Glutathione modulation influences methyl mercury induced neurotoxicity in primary cell cultures of neurons and astrocytes.
Methyl mercury (MeHg) is highly neurotoxic and may lead to numerous neurodegenerative disorders. In this study, we investigated the role of glutathione (GSH) and reactive oxygen species (ROS) in MeHg-induced neurotoxicity, using primary cell cultures of cerebellar neurons and astrocytes. To evaluate the effect of GSH on MeHg-induced cytotoxicity, ROS and GSH were measured using the fluorescent indicators chloro methyl derivative of di-chloro di-hydro fluorescein diacetate (CMH(2)DCFDA) and monochlorobimane (MCB). Cell-associated MeHg was measured with (14)C-radiolabeled MeHg. Mitochondrial dehydrogenase activity was detected by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. MTT timeline study was also performed to evaluate the effects of both the concentration and duration of MeHg exposure. The intracellular GSH content was modified by pretreatment with N-acetyl cysteine (NAC) or di-ethyl maleate (DEM) for 12 h. Treatment with 5 microM MeHg for 30 min led to significant (p<0.05) increase in ROS and reduction (p<0.001) in GSH content. Depletion of intracellular GSH by DEM further increased the generation of MeHg-induced ROS in both cell cultures. Conversely, NAC supplementation increased intracellular GSH and provided protection against MeHg-induced oxidative stress in both cell cultures. MTT studies also confirmed the efficacy of NAC supplementation in attenuating MeHg-induced cytotoxicity. The cell-associated MeHg was significantly (p<0.02) increased after DEM treatment. In summary, depletion of GSH increases MeHg accumulation and enhances MeHg-induced oxidative stress, and conversely, supplementation with GSH precursor protects against MeHg exposure in vitro.
Kaur
Parvinder
P
Department of Neuroscience, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
Aschner
Michael
M
Syversen
Tore
T
D000111:Acetylcysteine / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects*; D002452:Cell Count; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000502:physiology*; D020262:Mercury Poisoning, Nervous System / Q000209:etiology / Q000378:metabolism; D008767:Methylmercury Compounds / Q000378:metabolism / Q000633:toxicity*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D010088:Oxidoreductases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors
D004791:Enzyme Inhibitors; D008767:Methylmercury Compounds; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D010088:Oxidoreductases; C022616:thiazolyl blue; D005978:Glutathione; D005033:Ethylmaleimide; D000111:Acetylcysteine