RESUMEN
The biological membranes are important in cell function but, during development of diseases such as diabetes, they are impaired. Consequently, membrane-associated biological processes are impaired as well. The mitochondria are important organelles where oxidative phosphorylation takes place, a process closely related with the membranes. In general, it is accepted that the development process of diabetes decreases membrane fluidity. However, in some cases, it has been found to increase membrane fluidity of mitochondria but to decrease the Respiratory Control (RC) index. In this study we found an increase of membrane fluidity and an increase of the RC at an early phase of the development of a type 2 diabetes model. We measured the lipoperoxidation, analyzed the fatty acids composition by gas chromatography, and assessed membrane fluidity using three fluorescent monitors located at different depths inside the bilayer, dipyrenilpropane (DPyP), diphenylhexatriene (DPH), and trimethylammonium diphenylhexatriene (TMA-DPH). Our findings indicate that in the initial stage of diabetes development, when lipoperoxidation still is not significant, the membrane fluidity of liver mitochondria increases because of the increment in the unsaturated to saturated fatty acids ratio (U/S), thus producing an increase of the RC. The membrane fluidity is not the same at all depths in the bilayer. Contrary to the results obtained in mitochondria, the diabetes induced a decrease in the U/S fatty acids ratio of liver total lipids, indicating that the mitochondria might have an independent mechanism for regulating its fatty acids composition.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Fluidez de la Membrana , Mitocondrias Hepáticas/ultraestructura , Animales , Respiración de la Célula , Ácidos Grasos/análisis , Peróxidos Lipídicos/análisis , Mitocondrias Hepáticas/química , Membranas Mitocondriales , Fosforilación Oxidativa , Ratas WistarRESUMEN
Organoseleno-compounds have been investigated for its beneficial effects against methylmercury toxicity. In this way, diphenyl diselenide (PhSe)2 was demonstrated to decrease Hg accumulation in mice, protect against MeHg-induced mitochondrial dysfunction, and protect against the overall toxicity of this metal. In the present study we aimed to investigate if co-treatment with (PhSe)2 and MeHg could decrease accumulation of Hg in liver slices of rats. Rat liver slices were co-treated with (PhSe)2 (0.5; 5 µM) and/or MeHg (25 µM) for 30 min at 37 °C and Se and Hg levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) in the slices homogenate, P1 fraction, mitochondria and incubation medium. Co-treatment with (PhSe)2 and MeHg did not significantly alter Se levels in any of the samples when compared with compounds alone. In addition, co-treatment with (PhSe)2 and MeHg did not decrease Hg levels in any of the samples tested, although, co-incubation significantly increased Hg levels in homogenate. We suggest here that (PhSe)2 could exert its previously demonstrated protective effects not by reducing MeHg levels, but forming a complex with MeHg avoiding it to bind to critical molecules in cell.
Asunto(s)
Derivados del Benceno/farmacología , Hígado/química , Hígado/efectos de los fármacos , Mercurio/análisis , Compuestos de Metilmercurio/farmacología , Compuestos de Organoselenio/farmacología , Selenio/análisis , Animales , Derivados del Benceno/administración & dosificación , Masculino , Espectrometría de Masas , Compuestos de Metilmercurio/administración & dosificación , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Compuestos de Organoselenio/administración & dosificación , Ratas , Ratas WistarRESUMEN
The purpose of this work was to evaluate if the fat liver accumulation interferes with intracellular calcium fluxes and the liver glycogenolytic response to a calcium-mobilizing α(1)-adrenergic agonist, phenylephrine. The animal model of monosodium L-glutamate (MSG)-induced obesity was used. The adult rats develop obesity and steatosis. Calcium fluxes were evaluated through measuring the (45)Ca(2+) uptake by liver microsomes, inside-out plasma membrane, and mitochondria. In the liver, assessments were performed on the calcium-dependent glycogenolytic response to phenylephrine and the glycogen contents. The Ca(2+) uptake by microsomes and plasma membrane vesicles was reduced in livers from obese rats as a result of reduction in the Ca(2+)-ATPase activities. In addition, the plasma membrane Na(+)/K(+)-ATPase was reduced. All these matched effects could contribute to elevated resting intracellular calcium levels in the hepatocytes. Livers from obese rats, albeit smaller and with similar glycogen contents to those of control rats, released higher amounts of glucose in response to phenylephrine infusion, which corroborates these observations. Mitochondria from obese rats exhibited a higher capacity of retaining calcium, a phenomenon that could be attributed to a minor susceptibility of the mitochondrial permeability transition pore opening.