title: Increased oxidative stress with aging reduces chondrocyte survival: correlation with intracellular glutathione levels.
abstract: OBJECTIVE
  To examine the role of oxidative stress in mediating cell death in chondrocytes isolated from the articular cartilage of young and old adult human tissue donors.
  
  
  METHODS
  Cell death induced by the oxidant SIN-1 was evaluated in the alginate bead culture system using fluorescent probes to assess membrane integrity. Generation of peroxynitrite by the decomposition of SIN-1 was confirmed by positive immunostaining of treated cells for 3-nitrotyrosine. Determinations of oxidized glutathione (GSSG) and reduced glutathione (GSH) were performed in monolayer cultures using an enzyme- recycling assay. Cells were depleted of intracellular glutathione either by the addition of DL-buthionine-(S,R)-sulfoximine or by removal of L-cystine from the culture media. The activity of cellular antioxidant enzymes was determined spectrophotometrically by the decay of substrate from the reaction mixture.
  
  
  RESULTS
  More chondrocytes (>2-fold) from old donors (>/=50 years) died after exposure to 1 mM SIN-1 relative to those derived from young donors (18-49 years). Although autocrine production of insulin-like growth factor 1 (IGF-1) promotes chondrocyte survival, pretreatment with IGF-1 could not prevent the cell death induced by SIN-1 exposure. Cells isolated from old donors had a higher ratio of GSSG to GSH. Glutathione reductase is the principal enzyme involved in the regeneration of GSH from GSSG. Treatment of chondrocytes with SIN-1 to induce oxidative stress in vitro resulted in the decreased activity of glutathione reductase and thioredoxin reductase, but not catalase. Cells depleted of intracellular glutathione were more susceptible to cell death induced by SIN-1.
  
  
  CONCLUSIONS
  These results provide evidence that increased oxidative stress with aging makes chondrocytes more susceptible to oxidant-mediated cell death through the dysregulation of the glutathione antioxidant system. This may represent an important contributing factor to the development of osteoarthritis in older adults.
authors.affiliation: Rush Medical College of Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA.
authors.forename: Marcello Del
authors.initials: MD
authors.lastname: Carlo
authors.forename: Richard F
authors.initials: RF
authors.lastname: Loeser
mesh_terms: D000293:Adolescent; D000328:Adult; D000375:Aging / Q000378:metabolism*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008875:Middle Aged; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism
chemical_list: D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; C002385:linsidomine; D007334:Insulin-Like Growth Factor I; D008981:Molsidomine; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione; D019803:Glutathione Disulfide