Localization of superoxide dismutase activity in rat tissues

Superoxide anion radicals have been implicated in a variety of pathological processes. Under physiological conditions, superoxide dismutase (SOD) is effectively able to disproportionate superoxide anions into hydrogen peroxide and dioxygen. Until now, no techniques have been available to localize SOD activity within tissues. In the present study, SOD activity was detected in different rat tissues using a thin film of xanthine oxidase between the glass slide and the unfixed cryostat section and a medium containing hypoxanthine as a source of electrons for the production of superoxide anions. The incubation medium also contained cerium ions to precipitate the hydrogen peroxide product and polyvinyl alcohol to prevent leakage of soluble and/or loosely bound enzymes from the sections into the incubation medium. The cerium perhydroxides that are formed were visualized for the light microscope in a second step using an incubation medium consisting of 3,3'-diaminobenzidine, cobalt ions, and hydrogen peroxide, which results in oxidation of the diaminobenzidine to the final insoluble blue reaction product. By this methodology, high enzyme activity was found not only in endothelial cells of liver and kidney but also in hepatocytes of liver, myocytes of heart, smooth and striated cells of muscle, acinar cells of pancreas, epithelial cells of kidney ducts, and epithelial cells of the small intestine and colon. These findings were largely in agreement with immunohistochemical data obtained using antibodies against the Cu/Zn- and Mn-SODs. However, high activity was also detected extra-cellularly at the surface of epithelia of trachea, esophagus, small intestine, and colon and at the extracellular matrices, cartilage, and connective tissues. We conclude from these latter data that the activity of the extracellular form of the dismutase is localized. The present method allows the analysis of all three types of known SOD activity (Cu/Zn, Mn, and extracellular) in different tissues and cell compartments.