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Microsomas Hepáticos/metabolismo , Mitocondrias Hepáticas/metabolismo , Obesidad/metabolismo , Obesidad/patología , Adenosina Trifosfato/farmacología , Animales , Animales Recién Nacidos , Membrana Celular/efectos de los fármacos , Membrana Celular/patología , Glucogenólisis/efectos de los fármacos , Glucogenólisis/fisiología , Magnesio/análisis , Magnesio/metabolismo , Magnesio/farmacología , Masculino , Microsomas Hepáticos/química , Microsomas Hepáticos/efectos de los fármacos , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/efectos de los fármacos , Obesidad/inducido químicamente , Fenilefrina/farmacología , Ratas , Ratas Wistar , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Vesículas Secretoras/patología , Glutamato de Sodio , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismoRESUMEN
PURPOSE: The main aim of this study was to determine the influence of ischemic preconditioning (IPC) on rat liver cirrhosis. METHODS: Cirrhosis was induced in Wistar rats by occlusion of the hepatic duct. The animals were divided into four groups of six animals each: non-cirrhotic group (simulated operation only), cirrhotic control group (simulated operation in cirrhotic rats), I/R group (40-minute ischemia without IPC), and IPC group (cirrhotic rats with ischemia, previously submitted to IPC). The IPC procedure consisted of partial hepatic ischemia for five minutes, followed by 10 minutes of reperfusion. In the case of the IPC group, the animals were submitted to liver ischemia for 40 minutes after the preconditioning procedure, followed by 2 hours of reperfusion. Blood samples were collected for measurement of serum aminotransferases (ALT and AST). The respiratory control ratio (RCR), the mitochondrial membrane potential (MMP), and malondialdehyde (MDA) values in the hepatic tissue were analyzed. Nonparametric statistical analysis was used and a value of p<0.05 was considered statistically significant. RESULTS: Ischemia did not induce significant increase in ALT and AST levels. MDA values were significantly higher in cirrhotic animals. MMP did not significantly change in cirrhosis and liver ischemia. Mitochondrial RCR decreased in liver cirrhosis, accentuated upon liver ischemia, and did not significantly change with IPC. CONCLUSION: Ischemic preconditioning does not protect the liver from hepatic injury induced by the ischemia/reperfusion process.
Asunto(s)
Precondicionamiento Isquémico , Cirrosis Hepática Experimental/metabolismo , Hígado/irrigación sanguínea , Daño por Reperfusión/metabolismo , Animales , Hígado/química , Pruebas de Función Hepática , Masculino , Malondialdehído/análisis , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Hepáticas/química , Ratas , Ratas Wistar , Transaminasas/sangreRESUMEN
PURPOSE: The main aim of this study was to determine the influence of ischemic preconditioning (IPC) on rat liver cirrhosis. METHODS: Cirrhosis was induced in Wistar rats by occlusion of the hepatic duct. The animals were divided into four groups of six animals each: non-cirrhotic group (simulated operation only), cirrhotic control group (simulated operation in cirrhotic rats), I/R group (40-minute ischemia without IPC), and IPC group (cirrhotic rats with ischemia, previously submitted to IPC). The IPC procedure consisted of partial hepatic ischemia for five minutes, followed by 10 minutes of reperfusion. In the case of the IPC group, the animals were submitted to liver ischemia for 40 minutes after the preconditioning procedure, followed by 2 hours of reperfusion. Blood samples were collected for measurement of serum aminotransferases (ALT and AST). The respiratory control ratio (RCR), the mitochondrial membrane potential (MMP), and malondialdehyde (MDA) values in the hepatic tissue were analyzed. Nonparametric statistical analysis was used and a value of p<0.05 was considered statistically significant. RESULTS: Ischemia did not induce significant increase in ALT and AST levels. MDA values were significantly higher in cirrhotic animals. MMP did not significantly change in cirrhosis and liver ischemia. Mitochondrial RCR decreased in liver cirrhosis, accentuated upon liver ischemia, and did not significantly change with IPC. CONCLUSION: Ischemic preconditioning does not protect the liver from hepatic injury induced by the ischemia/ reperfusion process.
