RESUMO
Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) are found in ethylmalonic encephalopathy (EE), an inherited disorder associated with cerebral and cerebellar atrophy whose pathogenesis is poorly established. The in vitro and in vivo effects of EMA on bioenergetics and redox homeostasis were investigated in rat cerebellum. For the in vitro studies, cerebellum preparations were exposed to EMA, whereas intracerebellar injection of EMA was used for the in vivo evaluation. EMA reduced state 3 and uncoupled respiration in vitro in succinate-, glutamate-, and malate-supported mitochondria, whereas decreased state 4 respiration was observed using glutamate and malate. Furthermore, mitochondria permeabilization and succinate supplementation diminished the decrease in state 3 with succinate. EMA also inhibited the activity of KGDH, an enzyme necessary for glutamate oxidation, in a mixed manner and augmented mitochondrial efflux of α-ketoglutarate. ATP levels were markedly reduced by EMA, reflecting a severe bioenergetic disruption. Docking simulations also indicated interactions between EMA and KGDH and a competition with glutamate and succinate for their mitochondrial transporters. In vitro findings also showed that EMA decreased mitochondrial membrane potential and Ca2+ retention capacity, and induced swelling in the presence of Ca2+ , which were prevented by cyclosporine A and ADP and ruthenium red, indicating mitochondrial permeability transition (MPT). Moreover, EMA, at high concentrations, mildly increased ROS levels and altered antioxidant defenses in vitro and in vivo. Our data indicate that EMA-induced impairment of glutamate and succinate oxidation and MPT may contribute to the pathogenesis of the cerebellum abnormalities in EE.
Assuntos
Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glutamatos/metabolismo , Malonatos/toxicidade , Poro de Transição de Permeabilidade Mitocondrial , Succinatos/metabolismo , Animais , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Succinatos/farmacologiaRESUMO
OBJECTIVE: Alzheimer's disease and Parkinson's disease are two of several neurodegenerative disorders that affect the elderly. Although their aetiology remains uncertain, studies suggest that elevated aluminium or other metal ions in the brain directly influence the development of the histological abnormalities normally associated with these diseases; other investigations suggest that metal-ion-induced-dysfunction of mitochondria might be a critical factor. METHODS: In this study, the impact of elevated aluminum (Al3+), ferric (Fe3+), calcium (Ca2+) and magnesium (Mg2+) ions on brain histology and on the protein composition of brain mitochondria were evaluated. Rabbits were injected intra-cerebrally with 1.4% solutions of either aluminium chloride (AlCl3), ferric chloride (FeCl3), calcium chloride (CaCl2) or magnesium chloride (MgCl2) and sacrificed 10 days later RESULTS: Histological analysis revealed that Al3+ but not the other ions induced neurofibrillary degeneration within the midbrain and medulla. Alternatively, SDS-PAGE revealed that Fe3+, Ca2+ and Mg2+ but not Al3+ induced alterations to the distribution of brain mitochondrial proteins. Both Fe3+ and Ca2+ triggered decreased concentration of three low molecular weight proteins (-7-14 kd) but Ca2+ precipitated their total absence. Both ions led to increased concentration of a high molecular weight protein (-110 kd). In contrast, Mg2 led to the total absence of the protein of lowest molecular weight (-7 kd) and increased concentration of a -36 kd protein. CONCLUSION: These results suggest that elevation of some metal ions in the brain induces protein aggregation with the nature of the aggregation being highly ion dependent. The results also point toward major differences between the histopathological effect of Al3+ and other ions.
Assuntos
Encéfalo/metabolismo , Cloreto de Cálcio/farmacologia , Cloretos/farmacologia , Compostos Férricos/farmacologia , Cloreto de Magnésio/farmacologia , Proteínas Mitocondriais/metabolismo , Cloreto de Alumínio , Compostos de Alumínio/farmacologia , Animais , Encéfalo/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Proteínas Mitocondriais/efeitos dos fármacos , CoelhosRESUMO
OBJECTIVE: Alzheimer s disease and Parkinson s disease are two of several neurodegenerative disorders that affect the elderly. Although their aetiology remains uncertain, studies suggest that elevated aluminium or other metal ions in the brain directly influence the development of the histological abnormalities normally associated with these diseases; other investigations suggest that metal-ion-induced-dysfunction of mitochondria might be a critical factor. METHODS: In this study, the impact of elevated aluminum (Al3+), ferric (Fe3+), calcium (Ca2+) and magnesium (Mg2+) ions on brain histology and on the protein composition of brain mitochondria were evaluated. Rabbits were injected intra-cerebrally with 1.4% solutions of either aluminium chloride (AlCl3), ferric chloride (FeCl2), calcium chloride (CaCl2) or magnesium chloride (MgCl2) and sacrificed 10 days later. RESULTS: Histological analysis revealed that Al3+ but not the other ions induced neurofibrillary degeneration within the midbrain and medulla. Alternatively, SDS-PAGE revealed that Fe3+, Ca2+ and Mg2+ but not Al3+ induced alterations to the distribution of brain mitochondrial proteins. Both Fe3+ and Ca2+ triggered decreased concentration of three low molecular weight proteins (~7-14 kd) but Ca precipitated their total absence. Both ions led to increased concentration of a high molecular weight protein (~ 110 kd). In contrast, Mg2+ led to the total absence of the protein of lowest molecular weight (~7 kd) and increased concentration of a ~36 kd protein. CONCLUSION: These results suggest that elevation of some metal ions in the brain induces protein aggregation with the nature of the aggregation being highly ion dependent. The results also point toward major differences between the histopathological effect of Al3+ and other ions.
