears that the immunosuppressive effect of glucocorticoids is uncoupled from their metabolic effect under cholestatic disease conditions. In conclusion, we have shown that cholestatic liver disease-associated endogenous glucocorticoid overexposure does not increase atherosclerosis susceptibility in APOE-knockout mice. Our studies provide novel preclinical evidence for the observations that the hypercholesterolemia seen in cholestatic human subjects does not translate into a higher risk for atherosclerotic cardiovascular disease.
Assuntos
1-Naftilisotiocianato/efeitos adversos , Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Colestase/metabolismo , Glucocorticoides/metabolismo , 1-Naftilisotiocianato/administração & dosagem , Animais , Apolipoproteínas E/genética , Aterosclerose/diagnóstico , Aterosclerose/etiologia , Colestase/etiologia , Colesterol/sangue , Corticosterona/sangue , Modelos Animais de Doenças , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/etiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Risco , Especificidade da EspécieRESUMO
Plasma amino acid level changes occur in mild, moderate and severe stages of liver injury in human patients. In animal models, however, data are mainly restricted to severe liver injury models in rats. Here we present the characterization of a rat model of moderate liver dysfunction secondary to alpha-napthylisothiocyanate (ANIT)-induced cholestasis. Rats treated with 30 mg/kg/day ANIT for 3 weeks exhibited a time-dependent increase in plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin levels and a decrease in albumin concentration. According to a liver dysfunction evaluation based on the human Child-Pugh-Score, animals developed a moderate liver dysfunction in the first two weeks of ANIT treatment, while only a mild dysfunction was observed at the end of week 3 despite ongoing ANIT administration. Univariate analysis of branched-chain amino acid plasma levels indicated that reduced levels of branched chain amino acids were associated with the ANIT treatment. These data may set the stage for further research of amino acid disturbances and requirements in non-severe cholestasis.
Assuntos
1-Naftilisotiocianato/toxicidade , Aminoácidos de Cadeia Ramificada/sangue , Colestase/sangue , Colestase/induzido quimicamente , Modelos Animais de Doenças , Hepatopatias , 1-Naftilisotiocianato/administração & dosagem , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Colestase/complicações , Humanos , Hepatopatias/etiologia , Ratos Wistar , Albumina Sérica/metabolismo , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Promising biomarkers were identified in adult male Crl:CD (SD) rats for the screening of new chemical entities for their potential to cause liver injury. We examined the serum biochemistry, liver histopathology, and bile acid profiles by LC-MS/MS, and the mRNA expression of transporters and CYPs by an RT-PCR after the following treatments to male Crl:CD (SD) rats: (a) bile duct ligation (BDL); (b) a single oral dose of 150 mg/kg α-naphthylisothiocyanate (ANIT); and (c) repeated oral doses of a novel pyrrolidinecarboxylic acid derivative (abbreviated as PCA) at 30, 300, and 1000 mg/kg. The serum total bile acid levels and bilirubin concentrations were found to be elevated in all of the groups. However, the bile acid component profiles of the PCA group differed significantly from BDL and ANIT models: deoxycholic acid, lithocholic acid, and sulfated bile acids were upregulated in a dose-dependent manner only in the PCA group. In addition, the PCA group demonstrated high levels of hepatic heme oxygenase-1 expression, whereas the profiles of the mRNA levels of the hepatic transporters and CYPs of all groups were found to be similar. The histopathological findings, for both the BDL and ANIT groups, were of bile duct hyperplasia, hepatocyte degeneration and necrosis. In contrast, only bile duct hyperplasia and hepatocyte degeneration were observed in the PCA group, even at a lethal dose. These results indicated that PCA induced a cholestatic condition and the increase of oxidative stress markers implies that this will also lead hepatocellular injury. In conclusion, the serum bile acid components and sulfated bile acid levels, and the expression of oxidative stress markers could provide information that aids in the diagnosis of liver injury type and helps to elucidate the mechanisms of hepatotoxicity. These findings can be extrapolated into our clinical investigation. The analysis of these crucial biomarkers is likely to be a useful screening tool in the lead optimization phase of drug discovery.
