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.
Ishimoto
Nanako
N
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Nemoto
Takayuki
T
Nagayoshi
Keiko
K
Yamashita
Fumiyoshi
F
Hashida
Mitsuru
M
D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002412:Cations / Q000737:chemistry; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D003571:Cytochalasin B / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D007205:Indium Radioisotopes; D015227:Lipid Peroxidation / Q000187:drug effects; D008358:Mannose / Q000737:chemistry; D018613:Microscopy, Confocal; D010269:Paraquat / Q000633:toxicity; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors
D000975:Antioxidants; D002412:Cations; D007205:Indium Radioisotopes; D017382:Reactive Oxygen Species; D003571:Cytochalasin B; D013482:Superoxide Dismutase; D016650:Fluorescein-5-isothiocyanate; D008358:Mannose; D010269:Paraquat