26 research outputs found
Alpha glutathione S-transferase as novel parameter for hepatocellular damage in the isolated perfused rat liver
Alpha glutathione S-transferase as novel parameter for hepatocellular damage in the isolated perfused rat liver
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 compartment
The role of xanthine oxidase in
Oxygen radicals have been proposed to be
involved in the induction of liver cell damage during
reperfusion after ischemia. The role of xanthine oxidase
in this process and the potential of the antioxidant
system have been studied in a model of in vivo ischemia
of rat liver followed by 1 h reperfusion by the use of
enzyme histochemistry. Based on decreased lactate
dehydrogenase activity in certain areas of liver
parenchyma, cell damage could already be detected at
1 h reperfusion after ischemia. Incubations performed on
serial sections showed that the same areas contained
decreased activities of xanthine oxidoreductase, xanthine
oxidase, catalase and glucose-6-phosphate dehydrogenase.
Some individual cells in the undamaged liver
parenchyma expressed a very high glucose-6-phosphate
dehydrogenase, which suggests that these cells have a
good defence against oxidative stress. It is concluded
that oxygen radicals derived from xanthine oxidase do
not play a decisive role in the induction of cell damage
immediately at reperfusion after ischemia. However, it
cannot be excluded that xanthine oxidase present in the
blood stream can give rise to the development of
additional damage later on
In situ detection of constitutive superoxide anion production in granules of mast cells
3,3'-Diaminobenzidine, in the presence of manganese and cobalt ions, was applied for the detection of superoxide anions in unfixed cryostat sections of rat oesophagus, trachea, skin and intact mesenterium. In all connective tissues, a blue final reaction product was found in a granular form in mast cells. The amount of final reaction product formed after incubation with diaminobenzidine and cobalt ions was increased by the addition of manganese ions. Electron microscopical analysis revealed that the electron-dense final reaction product was exclusively present in the granules of mast cells and on elastin fibres. It was found that the constitutive spontaneous formation of final reaction product in mast cells was enzymatic and dependent on the presence of oxygen in the medium. Of all the enzyme inhibitors and free radical scavengers tested, only azide strongly reduced the amount of final reaction product. It was concluded that the reaction was partly caused by peroxidase activity, but that superoxide anions are also constitutively and spontaneously produced in mast cell granules. The exact enzymatic source could not be established. Whether this property of mast cell granules plays an antimicrobial role in connective tissues can only be speculate
A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver
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