Assuntos
1-Naftilisotiocianato/toxicidade , Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Fígado/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/toxicidade , 1-Naftilisotiocianato/administração & dosagem , Animais , Ácidos e Sais Biliares/sangue , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colestase/sangue , Colestase/induzido quimicamente , Colestase/genética , Colestase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Ácido Pirrolidonocarboxílico/administração & dosagem , Ácido Pirrolidonocarboxílico/análogos & derivados , Ratos , Ratos Sprague-DawleyRESUMO
Certain human diseases affecting the biliary tree can be modeled in rats by ingestion of the hepatobiliary toxin alpha-naphthylisothiocyanate (ANIT). Phosphorus magnetic resonance spectroscopy (MRS) allows the noninvasive monitoring of cell dynamics through detection of phosphodiesters (PDE) and phosphomonoesters (PME). Hepatic (31)P MRS techniques were therefore used to study the toxic effects of low-dose chronic ANIT ingestion, with a view toward providing biomarkers sensitive to hepatobiliary dysfunction and cholestatic liver injury. Rats were fed an ANIT supplemented diet at three doses (ANIT_0.05%, ANIT_0.04%, and ANIT_0.025%) for 2 weeks. Data from in vivo MRS were compared with results from pair-fed controls (PFCs). Blood and tissue samples were collected at 2 weeks for clinical chemistry, histology, and (1)H magic angle spinning MRS. Increases in PDE, relative to total phosphorus (tPh), were detected in both the ANIT_0.05% and ANIT_0.04% groups (0.07 ± 0.01 and 0.08 ± 0.01, respectively) relative to PFC groups (0.03 ± 0.01 and 0.05 ± 0.01, respectively). An increase in PME/tPh was observed in the ANIT_0.05% group only (0.17 ± 0.02) relative to PFC_0.05% (0.12 ± 0.01). Ex vivo (1)H MRS findings supported this, wherein measured phosphocholines (PCs) were increased in ANIT_0.05% and ANIT_0.04% groups. Increases in relative total choline (tCho) distinguished the ANIT_0.05% group from the ANIT_0.04% group. Markers of hepatotoxicity such as raised total bilirubin and alkaline phosphatase were found at all ANIT doses. Histological findings included a dose-related increase in both severity of biliary hyperplasia and focal hepatocellular necrosis. Here, we found that ANIT-induced moderate hepatobiliary dysfunction was associated with a relative increase in phosphodiesters in vivo and PCs ex vivo. Raised PME/tPh in vivo and tCho ex vivo were also present at high doses corresponding to a higher incidence of marked biliary hyperplasia and moderate hepatocellular necrosis.
Assuntos
1-Naftilisotiocianato/toxicidade , Fígado/efeitos dos fármacos , Espectroscopia de Ressonância Magnética/métodos , 1-Naftilisotiocianato/administração & dosagem , Análise de Variância , Animais , Peso Corporal , Comportamento Alimentar , Técnicas In Vitro , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Chronic cholestatic liver injury induced by cholestasis in rodents is associated with hepatic fibrin deposition, and we found evidence of fibrin deposition in livers of patients with cholestasis. Key components of the fibrinolytic pathway modulate cholestatic liver injury by regulating activation of hepatocyte growth factor. However, the exact role of hepatic fibrin deposition in chronic cholestasis is not known. We tested the hypothesis that fibrinogen (Fbg) deficiency worsens liver injury induced by cholestasis. Fbg-deficient mice (Fbgα(-/-) mice) and heterozygous control mice (Fbgα(+/-) mice) were fed either the control diet or a diet containing 0.025% α-naphthylisothiocyanate (ANIT), which selectively injures bile duct epithelial cells in the liver, for 2 weeks. Hepatic fibrin and collagen deposits were evident in livers of heterozygous control mice fed the ANIT diet. Complete Fbg deficiency was associated with elevated serum bile acids, periportal necrosis, and increased serum alanine aminotransferase activity in mice fed the ANIT diet. Fbg deficiency was associated with enhanced hepatic expression of the transcription factor early growth response-1 (Egr-1) and enhanced induction of genes encoding the Egr-1-regulated proinflammatory chemokines monocyte chemotactic protein-1, KC growth-regulated protein, and macrophage inflammatory protein-2. Interestingly, peribiliary collagen deposition was not evident near necrotic areas in Fbg-deficient mice. The results suggest that in this model of chronic cholestasis, fibrin constrains the release of bile constituents from injured intrahepatic bile ducts, thereby limiting the progression of hepatic inflammation and hepatocellular injury.