OBJETIVO: La enfermedad de Alzheimer y la enfermedad de Parkinson son dos de los varios trastornos neurodegenerativos que afectan al anciano. Aunque su etiologia sigue siendo incierta, los estudios sugieren que el aumento de los iones de aluminio, influyen directamente sobre el desarrollo de las anormalidades histológicas asociadas normalmente con estas enfermedades. Otras investigaciones sugieren que la disfunción de las mitocondrias, inducida por iones metálicos, pudiera ser un factor critico. MÃTODOS: Este estudio evalúa el impacto del aumento de los iones de aluminio (Al3+), los iones férricos (Fe3+), y los iones de calcio (Ca2+) y magnesio (Mg2+) sobre la histologia del cerebro y la composición proteica de las mitocondrias del cerebro. Un número de conejos recibieron inyecciones intracerebrales de soluciones al 1.4% de soluciones de cloruro de aluminio (AlCl3), cloruro ferroso (FeCl3), cloruro de calcio (CaCl2), o cloruro de magnesio (MgCl2), y fueron sacrificados después de 10 dÃas. RESULTADOS: El análisis histológico reveló que el Al3+ indujo una degeneración neurofibrilar dentro del mesencéfalo y la médula, Sin embargo, esto no ocurrió con los otros iones. Alternativamente, la técnica de electroforesis SDS-PAGE reveló que los iones Fe3+, Ca2+ y Mg2+, a diferencia del ión Al3+, inducÃan alteraciones de la distribución de las proteÃnas mitocondriales cerebrales. Tanto el Fe3+ como el Ca2+ desencadenaron una disminución de la concentración de tres proteÃnas de bajo peso molecular (~7-14 kd) pero Ca2+ precipitó su ausencia total. Ambos iones condujeron a un aumento de una proteÃna de peso molecular alto (~ 110 kd). En cambio, Mg2+ llevó a la ausencia total de la proteÃna de más bajo peso molecular (~7 kd) y al aumento de la concentración de una proteÃna de ~36 kd. CONCLUSIÃN: Estos resultados parecen sugerir que la elevación de algunos iones de metal en el cerebro induce la agregación de la proteÃna, siendo la naturaleza de la agregación altamente dependiente de los iones. Los resultados también apuntan a grandes diferencias entre el efecto histopatológico del Al3+ y otros iones.
Assuntos
Animais , Coelhos , Encéfalo/metabolismo , Cloreto de Cálcio/farmacologia , Cloretos/farmacologia , Compostos Férricos/farmacologia , Cloreto de Magnésio/farmacologia , Proteínas Mitocondriais/metabolismo , Compostos de Alumínio/farmacologia , Encéfalo/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Proteínas Mitocondriais/efeitos dos fármacosRESUMO
BACKGROUND: Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd(2) [S((-))C(2), N-dmpa](2) (µ-dppe)Cl(2)} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. METHODS: B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. RESULTS: Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. CONCLUSIONS: The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Mitocondriais/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Paládio/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Estrutura Molecular , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Paládio/química , Paládio/metabolismo , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Liver alcohol dehydrogenase (ADH) activity is decreased towards exogenous substrates after partial hepatectomy (PH), probably due to putative endogenous substrates acting as ADH inhibitors. Hence, retinoids could be suitable candidates as such endogenous substrates. Therefore, cytosolic ADH kinetic analysis using several substrates, liver cytosolic and mitochondrial aldehyde dehydrogenase (ALDH) activities, retinal and retinol content, as well as expression of proteins for ADH and CRBPI (a retinol carrier protein) were determined in liver samples, at two stages of liver regeneration (one- or two-thirds PH). The effect of inhibiting in vivo liver ADH by 4-methylpyrazole (4-MP) was also evaluated after 70%-PH. With 70%-PH, in vitro ADH activity towards exogenous alcohols and aldehydes was diminished, but retinol oxidation was increased and retinal reduction was decreased. These activities that be due to the participation of an ADH type which did not correlate with the amount of immunoreactive ADH protein. Cytosolic and mitochondrial ALDH activities oxidized actively retinal, whereas retinol and CBRP-I expression were reduced in these animals. With 30%-PH, these changes were less evident and sometimes opposite to those found with 70%-PH. In addition, retinol readily inhibited ADH-mediated ethanol oxidation. Interestingly, in vivo 4-MP administration inhibited ADH activity in a dose-dependent manner correlating with a progressive inhibition of liver regeneration. In conclusion, PH-induced inhibition of ADH (mainly type I) seems to be related to ADH-mediated retinoid metabolism during liver proliferation. Thus, results suggest a role of ADH in retinoid metabolism, which is apparently required during rat liver regeneration.