Journal of Cellular Biochemistry 101:1520–1531 (2007) Nitric Oxide From Both Exogenous and Endogenous Sources Activates Mitochondria-Dependent Events and Induces Insults to Human Chondrocytes Gong-Jhe Wu, 1,2 Tyng-Guey Chen, 3 Huai-Chia Chang, 3 Wen-Ta Chiu, 3 Chia-Chen Chang, 1,4 and Ruei-Ming Chen 1,4 * 1 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Fu-Jen Catholic University, Taipei, Taiwan 3 Core Laboratories, Wan-Fang Hospital, Taipei Medical University, Taipei, Taiwan 4 Topnotch Stroke Research Center, Taipei Medical University, Taipei, Taiwan Abstract During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon-g (IFN-g) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN-g in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time- dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S- nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism. J. Cell. Biochem. 101: 1520 – 1531, 2007. ß 2007 Wiley-Liss, Inc. Key words: human chondrocytes; nitric oxide; apoptosis; mitochondrial dysfunction; apoptotic factors; caspase activation Chondrocytes are responsible for preventing cartilage degeneration through maintaining the extracellular matrix [van der Kraan et al., 2002]. In cartilage subjected to trauma, chondrocytes may be damaged or even die [Quinn et al., 2001; Redman et al., 2004]. Studies on human osteoarthritic cartilage have revealed a direct correlation between disease severity and the degree of chondrocyte damage [Aigner and Kim, 2002; Murray et al., 2004]. Thus, chondrocyte insults are a critical factor in inducing joint degeneration. A variety of in- flammatory cytokines and reactive oxygen species (ROS) contribute to the pathophysi- ological regulation of chondrocytes [Mathy- Hartert et al., 2002; Kuhn et al., 2003]. Nitric oxide (NO), one of the ROS, has been implicated in the modulation of cartilage remodeling [Cake ß 2007 Wiley-Liss, Inc. This work was performed at the Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan. Grant sponsor: The National Science Council and Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan. *Correspondence to: Ruei-Ming Chen, PhD, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing St., Taipei 110, Taiwan. E-mail: rmchen@tmu.edu.tw Received 21 September 2006; Accepted 15 December 2006 DOI 10.1002/jcb.21268