Assuntos
Afibrinogenemia/complicações , Afibrinogenemia/metabolismo , Colestase/complicações , Colestase/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibrinogênio/metabolismo , Fígado/patologia , 1-Naftilisotiocianato/administração & dosagem , Afibrinogenemia/patologia , Idoso , Animais , Ductos Biliares/patologia , Colestase/induzido quimicamente , Colestase/patologia , Doença Crônica , Colágeno/metabolismo , Dieta , Modelos Animais de Doenças , Comportamento Alimentar , Feminino , Fibrina/deficiência , Fibrina/metabolismo , Regulação da Expressão Gênica , Humanos , Hiperplasia , Inflamação/complicações , Inflamação/genética , Inflamação/patologia , Fígado/metabolismo , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/metabolismo , XenobióticosRESUMO
Urinary metabolic fingerprinting with Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) was performed to monitor metabolic changes in an alpha-naphthylisothiocyanate (ANIT)-induced rat model of intrahepatic cholestasis and to investigate the relationships among metabolic changes, histopathology, and blood chemistry. ANIT was administered orally as a single dose of 100 mg/kg. Urine samples were collected predose (-31 to -24 hours) and postdose at 0-7, 7-24, 24-31, 31-48, 48-55, 55-72, and 72-96 hours, and serum samples were collected on days 1, 2, and 4 postdose. Increased levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin were seen on day 2. The negative ion profiles for urine samples collected after 7-24, 24-31, 31-48, and 48-55 hours differed from the predose profile based on principal component analysis. Onset of recovery was observed after 24-31 hours, when the urinary composition reverted toward the predose position. In conclusion, it is possible to monitor the progression of and recovery from drug-induced hepatotoxicity by urinary metabolic fingerprinting with FT-ICR MS and to search for potential biomarkers involved in intrahepatic cholestasis.
Assuntos
1-Naftilisotiocianato/toxicidade , Biomarcadores/urina , Colestase Intra-Hepática/induzido quimicamente , 1-Naftilisotiocianato/administração & dosagem , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Biomarcadores/química , Colestase Intra-Hepática/sangue , Colestase Intra-Hepática/patologia , Colestase Intra-Hepática/urina , Ciclotrons , Análise de Fourier , Fígado/patologia , Masculino , Espectrometria de Massas , Noxas/administração & dosagem , Noxas/toxicidade , Análise de Componente Principal , RatosRESUMO
There is an increasing body of evidence that the central nervous system is affected by cholestatic liver disorders. Cholestasis has been shown to result in a decreased seizure propensity which is believed to be mediated by an increased opioidergic tone and nitric oxide (NO) signaling pathway. In this study, we used a reversible chemically-induced cholestasis model in mice to investigate the changes in seizure susceptibility. The cholestasis was induced by intragastric administration of alpha-naphthylisothiocyanate (ANIT) (100 mg/kg) or vehicle (corn oil). The threshold to generalized clonic seizures induced by timed intravenous infusion of pentylenetetrazole (PTZ) was used as an index of seizure propensity. The role of opioid receptors and NO pathway in the changes of seizure threshold, and the responsiveness to the anticonvulsant effect of opioid agonist, morphine, during and after the resolution of cholestasis was studied in this reversible paradigm of cholestatic disease. A significant increase in cholestasis-related biochemical markers as well as in clonic seizure threshold was observed; it was maximal at day 3 after cholestasis induction and slowly decreased to normal thereafter. Seizure threshold rise was inhibited by chronic administration of the opioid antagonist naltrexone or acute administration of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO production. Co-administration of subeffective doses of L-NAME and naltrexone showed an additive effect. Injection of an anticonvulsant dose of morphine on day 7 after cholestasis induction did not increase seizure threshold, suggestive of a downregulation of receptors even after cholestasis resolution. These data shows that ANIT-induced cholestasis leads to a reversible increased resistance to PTZ-induced seizures through an opioid/NO-mediated pathway, and is probably accompanied by downregulation of opioid receptors.
