RESUMO
The mechanisms that mediate thallium (Tl) toxicity are still not completely understood. The exposure of rat pheochromocytoma (PC12) cells to Tl(I) or Tl(III) activates both mitochondrial (Tl(I) and Tl(III)) and extrinsic (Tl(III)) pathways of apoptosis. In this work we evaluated the hypothesis that the effects of Tl(III) may be mediated by the damage to lysosomes, where it might be incorporated following the route of iron uptake. PC12 cells exposed for 3 h to 100 µM Tl(III) presented marked endosomal acidification, effect that was absent when cells were incubated in a serum-free medium and that was fully recovered when the latter was supplemented with transferrin. After 6 h of incubation the colocalization of cathepsins D and B with the lysosomal marker Lamp-1 was decreased together with an increase in the total activity of the enzymes. A permanent damage to lysosomes after 18 h of exposure was evidenced from the impairment of acridine orange uptake. Cathepsin D caused the cleavage of pro-apoptotic protein BID that is involved in the activation of the intrinsic pathway of apoptosis. Supporting that, BID cleavage and the activation of caspase 3 by Tl(III) were fully prevented when cells were preincubated with cathepsin D inhibitor (pepstatin A) and only partially prevented when cathepsin B inhibitor (E64d) was used. None of these inhibitors affected BID cleavage or caspase 3 activation in Tl(I)-treated cells. Together, experimental results support the role of Tl(III) uptake by the acidic cell compartments and their involvement in the early steps of Tl(III)-mediated PC12 cells apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Endossomos/metabolismo , Lisossomos/metabolismo , Tálio/toxicidade , Animais , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina D/antagonistas & inibidores , Catepsina D/metabolismo , Compartimento Celular/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Células PC12/efeitos dos fármacos , Pepstatinas/farmacologia , Ratos , Tálio/farmacologiaRESUMO
Thallium (Tl) is highly toxic through yet poorly understood mechanisms. In this study, we comparatively investigated the effects of thallic (Tl(III)) cations on mitochondrial functionality and oxidative stress promotion, and results were compared to those obtained for thallous (Tl(I)) cation. PC12 cells were incubated between 1 and 72 h in the presence of a single dose of Tl(I) or Tl(III) (10-250 microM). A metal concentration- and time-dependent decrease in cell viability was observed evaluated by both MTT reduction and calcein fluorescence. After 24 h in culture, Tl(I) and Tl(III) significantly decreased mitochondrial membrane potential evaluated as the incorporation of rhodamine 123. Along the incubation period assessed, both Tl(I) and Tl(III) (50 and 100 microM) significantly increased mitochondrial H2O2 steady-state levels, being the magnitude of the effect: Tl(III)>Tl(I). Glutathione content, measured by reaction with monochlorobimane, was significantly reduced in Tl-treated cells. Finally, higher oxidant species content in cells cytoplasm was found, which positively correlated with mitochondrial H2O2 content. Together, these results indicate that both ionic species of Tl enhance cells reactive oxygen species production, decreasing mitochondrial functionality. These effects could partially be responsible for the loss of cell viability, and account for the metabolic alterations found in Tl intoxication.
Assuntos
Peróxido de Hidrogênio/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxidantes/metabolismo , Tálio/farmacologia , Algoritmos , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oxirredução , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Sais de Tetrazólio , TiazóisRESUMO
The possibility that Tl(OH)3, the main Tl3+ specie present in water solutions, could interfere with the normal functioning of the glutathione-dependent antioxidant defense system was investigated. For this purpose, we used both the purified components of this system and rat brain cytosolic fractions. Tl(OH)3 (1-25 microM) significantly decreased the content of reduced glutathione (GSH) in both experimental systems, caused by GSH oxidation. In the same range of concentrations Tl(OH)3 inhibited glutathione peroxidase (GPx) activity in both models, using cumene hydroperoxide as the substrate. No alterations in the capacity of GPx activity to metabolize H2O2 were observed. Both in purified GR as well as in the cytosolic fraction, Tl(OH)3 (1-5 microM) inhibited GR activity, with a partial recovery of the activity at higher concentrations. While Tl(OH)3 inhibited the GR diaphorase activity of purified GR, in a concentration (1-25 microM) dependent manner, this effect was only observed in the cytosolic fractions at the highest concentration assessed (25 microM). Results indicate that, similarly to previous findings for Tl+ and Tl3+, Tl(OH)3 also alters the glutathione-dependent antioxidant defense system. The observed alterations of this important antioxidant protective pathway by the major Tl3+ specie in water solutions could be one mechanism involved in the oxidative stress associated to Tl-intoxication.
Assuntos
Citosol/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Glutationa/metabolismo , Tálio/toxicidade , Animais , Encéfalo , Química Encefálica , Catalase/metabolismo , Fracionamento Celular , Citosol/enzimologia , Relação Dose-Resposta a Droga , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , NADP/metabolismo , Oxirredução , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
We investigated the hypothesis that thallium (Tl) interactions with the glutathione-dependent antioxidant defence system could contribute to the oxidative stress associated with Tl toxicity. Working in vitro with reduced glutathione (GSH), glutathione reductase (GR) or glutathione peroxidase (GPx) in solution, we studied the effects of Tl+ and Tl3+ (1-25 microM) on: (a) the amount of free GSH, investigating whether the metal binds to GSH and/or oxidizes it; (b) the activity of the enzyme GR, that catalyzes GSH regeneration; and (c) the enzyme GPx, that reduces hydroperoxide at expense of GSH oxidation. We found that, while Tl+ had no effect on GSH concentration, Tl3+ oxidized it. Both cations inhibited the reduction of GSSG by GR and the diaphorase activity of this enzyme. In addition, Tl3+ per se oxidized NADPH, the cofactor of GR. The effects of Tl on GPx activity depended on the metal charge: Tl+ inhibited GPx when cumene hydroperoxide (CuOOH) was the substrate, while Tl(3+)-mediated GPx inhibition occurred with both substrates. The present results show that Tl interacts with all the components of GSH/GSSG antioxidant defence system. Alterations of this protective pathway could be partially responsible for the oxidative stress associated with Tl toxicity.