title: Reactive oxygen species involved in the glutamate toxicity of C6 glioma cells via xc antiporter system.
abstract: We recently demonstrated that continuous L-glutamate exposure led to cell death in C6 glioma cells over a period of 24-36 h, due to inhibition of cystine uptake through the cystine/glutamate (XC) antiporter. The antioxidant vitamin E provided protection against this effect, supporting the hypothesis that depletion of glutathione might be responsible, resulting from insufficient cystine uptake. To clarify the content of oxidative stress after glutathione depletion, the present study was done to investigate accumulation and target molecules of reactive oxygen species induced by glutamate treatment. The accumulation of reactive oxygen species was increased three-fold as compared to a control culture. Membrane oxidation, as judged by lipid peroxidation, was increased two-fold after glutamate treatment. Cellular ATP content was significantly reduced by glutamate exposure. For the two cytosolic enzymes examined, activity of glyceraldehyde 3-phosphate dehydrogenase was slightly enhanced by glutamate treatment, while activity of glutamine synthetase was not changed. Impairment of nuclear DNA after glutamate exposure was also revealed by nuclear chromatin condensation with DNA fragmentation. Thus, the multiple targets (membrane, cytoplasm and nuclei) of oxygen radicals in glutamate toxicity through the xc antiporter system were evaluated for the first time. Furthermore, prevention from cell death and from cellular toxicity induced by oxygen radicals could be seen using three specific oxygen radical scavengers, catalase, 3,3,5,5-tetramethyl-pyrroline N-oxide and alpha-phenyl-N-t-butylnitrone, without restoring the glutathione deficit. This indicates that radical scavengers did not interact with the xc antiporter system, but directly scavenged the oxygen radicals. Taken together, the data strongly suggest that O2-, H2O2 and OH accumulate in response to oxidative stress after glutathione depletion, resulting in glutamate cell death of C6 glioma cells.
authors.affiliation: Department of Laboratory Sciences, University of Kanazawa Faculty of Medicine, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Mawatari
authors.forename: Y
authors.initials: Y
authors.lastname: Yasui
authors.forename: K
authors.initials: K
authors.lastname: Sugitani
authors.forename: T
authors.initials: T
authors.lastname: Takadera
authors.forename: S
authors.initials: S
authors.lastname: Kato
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017920:Antiporters / Q000502:physiology*; D001932:Brain Neoplasms / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003553:Cystine / Q000378:metabolism; D004249:DNA Damage; D016166:Free Radical Scavengers / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D017920:Antiporters; D016166:Free Radical Scavengers; D008054:Lipid Peroxides; D017382:Reactive Oxygen Species; D018698:Glutamic Acid; D003553:Cystine; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005974:Glutamate-Ammonia Ligase; D005978:Glutathione