OBJETIVO: O objetivo deste estudo foi determinar a influência do pré-condicionamento isquêmico (IPC) em fígados de ratos cirróticos. MÉTODOS: A cirrose hepática foi induzida em ratos Wistar machos (250 a 300g) por oclusão, durante 30 dias, do ducto hepático comum.A seguir, os animais cirróticos foram divididos em três grupos de seis; Grupo controle cirrótico (operação simulada para isquemia/reperfusão/pré-condicionamento), Grupo I/R, isquemia de 40 minutos sem pré-condicionamento (IPC) e grupo IPC com isquemia precedida por IPC. O IPC consistiu de uma isquemia parcial por cinco minutos, seguida por 10 minutos de reperfusão. No grupo IPC, após o pré-condicionamento, os animais foram submetidos à isquemia hepática de 40 minutos seguida de 2 horas de reperfusão. Foram colhidas amostras de sangue para dosagem sérica de aminotransferases (ALT e AST). Razão de controle respiratório (RCR), potencial de membrana das mitocôndrias (PMM) e dosagem de malondialdeído (MDA) foram analisadas no tecido hepático. Analise estatística não paramétrica foi utilizada com nível de significância de 5 por cento para as comparações entre grupos. RESULTADOS: A isquemia não induziu aumento significativo das aminotransferases. Houve aumento significativo de MDA nos animais cirróticos.O PMM não se alterou significativamente na cirrose e na isquemia hepática. A RCR mitocondrial diminuiu na cirrose hepática e acentuou-se com a isquemia do fígado e não se alterou significativamente com o IPC. CONCLUSÃO: O pré-condicionamento isquêmico não protegeu o fígado das lesões hepáticas induzidas pelo processo de isquemia/reperfusão.
Asunto(s)
Animales , Masculino , Ratas , Precondicionamiento Isquémico , Cirrosis Hepática Experimental/metabolismo , Hígado/irrigación sanguínea , Daño por Reperfusión/metabolismo , Modelos Animales de Enfermedad , Pruebas de Función Hepática , Hígado/química , Malondialdehído/análisis , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Hepáticas/química , Ratas Wistar , Estadísticas no Paramétricas , Transaminasas/sangreRESUMEN
Atherosclerotic disease remains a leading cause of death in westernized societies, and reactive oxygen species (ROS) play a pivotal role in atherogenesis. Mitochondria are the main intracellular sites of ROS generation and are also targets for oxidative damage. Here, we show that mitochondria from atherosclerosis-prone, hypercholesterolemic low-density lipoprotein (LDL) receptor knockout mice have oxidative phosphorylation efficiency similar to that from control mice but have a higher net production of ROS and susceptibility to develop membrane permeability transition. Increased ROS production was observed in mitochondria isolated from several tissues, including liver, heart, and brain, and in intact mononuclear cells from spleen. In contrast to control mitochondria, knockout mouse mitochondria did not sustain a reduced state of matrix NADPH, the main source of antioxidant defense against ROS. Experiments in vivo showed faster liver secretion rates and de novo synthesis of triglycerides and cholesterol in knockout than in control mice, suggesting that increased lipogenesis depleted the reducing equivalents from NADPH and generated a state of oxidative stress in hypercholesterolemic knockout mice. These data provide the first evidence of how oxidative stress is generated in LDL receptor defective cells and could explain the increased LDL oxidation, cell death, and atherogenesis seen in familiar hypercholesterolemia.