Assuntos
Colestase/fisiopatologia , Óxido Nítrico/fisiologia , Receptores Opioides/fisiologia , Convulsões/fisiopatologia , 1-Naftilisotiocianato/administração & dosagem , 1-Naftilisotiocianato/toxicidade , Fosfatase Alcalina/sangue , Animais , Bilirrubina/sangue , Biomarcadores/sangue , Colestase/induzido quimicamente , Colestase/prevenção & controle , Óleo de Milho/administração & dosagem , Óleo de Milho/química , Modelos Animais de Doenças , Suscetibilidade a Doenças/fisiopatologia , Relação Dose-Resposta a Droga , Infusões Intravenosas , Intubação Gastrointestinal , Masculino , Camundongos , Morfina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , NG-Nitroarginina Metil Éster/uso terapêutico , Naltrexona/farmacologia , Antagonistas de Entorpecentes , Óxido Nítrico/antagonistas & inibidores , Pentilenotetrazol/administração & dosagem , Pentilenotetrazol/toxicidade , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Fatores de TempoRESUMO
Many naturally occurring and synthetic isothiocyanates can inhibit chemical carcinogenesis in animal models. Recently, we found that alpha-naphthyl isothiocyanate (1-NITC) inhibited P-glycoprotein- and multidrug resistance associated protein 1-mediated efflux, indicating the potential application of 1-NITC as a chemosensitizing agent for cancer chemotherapy. The objective of this study was to explore the pharmacokinetic characteristics of 1-NITC in rats. A single dose of 10, 25, 50, or 75 mg/kg of 1-NITC was administered intravenously or orally to female Sprague-Dawley rats (n = 4 for each group). Dose-normalized concentration-time profiles were not superimposable following intravenous or oral dosing, indicating that the disposition of 1-NITC in rats was nonlinear. As doses increased from 10 to 75 mg/kg following iv administration, the total clearance decreased from 2.2 +/- 0.9 to 0.8 +/- 0.3 L/h/kg; oral availability averaged 0.46 for oral doses of 10-75 mg/kg. A nonlinear two-compartment open model with capacity-limited absorption and capacity-limited elimination from the central compartment best fit the data, based on goodness-of-fit criteria. The mechanism underlying the nonlinear elimination of 1-NITC in rats is most likely due to the capacity-limited metabolism of 1-NITC. This study represents the first report of the pharmacokinetics of 1-NITC.
Assuntos
1-Naftilisotiocianato/farmacocinética , 1-Naftilisotiocianato/administração & dosagem , 1-Naftilisotiocianato/sangue , Administração Oral , Animais , Área Sob a Curva , Feminino , Meia-Vida , Injeções Intravenosas , Taxa de Depuração Metabólica , Modelos Biológicos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND AIMS: alpha-Naphthylisothiocyanate (ANIT) is known to cause cholestasis due to injury of the bile duct epithelial cells. The aim of the present study was to examine the effect of a single dose of ANIT on the biliary excretion of various cholephilic compounds and on the amount of canalicular transporters. METHODS: Twenty-four hours after the oral administration of ANIT (100 mg/kg), the biliary excretion of taurocholate, leukotriene C(4), pravastatin and vinblastine was studied. The protein levels of the bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver were also examined. RESULTS: The ANIT treatment markedly decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C(4), pravastatin and vinblastine. The biliary excretory maximum of taurocholate was also markedly decreased after ANIT treatment. The ANIT treatment had no effect on the protein levels of bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver. CONCLUSIONS: These findings support canalicular transporters having little effect on the marked impairment of biliary excretion of cholephilic compounds in ANIT-induced cholestasis.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Ácidos e Sais Biliares/metabolismo , Bile/metabolismo , Colagogos e Coleréticos/farmacocinética , Colestase/metabolismo , Ácido Taurocólico/farmacocinética , Vimblastina/farmacocinética , 1-Naftilisotiocianato/administração & dosagem , 1-Naftilisotiocianato/toxicidade , Administração Oral , Animais , Ânions , Transporte Biológico/efeitos dos fármacos , Colestase/induzido quimicamente , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: and aims: Primary sclerosing cholangitis is a Th1 cytokine driven disease with a poor clinical responsiveness to glucocorticoid therapy. We have previously documented elevated circulating glucocorticoid levels in cholestatic rats and in addition have noted increased hepatic expression of the Th1 cytokine interferon gamma (IFN-gamma) in a rat model of cholangitis. Therefore, we examined the relationship between circulating glucocorticoid levels, hepatic IFN-gamma expression, and hepatic T cell glucocorticoid receptor (GR) expression in a rat model of cholangitis to provide insight into the possible mechanism underlying