title: The responses of Ht22 cells to oxidative stress induced by buthionine sulfoximine (BSO).
abstract: BACKGROUND
  glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells. It directly reacts with reactive oxygen species (ROS), functions as a cofactor of antioxidant enzymes, and maintains thiol redox potential in cells. GSH depletion has been implicated in the pathogenesis of neurological diseases, particularly to Parkinson's disease (PD). The purpose of this study was to investigate the change of cellular antioxidant status and basic cell functions in the relatively early stages of GSH depletion.
  
  
  RESULTS
  in this study, GSH was depleted by inhibition of glutamylcysteine synthetase using buthionine sulfoximine (BSO) treatment in Ht22, a neuronal cell line derived from mouse hippocampus. Treatment with BSO produced dose-dependent decreases in total GSH level, Fe3+-reducing ability (FRAP assay), Cu2+-reducing ability (Antioxidant Potential, AOP assay), and ABTS free radical scavenging ability (ABTS assay) of the cells, but the sensitivity of these indicators to dosage varied considerably. Most of the changes were completed during the first 8 hours of treatment. Cell viability was tested by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay, and cells at lower density in culture were found to be more sensitive to GSH depletion. The activity of antioxidant enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), and copper/zinc superoxide dismutase (Cu/Zn-SOD) were affected by GSH depletion. A cDNA expression array assay of the effects of BSO treatment showed significantly decreased mRNA level for 3 genes, and significantly increased mRNA level for 10 genes, including the antioxidant enzymes Cu/Zn-SOD and thioredoxin peroxidase 2 (TPxII).
  
  
  CONCLUSIONS
  the study suggests that there are BSO-sensitive and BSO-resistant pools of GSH in Ht22 cells, and that different categories of antioxidant react differently to GSH depletion. Further, the effect of GSH status on cell viability is cell density dependent. Finally, the alterations in expression or activity of several antioxidant enzymes provide insight into the various cellular responses to GSH depletion.
authors.affiliation: Department of Cell & Molecular Biology, John A Burns School of Medicine, University of Hawaii, 1960 East West Rd, Honolulu, HI 96822, USA. junchen@hawaii.edu
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Andrea
authors.initials: A
authors.lastname: Small-Howard
authors.forename: Amy
authors.initials: A
authors.lastname: Yin
authors.forename: Marla J
authors.initials: MJ
authors.lastname: Berry
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology
chemical_list: D000975:Antioxidants; D019328:Buthionine Sulfoximine; D005978:Glutathione