Asunto(s)
Antioxidantes/metabolismo , Arteriosclerosis/metabolismo , Mitocondrias/química , Estrés Oxidativo/fisiología , Animales , Arteriosclerosis/patología , Encéfalo/metabolismo , Femenino , Hipercolesterolemia , Canales Iónicos/química , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/química , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Miocardio/química , Especies Reactivas de Oxígeno/metabolismo , Receptores de LDL/deficiencia , Bazo/citologíaRESUMEN
Methylene Blue (MB) has well-established photochemical properties and has been used in a variety of photochemical applications including photodynamic therapy. Despite the fact that most of MB's cytotoxic effects in cells are attributed to mitochondrial damage, the interactions of this dye with mitochondria and the consequent effects on photochemical properties have not yet been fully determined. We monitored MB binding, aggregation and its ability to release singlet oxygen (1O2) on irradiation when interacting with mitochondrial suspensions. MB actively binds to mitochondria and enters the matrix in a manner stimulated by the mitochondrial proton potential and by the increase in mitochondrial concentrations. The greater accumulation of MB in mitochondria with elevated proton potentials or those treated with high concentrations of MB results in the formation of MB dimers, previously shown to be less effective generators of 1O2. Accumulation of MB within mitochondria with high membrane potentials also results in the reduction of MB to the photochemically inactive leuco-MB. Indeed, irradiation of mitochondria with high proton potentials in the presence of MB results in the generation of approximately half the quantity of 1O2 compared with 1O2 generated in mitochondria with low proton potentials. These differences in photochemical properties should influence the cytotoxic effects of photodynamic treatment in the presence of MB.
Asunto(s)
Azul de Metileno/análogos & derivados , Azul de Metileno/metabolismo , Mitocondrias Hepáticas/metabolismo , Oxígeno Singlete/metabolismo , Animales , Carbonil Cianuro m-Clorofenil Hidrazona , Dimerización , Luz , Potenciales de la Membrana , Azul de Metileno/química , Azul de Metileno/efectos de la radiación , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/ultraestructura , Oxígeno/metabolismo , Fotoquímica , Fotoquimioterapia , Ratas , Ratas Wistar , Oxígeno Singlete/análisis , Oxígeno Singlete/química , Factores de TiempoRESUMEN
In this work, the mitochondrial transmembrane electric potential (delta psi) of isolated mitochondria was used to evaluate the toxicity of some chemicals (endosulfan, 3,4-dichloroaniline, parathion, tributyltin and cadmium) and wastewater. Mitochondria were isolated from rat liver, and the delta psi measured in a suitable assay medium, using a sensitive tetraphenylphosphonium (TPP+) electrode. The test substance was pre-incubated in a rotenone-containing medium during 3 min with 1.0 mg of mitochondrial protein. Mitochondria were energised with succinate and after the establishment of a constant maximal potential ADP was added to induce the phosphorylative cycle. Chosen endpoints were the membrane potential from mitochondria oxidising succinate and the depolarisation induced by ADP. After the appropriate transformations the EC50 (effective concentration) was calculated for each toxicant. Even very low concentrations of a toxicant were able to affect the delta psi, thus showing its suitability as a biosensor in ecotoxicology and results were reproducible between tests. The utilisation of delta psi in screening tests of pure substances and wastewater seems to be very effective and can be carried out routinely.
Asunto(s)
Técnicas Biosensibles , Membranas Intracelulares/química , Mitocondrias Hepáticas/química , Contaminantes Químicos del Agua/análisis , Compuestos de Anilina/química , Compuestos de Anilina/toxicidad , Animales , Cadmio/química , Cadmio/toxicidad , Endosulfano/química , Endosulfano/toxicidad , Insecticidas/química , Insecticidas/toxicidad , Masculino , Potenciales de la Membrana/fisiología , Paratión/química , Paratión/toxicidad , Ratas , Ratas Wistar , Aguas del Alcantarillado/análisis , Compuestos de Trialquiltina/química , Compuestos de Trialquiltina/toxicidad , Abastecimiento de Agua/análisisRESUMEN
5-Nitroindole (NI), a mutagenic nitroarene, was assayed for cytotoxic effects on rat hepatocytes. After incubation with 25-100 microM NI, the adenylate energy charge of the hepatocytes decreased significantly as a result of the decrease in ATP and the increase in AMP. ATP depletion correlated well with the effects of NI on mitochondrial electron transfer and energy transduction in hepatocytes. Thus, NI (a) inhibited the antimycin-sensitive hepatocyte respiration; (b) inhibited NADH oxidation by disrupted hepatocyte mitochondria; (c) inhibited L-malate-L-glutamate oxidation by ADP-supplemented mitochondria; (d) in the absence of ADP, stimulated the same substrates and also succinate oxidation by mitochondria; (e) released the latent ATPase activity of mitochondrial F1F0-ATP synthase; (f) shifted the redox level of reduced cytochromes (c + c1) and b towards the oxidized state; (g) inhibited NADH oxidation by disrupted mitochondria in the vicinity of the NADH-dehydrogenase flavoprotein; (h) inhibited Ca2+ uptake by mitochondria using L-malate-L-glutamate as an energy source; (i) inhibited valinomycin-induced, endogenously energized K+ uptake, with little effect on the ATP-induced uptake; and (j) inhibited the MgATP-dependent contraction of Ca(2+)-swollen mitochondria. NI inhibited lipid peroxidation in hepatocytes and also in substrate-supplemented liver microsomes and mitochondria, thus ruling out hydroperoxides as a cause of NI cytotoxicity. Long-term incubation with NI produced loss of hepatocyte viability, as indicated by lactate dehydrogenase leakage.
