Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis.
OBJECTIVE
Osteoarthritis (OA) is associated with increased levels of reactive nitrogen and oxygen species and pro-inflammatory cytokines, such as interleukin-1 (IL-1). Nitric oxide (NO) can mediate a number of the catabolic effects of IL-1 in articular cartilage. The aims of this study were to determine if OA cartilage shows evidence of DNA damage, and if IL-1 could induce DNA damage in non-OA cartilage by increasing NO or superoxide.


METHODS
Articular chondrocytes were isolated from porcine femoral condyles and embedded in 1.2% alginate. The effects of 24h incubation with IL-1, the nitric oxide synthase 2 (NOS2)-selective inhibitor, the free radical scavenger superoxide dismutase (SOD), the NO donor NOC18, or the combined NO and peroxynitrite donor SIN-1 on DNA damage were tested, using the "comet" assay. NO production was measured using the Griess assay. The type of oxidative damage present was assessed using a modified comet assay.


RESULTS
OA cartilage had significantly more DNA damage than non-OA cartilage (P<0.001). IL-1 caused an increase in DNA damage (P<0.01), which was associated with increased NO production (P<0.01). Both oxidative DNA strand breaks and base modifications of purines and pyrimidines were observed. IL-1-induced DNA damage was inhibited by an NOS2 inhibitor or by SOD (P<0.01). Furthermore, NOC18 or SIN-1 caused DNA damage (P<0.001).


CONCLUSIONS
Our work shows chondrocytes in osteoarthritic cartilage exhibit DNA damage, and that IL-1 induces DNA damage and reactive oxygen and nitrogen species in non-OA chondrocytes in alginate.
Davies
C M
CM
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Guilak
F
F
Weinberg
J B
JB
Fermor
B
B
D000818:Animals; D002358:Cartilage, Articular / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D005260:Female; D053582:Interleukin-1alpha / Q000494:pharmacology*; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D034421:Sus scrofa
D053582:Interleukin-1alpha; C093285:NOC 18; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; C002385:linsidomine; D008981:Molsidomine