Improved anti-oxidant activity of superoxide dismutase by direct chemical modification

Chemically modified derivatives of superoxide dismutase (SOD), i.e., cationized (Cat-SOD) and mannosylated SOD (Man-SOD), were designed to improve an ability of SOD to suppress reactive oxygen species (ROS)-mediated injury in the alveolar epithelium. To evaluate their effectiveness, an in vitro model of paraquat poisoning was developed with primary cultured rabbit alveolar type II cells. Despite a 5.6-fold higher cellular association than native SOD, Man-SOD did not protect cell injury due to paraquat following evaluation by MTT assay. In contrast, Cat-SOD exhibited a 140-fold higher cellular association than native SOD and greatly suppressed paraquat-induced cell injury, as well as lipid peroxidation. Incubation with 300 U/ml Cat-SOD for 2 h increased intracellular SOD activity 5.3-fold. The increase in intracellular SOD activity was significantly inhibited in the presence of cytochalasin B, an endocytosis inhibitor. Internalization of Cat-SOD was also confirmed by confocal laser scanning fluorescein microscopy. In addition, the protective effect of Cat-SOD against paraquat-induced cell injury was completely abolished by the presence of cytochalasin B. In conclusion, this study demonstrated that cationization of SOD greatly enhances its intracellular delivery and, as a consequence, produces a significant protective effect against ROS-mediated injury of the alveolar epithelium.