hepatic T cell glucocorticoid resistance in cholangitic diseases. METHODS: Cholangitis was induced in male Sprague-Dawley rats by oral administration of low dose alpha-naphthylisothiocyanate (ANIT). On day 14, ANIT fed and control rats were sacrificed, serum collected, and hepatic, splenic, and peripheral blood T lymphocytes isolated for GR expression, as determined by reverse transcription-polymerase chain reaction and western blotting. RESULTS: Circulating glucocorticoid levels were markedly elevated in ANIT fed rats. Hepatic T lymphocyte GR mRNA and protein levels were significantly reduced in ANIT treated rats compared with controls. In contrast, GR mRNA and protein expression in splenic and circulating T lymphocytes was similar in both groups. Furthermore, reduced hepatic T cell GR expression in ANIT fed rats was associated with reduced hepatic CD4(+) T cell sensitivity to dexamethasone inhibitory effects (that is, inhibition of interleukin 2 receptor expression). CONCLUSION: We conclude that hepatic T lymphocyte resistance to elevated endogenous glucocorticoid levels in rats with experimental cholangitis appears, in part, to be mediated by decreased GR expression.
Assuntos
Colangite Esclerosante/metabolismo , Corticosterona/sangue , Desidroepiandrosterona/sangue , Receptores de Glucocorticoides/metabolismo , Linfócitos T/metabolismo , 1-Naftilisotiocianato/administração & dosagem , 1-Naftilisotiocianato/efeitos adversos , Animais , Colangite Esclerosante/induzido quimicamente , Doença Crônica , Modelos Animais de Doenças , Regulação para Baixo , Citometria de Fluxo , Interferon gama/metabolismo , Interleucina-4/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Chronic cholestasis is characteristic of many human liver diseases. Renal injury has been often associated with this type of disease. The aim of this study was to evaluate the effect of cholestasis on kidney mitochondrial bioenergetics following in vivo chronic administration of alpha-naphthyl-isothiocyanate (ANIT), a known cholestatic agent. Serum markers of renal injury, kidney morphology and endogenous adenine nucleotides were measured in ANIT-treated rats (80 mg/kg per week s.c. for 16 weeks). Changes in membrane potential and mitochondrial respiration as well as alterations in mitochondrial calcium homeostasis were monitored. Cholestatic animals shown no alterations in renal morphology when compared with control. Additionally, following chronic ANIT administration, no significant alterations in mitochondrial respiratory function have been shown. The phosphorylation capacity of cholestatic kidney mitochondria was enhanced. Associated with these parameters, mitochondria from treated animals exhibited a decreased susceptibility to disruption of mitochondrial calcium homeostasis, due to permeability transition induction. These data suggest that, despite being submitted to chronic treatment with ANIT, kidney mitochondria from cholestasis-induced rats present some defense mechanisms to circumvent this aggression. They show improved phosphorylative capacity and, moreover, a decreased susceptibility to mitochondrial permeability transition induction, probably due to adaptative mechanisms of calcium transport.
Assuntos
1-Naftilisotiocianato/toxicidade , Colestase Intra-Hepática/induzido quimicamente , Metabolismo Energético/efeitos dos fármacos , Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , 1-Naftilisotiocianato/administração & dosagem , Nucleotídeos de Adenina/metabolismo , Animais , Cálcio/metabolismo , Colestase Intra-Hepática/metabolismo , Colestase Intra-Hepática/patologia , Modelos Animais de Doenças , Feminino , Injeções Intraperitoneais , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Rim/metabolismo , Rim/patologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Fosforilação , Ratos , Ratos WistarRESUMO
As part of a wider metabonomic investigation into the early detection and discrimination of site-specific hepatotoxicity, male Sprague-Dawley rats were dosed with the model hepatotoxins allyl formate, ethionine and alpha-naphthylisothiocyanate (ANIT). Urine samples collected pre- and post-dose were examined by (1)H nuclear magnetic resonance (NMR) spectroscopy and the toxin-induced changes in urinary taurine and creatine excretion were quantified. Hypertaurinuria and hypercreatinuria were observed following allyl formate dosing, hypertaurinuria with no change in creatine excretion was observed after ethionine dosing, and hypotaurinuria and hypercreatinuria were observed after ANIT dosing. These changes are indicative of different effects on liver and it has been previously suggested that some hepatotoxin-induced changes in urinary taurine excretion may be due to altered hepatic cysteine utilisation. A related hypothesis is now presented that would explain the selective hypercreatinuria in terms of increased cysteine synthesis.