Asunto(s)
Adenosina Monofosfato/química , Indoles/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Adenosina Trifosfato/análisis , Animales , Calcio/metabolismo , Células Cultivadas , Hígado/metabolismo , Masculino , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/efectos de los fármacos , Nitrofuranos/farmacología , ATPasas de Translocación de Protón/metabolismo , Ratas , Ratas Wistar , EspectrofotometríaRESUMEN
The present study was carried out to investigate the biochemical and morphological changes in the liver after ligation of the hepatic artery (HA) in the presence and in the absence of extrahepatic cholestasis (EHC). The study was conducted on 100 rats divided into four groups of 25 animals each: group 1, sham operation; group 2, hepatic artery ligation (HAL); group 3, bile duct ligation (BDL); and group 4, HAL plus BDL. All animals were sacrificed 7 days after surgery when total bilirubin and fractions, alkaline phosphatase (AP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in serum and on the inner hepatocyte mitochondrial membrane (IHMM); the incidence of necrosis and the volume fractions of vessels, bile ducts and hepatocytes in the liver were also determined. HAL reduces the relative volumes of bile ducts, with no changes in levels of bilirubin and fractions, AP, ALT, AST and IHMM, but HAL associated with EHC reduces duct proliferation and the liver becomes more vulnerable to necrosis. In conclusion, the normal liver depends on HA flow and this dependence is more evident in the presence of EHC.
Asunto(s)
Colestasis/patología , Hígado/patología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Bilirrubina/análisis , Arteria Hepática , Ligadura , Hígado/química , Circulación Hepática , Membranas/química , Mitocondrias Hepáticas/química , Necrosis , Ratas , Ratas WistarRESUMEN
Phospholipids extracted from liver microsomes and mitochondria of ethanol-fed rats retained the resistance to membrane disordered by ethanol which is observed in the intact isolated membranes. The lipid extracts were separated into the major phospholipid classes (phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol from microsomes and phosphatidylcholine, phosphatidylethanolamine and cardiolipin from mitochondria) by preparative TLC. The extent of membrane disordering by ethanol of phospholipid vesicles composed of a mixture of phospholipids from ethanol-fed rats and controls was determined from the reduction of the order parameter of the spin-probe 12-doxyl-stearate. In contrast to previous reports, we found that all phospholipid classes from ethanol-fed rats confer resistance to disordering by ethanol. To a first approximation the extent of resistance was proportional to the fraction of lipids from ethanol-fed rats, regardless of the phospholipid head-group. Subtle differences between phospholipid classes may exist but were too small to measure accurately. Except for phosphatidylethanol, incorporation of anionic phospholipids did not have a significant effect on the sensitivity of phospholipid vesicles to the disordering effect of ethanol. Vesicles prepared from mixtures of various dioleoyl phospholipids and natural phospholipids did not indicate a clear effect of fatty acid saturation on the sensitivity to disordering by ethanol. Although the precise molecular changes that occur in phospholipids from ethanol-fed rats have not been fully characterized it appears that subtle changes in all phospholipid classes contribute to the resistance to ethanol disordering of these membranes.