This revised proposal seeks support to study pulmonary generation of oxygen radicals in a new model system -- the transendothelially perfused rat lung -- using three novel biochemical and histochemical methods to detect and quantify reactive oxygen species directly. The first employs dimethyl sulfoxide (DMSO) as a quantitative molecular probe for hydroxyl radicals (HO.), which oxidize DMSO to the stable, non-radical compound, methane sulfinic acid (MSA). MSA is then recovered from effluent lung perfusate and measured spectrophotometrically. The second method is an adaptation of Karnovsky's manganese technique for histochemical demonstration of superoxide generation in leukocytes. Superoxide oxidizes Mn++ to Mn+++, which in turn causes oxidation of diaminobenzidine to an extremely well localized, osmiophilic polymer, observable by both light and electron microscopy. The third is a high iron/diaminobenzidine histochemical technique, in which hydrogen peroxide oxidizes diethylenetriaminepentaacetate-chelated Fe++ to form intermediate species, which in turn oxidize DAB similarly to Mn+++. These measures of oxygen radical formation will be correlated with LDH release and standard electron microscopy as indices of functional and structural lung injury. Preliminary results with these methods indicate substantial (= 100 mu mole/kg fresh lung) HO. generation in postischemic lung during the first 5 min of reoxygenation, with localization of oxygen radical production to pulmonary vascular endothelial cells. Histochemically, superoxide dismutase strongly inhibited Mn++/DAB reaction produce formation and catalase strongly inhibited Fe++/DAB reaction produce formation, providing direct chemical and visual evidence of the existence of the heretofore putative burst of reactive oxygen species in intact, postischemic lung. The focus future of research will be evaluation of the oxygen radical hypothesis of pulmonary reperfusion injury. Specifically, we propose (1) to characterize the time course of oxidant formation after ischemia using our three novel methods -- the biochemical DMSO trapping technique for HO. and the histochemical Mn/diamine and Fe/diamine techniques for O2. and H2O2, (2) to examine the effects of added granulocytes on the time course of oxygen radical formation, (3) to correlate radical production with conventional measures of tissue injury in the presence of varying oxygen tensions during re-oxygenation after varying durations of ischemia, and (4) to compare oxygen radical generation in lung tissue after ischemia with that in perfused rat heart, kidney, and liver. These studies will allow us to test and elaborate the free radical hypothesis of lung injury associated with ischemia and reperfusion, using new methods that directly demonstrate the generation of reactive oxygen species in tissues.
