Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-dependent neurotoxicity remains far from clear, overwhelming data give credence to a mediatory role for astrocytes, a major cell type that preferentially accumulates MeHg. To extend our recent findings of MeHg-induced increase in ROS formation (G. Shanker, J.L. Aschner, T. Syversen et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128 (2004) 48-57), the present studies were designed to assess the effect of modulating intracellular glutathione (GSH) content, on ROS generation, in the absence and presence of MeHg. Intracellular GSH was reduced by treatment with 100 μM buthionine-l-sulfoxane (BSO) for 24 h, and increased by treatment with 1 mM l-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 h. Additionally, the effects of the selective antioxidants, catalase (1000 U/ml for 1 h), an H2O2 scavenger, and n-propyl gallate (100 μM for 1 h), a superoxide radical ( •O2-) and possibly hydroxyl radical ( •OH) scavenger on MeHg-induced ROS formation were examined. After these treatments, astrocytes were exposed to ±10 μM MeHg for 30 min, following which the fluorescent probes, CM-H2DCFA and CM-H 2XRos were added; 20 min later, laser scanning confocal microscopy (LSCM) images were obtained. Exposure of astrocytes for 24 h to 100 μM BSO, a GSH synthesis inhibitor, led to a significant increase in mitochondrial ROS (i.e., •O2-, •NO, and ONOO-) formation, as assessed with CM-H2XRos mitotracker red dye. Similarly, BSO increased ROS formation in various intracellular organelles, as assessed with CM-H2DCFDA. BSO in combination with MeHg increased fluorescence levels in astrocytes to levels above those noted with BSO or MeHg alone, but this effect was statistically indistinguishable from either of these groups (BSO or MeHg). Pretreatment of astrocytes for 24 h with 1 mM OTC abolished the MeHg-induced increase in ROS. Results similar to those obtained with OTC were observed with the free radical scavenger, n-propyl gallate (n-PG). The latter had no significant effects on astrocytic fluorescence when administered alone. This •O2- and possibly •OH radical scavenger significantly attenuated MeHg-induced ROS formation. Catalase, an H2O2 scavenger, was less effective in reducing MeHg-induced ROS formation. Taken together, these studies point to the important protective effect of adequate intracellular GSH content as well as antioxidants against MeHg-triggered oxidative stress in primary astrocyte cultures.
- Laser scanning confocal microscopy
- Reactive oxygen species
- Redox status
- n-Propyl gallate
ASJC Scopus subject areas
- Molecular Biology
- Cellular and Molecular Neuroscience