Resumen

When the balance between the intracellular levels of oxidants and antioxidants is upset, reactive oxygen species (ROS) are produced in excess and cells reach the state of oxidative stress. As it is known, ROS levels can increase in response to a wide variety of stimuli and through the action of different oxidants. We investigated then the effect of different H2O2 concentrations (0.2 – 1.0 mM) on cell viability measured through the MTT assay. In 3h incubations with 1.0 mM H2O2, 75% of cell death was observed and apoptosis was detected by Hoechst 33258 dye staining, at 0.5 and 1.0 mM H2O2. Besides, apoptotic cell death was demonstrated measuring caspase-3-like activity employing Ac-DEVD-pNA as the substrate. Caspase 3 specific activity increased in a concentration-time-dependent manner. Moreover, a relatively specific caspase-3-like protease inhibidor, Ac-DEVD-cmk, was able to reduce caspase-3-like protease activation although not totally. Further, we also investigated whether caspase 8 are activated during H2O2-mediated apoptosis in cultured astrocytes. Caspase 8 specific activity significantly enhanced (80 – 120%) during 3h incubations with 1.0 mM H2O2. Procaspase 8 and activated caspase 8 were also detected through Western blots. Incubation with specific antioxidants demonstrated that NAC (10 mM) reduces both activated caspases to basal levels whereas GSH produces a decrease of 70% and 35% in caspase 3 and caspase 8 activities respectively. Our results suggest that caspase 8 and caspase 3 play a major role in H2O2-induced apoptosis in astrocytes. Supported by grants from UBA (TX-36) and CONICET (PEI 089).

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2007-01-31   |   1,364 visitas   |   Evalua este artículo 0 valoraciones

Vol. 1 Núm.1. Abril 2002 Pags. Qviva 2002; 1(1)