Assuntos
1-Naftilisotiocianato/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/urina , Creatinina/urina , Cisteína/biossíntese , Etionina/toxicidade , Ésteres do Ácido Fórmico/toxicidade , 1-Naftilisotiocianato/administração & dosagem , Administração Oral , Animais , Modelos Animais de Doenças , Etionina/administração & dosagem , Ésteres do Ácido Fórmico/administração & dosagem , Injeções Intraperitoneais , Espectroscopia de Ressonância Magnética , Masculino , Ratos , Ratos Sprague-Dawley , Taurina/urinaRESUMO
OBJECTIVES: A growing number of reports in the literature have been correlating cardiovascular alterations with the presence of chronic liver disease, such as cirrhosis and cholestasis. The objective of this work was to compare mitochondrial bioenergetics and calcium loading capacity in the hearts of rats injected with alpha-naphthylisothiocyanate (ANIT), a compound used to induce cholestasis in animal models. METHODS: Female Wistar rats (n = 12) were randomly divided into two groups. One of the groups was injected with six weekly doses of ANIT. Each group was evaluated in terms of mitochondrial bioenergetic capacity and susceptibility to the mitochondrial permeability transition (MPT), a deleterious phenomenon associated with oxidative stress and excessive mitochondrial calcium accumulation. RESULTS: Our data showed unequivocally that cardiac mitochondria of rats chronically injected with ANIT lost their ability to accumulate calcium, in a cyclosporin A sensitive manner. This was reflected in a higher calcium-dependent swelling rate and amplitude (p < 0.01). The RCI value, an index of mitochondrial integrity, was also lower in the ANIT-treated group (p < 0.05). CONCLUSIONS: This suggests that during cholestasis development, cardiac mitochondria lose their normal ability to control cytosolic calcium due to increased susceptibility to the MPT. Our results may suggest an explanation for the occurrence of cardiomyopathies associated with cholestatic disease, which may persist even in the absence of serum markers for liver disease.
Assuntos
Cálcio/metabolismo , Colestase/fisiopatologia , Mitocôndrias Cardíacas/fisiologia , 1-Naftilisotiocianato/administração & dosagem , Animais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Colestase/induzido quimicamente , Colestase/complicações , Doença Crônica , Ciclosporina/farmacologia , Modelos Animais de Doenças , Feminino , Fibrose/complicações , Fibrose/fisiopatologia , Hepatopatias/complicações , Hepatopatias/fisiopatologia , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
BACKGROUND: Hepatocyte dysfunction caused by impaired mitochondrial function has been pointed out as a probable leading cause of cholestatic liver injury. The aim of this study was to evaluate liver mitochondrial bioenergetics that followed repeated in vivo administration of alpha-naphthylisothiocyanate, a known cholestatic agent. METHODS: Serum markers of liver injury and endogenous adenine nucleotides were measured in alpha-naphthylisothiocyanate-treated rats (intraperitoneally, 100 mg/Kg/wk x 6 wk). Changes in membrane potential, mitochondrial respiration, as well as alterations in mitochondrial calcium homeostasis were monitored. RESULTS: In rats injected with alpha-naphthylisothiocyanate, liver injury with cholestasis developed within 48 hours, as indicated by both serum enzyme activities and total bilirubin concentration. However, 1 week after the last injection, serum enzyme activity returned to control levels. In addition, after chronic alpha-naphthylisothiocyanate administration, no alterations in mitochondrial respiratory function and membrane potential were observed. Associated with these parameters, mitochondria from treated animals exhibited increased susceptibility to disruption of mitochondrial calcium homeostasis by calcium phosphate and by bile acids, which was probably caused by induction of permeability transition pore. CONCLUSIONS: Our data suggest that chronic cholestasis in rats leads to impaired mitochondrial function due to the disruption of mitochondrial calcium homeostasis. The initiating event is the induction of a cyclosporine A-sensitive release of calcium. This event may be an important determinant of the progression of cholestatic liver injury and associated liver cirrhosis. In addition, in the present study we observed that impairment of mitochondrial function is potentiated by chenodeoxycholate, a bile acid that is known to be toxic. Ursodeoxycholate (the beta- epimer of chenodeoxycholate) is approved for the treatment of chronic cholestatic liver disease. Interestingly, we show that the susceptibility to the cyclosporine A-sensitive release of calcium was increased by the combination of both bile acids. These results indicate that the reported improvement of biochemical parameters in cholestatic patients treated with ursodeoxycholate would not prevent the associated mitochondrial dysfunction. This may explain the progression of the histological stage and the maintenance of symptoms during cholestasis.
