Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species.
OBJECTIVE
Chondrocyte cell death, possibly related to increased production of endogenous nitric oxide (NO), has been observed during the pathogenesis of osteoarthritis and rheumatoid arthritis. The purpose of this study was to investigate the potential role of NO in causing chondrocyte cell death and to determine the contribution of other reactive oxygen species (ROS).


METHODS
Cell death and cytotoxicity were evaluated in human articular chondrocytes in response to various NO donor compounds with and without agents that stimulate or inhibit the production of additional ROS using both the alginate bead and the monolayer culture systems. Cell death was quantified by a total cell count with fluorescent labels, and cytotoxicity was measured as a function of cellular NADH- and NADPH-dependent dehydrogenase activity. To determine if the redox status of the chondrocyte could influence the observed effect of NO, cells were preincubated for 24 hours in L-cystine- and glutathione (GSH)-depleted media to reduce intracellular GSH levels, a major defense mechanism against oxidative stress. Apoptosis was analyzed with the quantification of histone-associated DNA fragments.


RESULTS
Treatment of chondrocytes with peroxynitrite (ONOO-), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) resulted in apoptotic cell death at concentrations of 0.5 mM, 1.0 mM, and 0.5 mM, respectively. In contrast, treatment of chondrocytes with diazeniumdiolates (or the "NOC" compounds, NOC-5 and NOC-12) at concentrations as high as 2.0 mM did not cause cell death. Furthermore, NOC-5 and NOC-12, at all concentrations tested (0.125-2.0 mM), could prevent cell death caused by oxidative stress. Selective ROS scavengers protected against cell death caused by either SIN-1 or ONOO-; however, no protection could be afforded against the cytotoxicity of SNP with any of the ROS scavengers tested.


CONCLUSIONS
These results show that NO by itself is not cytotoxic to cultured chondrocytes and can even be protective under certain conditions of oxidative stress. Chondrocyte cell death from NO occurs under conditions where other ROS are also generated.
Del Carlo
Marcello
M
College of Rush-Presbyterian-St. Luke's Medical Center, 1725 West Harrison Avenue, Chicago, IL 60612, USA.
Loeser
Richard F
RF
D000464:Alginates; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008863:Microspheres; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009573:Nitrites / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014226:Triazenes / Q000494:pharmacology
C114535:1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene; D000464:Alginates; C118603:NOC 12; D009566:Nitrates; D020030:Nitric Oxide Donors; D009573:Nitrites; D009603:Nitroso Compounds; D017382:Reactive Oxygen Species; D014226:Triazenes; D009243:NAD; D009599:Nitroprusside; D009569:Nitric Oxide; D009249:NADP; C002385:linsidomine; D008981:Molsidomine