Oxidative DNA damage in osteoarthritic porcine articular cartilage

Abstract

Osteoarthritis (OA) is associated with increased levels of reactive oxygen species. This study investigated if increased oxidative DNA damage accumulates in OA articular cartilage compared with non-OA articular cartilage from pigs with spontaneous OA. Additionally, the ability of nitric oxide (NO) or peroxynitrite (ONOO(-)) induced DNA damage in non-OA chondrocytes to undergo endogenous repair was investigated. Porcine femoral condyles were graded for the stage of OA, macroscopically by the Collins Scale, and histologically by the modified Mankin Grade. Levels of DNA damage were determined in non-OA and OA cartilage, using the comet assay. For calibration, DNA damage was measured by exposing non-OA chondrocytes to 0-12 Gray (Gy) of X-ray irradiation. Non-OA articular chondrocytes were treated with 0-500 microM of NO donors (NOC-18 or SIN-1), and DNA damage assessed after treatment and 5 days recovery. A significant increase (P < 0.01) in oxidative DNA damage occurred in OA chondrocytes in joints with Mankin Grades 3 or greater, compared to non-OA chondrocytes. The percentage of nuclei containing DNA damage increased significantly (P < 0.001) from early to late grades of OA. An increase of approximately 0.65-1.7 breaks/1,000 kb of DNA occurred in OA, compared to non-OA nuclei. NOC-18 or SIN-1 caused significant DNA damage (P < 0.001) in non-OA chondrocytes that did not undergo full endogenous repair after 5 days (P < 0.05). Our data suggest significant levels of oxidative DNA damage occur in OA chondrocytes that accumulates with OA progression. Additionally, DNA damage induced by NO and ONOO(-) in non-OA chondrocytes does not undergo full endogenous repair.

Figure 1

Levels of nuclear DNA damage in pig articular cartilage associated with different histological grades of OA, as measured by the modified Mankin Score, (a) Representative histology, (b) representative comets and (c) Olive Tail Moment (OTM). Mean ± SEM, N ≥ 3 (Grade 0, N = 3; Grades 1-2, N = 9; Grades 3-5, N = 4; Grades 6-7, N = 7), n = 50/group. * = p<0.05, ** = p<0.01, *** = p<0.001.

Figure 2

Determination of oxidative nuclear DNA damage in OA. DNA repair enzymes Endonuclease III (EndoIII), formamidopyrimidine (Fpg), or oxoguanine glycosylase-1 (Ogg1) incubated with OA nuclei in the modified comet assay determined the presence of oxidative DNA damage in OA. Mean ± SEM, N = 3, n = 50/group. *** = p<0.001.

Figure 3

Calibration of DNA damage levels with x-ray damage. Non-OA porcine articular chondrocytes were exposed to 0-12 Gy of x-ray irradiation and the levels of DNA damage were measured by OTM. OTM = 1.4996 Gy + 2.738, r²= 0.985. Mean ± SEM, N = 3, n = 50/group.

Figure 4

Levels of increased DNA damage and incomplete endogenous DNA repair in articular chondrocytes in alginate treated with either NOC-18 or SIN-1 for 24 hrs, followed by 5 days recovery without NO donors in the media. Mean ± SEM, N = 3, n = 50/group. * = p<0.05, ** = p<0.01, *** = p<0.001.