Assuntos
Cálcio/metabolismo , Colestase/metabolismo , Mitocôndrias Hepáticas/metabolismo , 1-Naftilisotiocianato/administração & dosagem , 1-Naftilisotiocianato/toxicidade , Nucleotídeos de Adenina/metabolismo , Alanina Transaminase/sangue , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ácido Quenodesoxicólico/toxicidade , Colestase/induzido quimicamente , Metabolismo Energético/efeitos dos fármacos , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Homeostase/fisiologia , Injeções Intraperitoneais , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The hepatoprotective effects of Tao-shang-tsao, Ixeris laevigata Sch.-Bip. var. oldhami Kitam., were studied on cholestatic hepatitis induced by alpha-naphthylisothiocyanate (ANIT, 100 mg/10 ml/kg, in olive oil, i.p.) and acute hepatitis induced by carbon tetrachloride (20% CCl4/olive oil, 1.5 ml/kg, i.p.) in rats by post-treatment with the crude methanolic extracts of I. laevigata (0.3, 1.0, and 2.0 g/kg) orally in the therapeutic model. Hepatoprotective activity was monitored by estimating the serum transaminases concentrations and histopathological changes in the livers of experimental rats. It was found that the I. laevigata extract significantly decreased the acute elevation of serum transaminases by biochemical examination. According to pathohistological studies in the liver of experimental rats, the crude I. laevigata extract ameliorates the central necrosis, fatty change or proliferation of bile duct epithelium focal necrosis caused by ANIT or CCl4-induced hepatitis rats.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Hepatopatias/tratamento farmacológico , Fígado/efeitos dos fármacos , 1-Naftilisotiocianato/administração & dosagem , Animais , Tetracloreto de Carbono/farmacologia , Colangite/induzido quimicamente , Hiperbilirrubinemia/induzido quimicamente , Masculino , Extratos Vegetais , Ratos , Ratos WistarRESUMO
An association between lipid peroxidation and alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). The severity of liver injury was estimated 12, 24, 48, and 72 h after ANIT injection. Liver injury appeared 24 h after ANIT injection, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Serum lipid peroxide (LPO) concentration increased 24 h after ANIT injection and further increased at 48 h, but this increase was attenuated at 72 h. In contrast, liver LPO content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Similarly, myeloperoxidase (MPO) activity, an index of neutrophil infiltration, in the liver tissue increased 12 h after ANIT injection and further increased at 24 and 48 h, but this increase was attenuated at 72 h. Either serum LPO concentration or liver LPO content was significantly correlated with liver MPO activity (r = 0.661 for serum LPO concentration; r = 0.585 for liver LPO content). These results suggest that lipid peroxidation might be associated with ANIT-induced liver injury in rats and that this lipid peroxidation might occur via oxygen radicals derived from neutrophils infiltrated into the liver tissue of ANIT-intoxicated rats.
