Methylmercury-induced reactive oxygen species-dependent and independent dysregulation of MAP kinase-related signaling pathway in cultured normal rat cerebellar astrocytes

Abstract

Methylmercury (MeHg), a global environmental pollutant, could seriously damage the central nervous system (CNS) and cause neurological disorders such as cerebellar symptoms. Although numerous studies have revealed detailed toxicity mechanisms of MeHg in neurons, toxicity in astrocytes is barely known. Here, we tried to shed light on the toxicity mechanisms of MeHg exposure in cultured normal rat cerebellar astrocytes (NRA), focusing on the involvement of reactive oxygen species (ROS) in MeHg toxicity by assessing the effects of major antioxidants Trolox, a free-radical scavenger, N-acetyl-L-cysteine (NAC), a potent thiol-containing antioxidant, and glutathione (GSH), an endogenous thiol-containing antioxidant. Exposure to MeHg at just approximately 2 µM for 96 h increased cell viability, which was accompanied by the increase in intracellular ROS level and at ≥ 5 µM induced significant cell death and lowered ROS level. Trolox and NAC suppressed 2 µM MeHg-induced increases in cell viability and ROS level corresponding to control, although GSH with 2 µM MeHg induced significant cell death and ROS increase. On the contrary, against 4 µM MeHg-induced cell loss and ROS decrease, NAC inhibited both cell loss and ROS decrease, Trolox inhibited cell loss and further enhanced ROS decrease, and GSH moderately inhibited cell loss and increased ROS level above the control level. MeHg-induced oxidative stress was suggested by increases in the protein expression levels of heme oxygenase-1 (HO-1), Hsp70, and Nrf2, except for the decrease in SOD-1 and no change in catalase. Furthermore, MeHg exposure dose-dependently induced increases in the phosphorylation of MAP kinases (ERK1/2, p38MAPK, and SAPK/JNK) and phosphorylation and/or expression levels of transcription factors (CREB, c-Jun, and c-Fos) in NRA. NAC successfully suppressed 2 µM MeHg-induced alterations in all of the above-mentioned MeHg-responsive factors, whereas Trolox suppressed some MeHg-responsive factors but failed to suppress MeHg-induced increases in the protein expression levels of HO-1 and Hsp70 and increase in p38MAPK phosphorylation. Protein expression analyses in NRA exposed to 2 µM MeHg and GSH were excluded because of devastating cell death. These results suggested that MeHg could induce aberrant NRA activation, and ROS must be substantially involved in the toxicity mechanism of MeHg in NRA; however, other factors should be assumed.