Free radical fonnation in cerebral cortical astrocytes in culture induced by methylmercury

Oxidative stress has been implicated in neurotoxic damage associated with various metals, including methylmercury (MeHg). Although the mechanism(s) of MeHg-induced neurotoxicity remains unclear, evidence supports a mediatory role for astrocytes, a cell type that preferentially accumulates MeHg. Using scanning confocal microscopy (LSCM), the present study was undertaken to examine the role of astrocytes as the site of reactive oxygen species (ROS). Three redox-sensitive fluorescent probes were used for ROS analysis, (a) CM-H(2)DCFDA (chloromethyl derivative of dichlorodihydrofluorescein diacetate), a probe for intracellular hydrogen peroxide (H2O2); (b) hydroethidine (HETH), a probe for superoxide anion (.O-2(-)), and (c) CM-H(2)XRos (chloromethyl derivative of dihydro X-rosamine), and a probe that is selective for mitochondrial reactive oxygen intermediates. Astrocytes were treated with 10 muM MeHg for 30 min, following which the various fluorescent probes were added; 20 min later LSCM images were collected. Astrocytes loaded with CM-H(2)DCFDA and HE demonstrated a significant MeHg-induced increase in fluorescence intensity indicative of increased intracellular H2O2 and .O-2, respectively. Similar results were obtained with the mitotracker dye, CM-H(2)XRos. Additionally, exposure of astrocytes for 24 h to 100 muM buthionine-L-sulfoxane (BSO), a glutathione (GSH) synthesis inhibitor, caused a significant increase in ROS formation. Furthermore, BSO pretreatment significantly enhanced the MeHg-induced formation of .O-2(-), indicating an important role for GSH in the maintenance of optimal cellular redox status. Time-course experiments performed in the simultaneous presence of CM-H(2)XRos and CM-H(2)DCFDA demonstrated that the MeHg-induced CM-H(2)XRos fluorescence changes preceded those of CM-H(2)DCFDA, suggesting that the mitochondria represent an early primary site for ROS formation. Taken together, these studies illustrate that MeHg induces the generation of astrocyte-derived ROS and support a role for astrocytic ROS in MeHg-associated neurotoxic damage. (C) 2004 Elsevier B.V. All rights reserved.