Assuntos
1-Naftilisotiocianato/efeitos adversos , Colestase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , 1-Naftilisotiocianato/administração & dosagem , Alanina Transaminase/sangue , Alanina Transaminase/efeitos dos fármacos , Animais , Ácidos e Sais Biliares/sangue , Bilirrubina/sangue , Peso Corporal/efeitos dos fármacos , Colestase/induzido quimicamente , Colestase/patologia , Injeções Intraperitoneais , Peróxidos Lipídicos/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Ratos , Ratos Wistar , Estatística como Assunto , gama-Glutamiltransferase/sangue , gama-Glutamiltransferase/efeitos dos fármacosRESUMO
The change in hepatic antioxidant defense system with the development of alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). Liver injury with cholestasis did not occur 12 h after ANIT injection, but appeared at 24 h, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Liver lipid peroxide content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Liver superoxide dismutase and catalase activities decreased 24 and 48 h, respectively, after ANIT injection, although the catalase activity increased at 12 h, but these decreases were attenuated at 72 h. Liver Se-glutathione peroxidase activity remained unchanged 24, 48, and 72 h after ANIT injection, although the activity increased at 12 h. Liver reduced glutathione content increased 24 h after ANIT injection, but the increase was reduced time dependently thereafter. Liver ascorbic acid content increased 12 h after ANIT injection and further increased at 24 h, but the increase was reduced time dependently thereafter. These results indicate that the change in hepatic antioxidant defense system occurs before and with the development of ANIT-induced liver injury in rats, and suggest that the reduction of hepatic antioxidant defense system mediated by SOD and catalase could contribute to the liver injury development through an enhancement of hepatic lipid peroxidation.
Assuntos
1-Naftilisotiocianato/administração & dosagem , 1-Naftilisotiocianato/toxicidade , Antioxidantes/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Ácido Ascórbico/sangue , Ácido Ascórbico/metabolismo , Ácidos e Sais Biliares/sangue , Bilirrubina/sangue , Bovinos , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colangite/sangue , Colangite/induzido quimicamente , Colangite/enzimologia , Colangite/metabolismo , Esquema de Medicação , Glutationa/sangue , Glutationa/metabolismo , Injeções Intraperitoneais , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Masculino , Ratos , Ratos Wistar , Selênio/metabolismoRESUMO
The importance of the bioactivation of 1-naphthylisothiocyanate was studied. Forty minutes after 1-naphthylisothiocyanate administration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1-naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1-naphthyl isocyanate, 1-naphthylamine and the parent compound, 1-naphthylisothiocyanate. When rats were given cycloheximide, which attenuates 1-naphthylisothiocyanate toxicity, 30 min before 1-naphthylisothiocyanate (300 mg/kg), 1-naphthyl isocyanate concentration was significantly lower than in rats receiving only 1-naphthylisothiocyanate. The appearance of 1-naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepatotoxicity, enhanced 1-naphthyl isocyanate and 1-naphthylamine formation. It is suggested that 1-naphthyl isocyanate, 1-naphthylamine and the highly reactive sulfur released from 1-naphthylisothiocyanate might be involved in the hepatotoxic effect of 1-naphthylisothiocyanate.
Assuntos
1-Naftilamina/metabolismo , 1-Naftilisotiocianato/farmacocinética , Bile/metabolismo , Fígado/metabolismo , 1-Naftilisotiocianato/administração & dosagem , Animais , Bile/efeitos dos fármacos , Biotransformação , Cicloeximida/farmacologia , Fígado/efeitos dos fármacos , Masculino , Fenobarbital/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
The blood-brain-barrier plays an essential role in regulating the entrance of substances into the brain. To date, permeability of the blood-brain barrier has not been studied in models of cholestatic liver injury, although levels of substances known to enhance vascular permeability (bile acids, substance P, histamine) are elevated in cholestasis. Two rat models of cholestasis were studied: bile duct resection (5 days after surgery) and alpha-naphthylisothiocyanate treatment (45 mg/kg/day for 7 days). The mean value for whole brain blood-to-brain transfer constant in bile duct resection rats was about 50% less than corresponding values in sham-operated and unoperated control rats (p < or = 0.05, respectively). Reductions in blood-to-brain transfer constant of similar magnitude were found in the caudate nuclei, cortexes and hippocampi of bile duct-resected rats. Blood-to-brain transfer constant values in alpha-naphthylisothiocyanate-treated rats were also about 50% less in whole brain and specific brain regions than corresponding control values. A precedent for a decrease in blood-to-brain transfer constant is the dexamethasone-treated rat, in which the phenomenon has been attributed to a decrease in cerebral capillary endothelial cell membrane fluidity. We confirmed that blood-to-brain transfer constant values are reduced by about 50% in dexamethasone-treated rats. A decrease in membrane fluidity affords a rational explanation for a decrease in blood-to-brain transfer constant in cholestasis as a consequence of the dynamic equilibrium between elevated plasma levels of cholesterol in cholestasis and cell membranes exposed to the circulation.