Cytotoxicity of Superoxide and Nitric Oxide (NO)

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1. MEASUREMENT OF SUPEROXIDE,NITRIC OXIDE (NO) and ONOO^-The generation of superoxide, NO and ONOO^- from human polymorphonuclear leukocytes were measured. For superoxide cytochrome c reduction and MCLA chemiluminescence methods were used. For NO,end uprodct of NO,nitrite+nitrate was measured by Griess method after reducing nitrite to nitrate. For ONOO^- we established a stable method by modifying the previously reported HPA-NO_2-HPLC method. Using these methods, the roles of these active molecules in various diseases were investigated and we found that the plausible cytotoxic molecules in polymorphonuclear leukocytes-mediated injury appears to be ONOO^- when the target of leukocytes is cardiac myocytes but superoxide is the major cytotoxic molecule when the target of leukocytes is endothelial cells.2. REAL-TIME MEASUREMENT OF SUPEROXIDE,NO and ONOO^-The real-time measurement of above mentioned molecules from isolated organs were also performed to investigate the cytotoxic mechanisms u … More nderlying the ischemia-reperfusion injury. Among several methods applied, we found that the oxymyoglobin method was applicable for that purpose.3. DEMONSTRATION OF ONOO^--MEDIATED INJURYTo demonstrate ONOO^--mediated tissue damage in various diseases such as liver cirrhosis, stomach ulcer and intestinal cancer, nitrotyrosine which is the reaction product of ONOO^- and tyrosine residues of protein was measured. Plasma and tissue concentration of nitrite+nitrate were also measured. The immunohistological staining using nitrotyrosine monoclonal antibodies was also performed for tissue samples obtained at the time of resection or through biopsy. The nitrotyrosine and nitrite+nitrate concentrations were significantly elevated in ulcer tissue. The increase correlated with severity ulcer. In contrast these levels remained low in the center of cancer and slightly elevated in the marginal portion of cancer. The nitrotyrosine staining method gave some positive staining in diseased portion but the specificity was shown to need further improvement. Less