Salicylate as an in vivo free radical trap: studies on ischemic insult to the rat intestine.

Ischemia of rat intestine was induced in vivo by occlusion of the superior mesenteric artery (SMA) for 15 min. Sodium salicylate, 100 mg/kg, given IP, 30 min prior to the ischemic event served as a specific trap for hydroxyl radicals. Portions of the bowel were sequentially isolated and removed--2 min prior to ischemia, 2 min prior to declamping of the SMA, and 10 min following reperfusion. The bowel segments were homogenized in 3% TCA. The homogenate was centrifuged and filtrated through a 0.22 mu filter. The hydroxylation products of salicylate, dihydroxybenzoic acid (DHBA) derivatives, were isolated, identified, and quantified by HPLC coupled with electrochemical detection (ECD). The level of 2,5-DHBA (M +/- SE, ng/g tissue) in the preischemic bowel (N = 21) was 241.8 +/- 10.0. In the ischemic specimen the level of 2,5-DHBA increased significantly to 313.3 +/- 15.5 (p = 0.0129), and remained unchanged in the reperfusion period (322.8 +/- 15.5). The histological examination correlated well with these levels: mild villi damage in the ischemic period with no further exacerbation during the reperfusion period. This study in an in vivo animal model of intestinal ischemia-reperfusion provides direct evidence for the involvement of free radicals during the ischemic insult.     

Gallium-desferrioxamine protects the cat retina against injury after ischemia and reperfusion

This study sought to determine whether gallium-desferrioxamine (Ga/DFO) can curb free radical formation and mitigate biochemical and electrophysiological parameters of injury in the cat retina subjected to ischemia followed by reperfusion.For the biochemical studies, cat eyes were subjected to 90 min of retinal ischemia followed by 5 min of reperfusion, and enucleation of one eye of each cat was used to measure retinal reperfusion injury. Before enucleation of fellow eyes, 2.5 mg/kg Ga/DFO was injected intravenously 5 min before reperfusion. The flux of hydroxyl radicals, as measured directly by conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA), was significantly lower in Ga/DFO-treated eyes. The mean normalized level of 2,3-DHBA (considered a specific marker of hydroxyl radicals) was 3.5 times higher in untreated eyes. Ga/DFO caused a significant reduction, by 2.56-fold, in lipid peroxidation, as reflected by levels of malondialdehyde. Ascorbic acid, a natural antioxidant present in the retina, is severely depleted in untreated eyes. In contrast, in Ga/DFO-treated eyes, levels were 10 times higher than the control. Energy charge was 2.38 times higher in treated eyes. Levels of purine catabolites (hypoxanthine, xanthine, and uric acid) that reflect excessive metabolism of purine nucleotides were approximately twice higher in untreated retinas. Electroretionographic studies, performed on a different subset of animals, substantiated the biochemical results. In Ga/DFO-treated eyes the amplitude of the mixed cone-rod response b-wave (as compared with fellow nonischemic eyes) fully recovered within 24 h after ischemia (b-wave ratio 1.04 +/- 0.09, [mean +/- SEM]) whereas ischemic/reperfused and nontreated eyes recovered to only 0.33 +/- 0. 05. The results show that severe biochemical and functional retinal injury occurs in cat eyes subjected to ischemia and reperfusion. These severe changes were significantly reduced by a single administration of Ga/DFO just before reperfusion. We hypothesize that the protection afforded by Ga/DFO is due to a combined effect of "Push-Pull" mechanisms interfering with transition metal-dependent and free radical-mediated injurious processes.     

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron - providing protection to the ischemic heart via preconditioning (PC). PC was accomplished by subjecting isolated rat hearts to three episodes of 2 min ischemia separated by 3 min of reperfusion. Prolonged ischemia followed the PC phase. PC hearts (group I) were compared to hearts subjected to normal perfusion (group II, no ischemia) and to ischemia without PC (group III). Group I showed a marked improvement in the recovery of hemodynamic function vs. group III. Biochemical parameters further substantiated the PC protection provided to group I against prolonged ischemia. Correspondingly, group I presented markedly lower re-distribution and mobilization of iron and copper into the coronary flow, following prolonged ischemia, as evinced from the decrease in total levels, and in the 'free' fraction of iron and copper. During the PC phase no loss of cardiac function was observed. A small wave of re-distribution and mobilization of iron (typically less than 4-8% of the value of 35 min ischemia) was recorded. The cellular content of ferritin (Ft) measured in the heart was significantly higher in group I than in group III (0.90 and 0.54 microg/mg, respectively). Also, iron-saturation of Ft was significantly lower for PC hearts, compared to both groups II and III (0.22 vs. 0.32 and 0.31 microg/mg, for 35 min ischemia, respectively). These findings are in accord with the proposal that intracellular re-distribution and mobilization of small levels of iron, during PC, cause rapid accumulation of ferritin - the major iron-storage protein. It is proposed that iron play a dual role: (i) It serves as a signaling pathway for the accumulation of Ft following the PC phase. This iron is not involved in cardiac injury, but rather prepares the heart against future high levels of 'free' iron, thus reducing the degree of myocardial damage after prolonged ischemia. (ii) High levels of iron (and copper) are mobilized following prolonged ischemia and cause tissue damage.     

Time window of nitroxide effect on myocardial ischemic-reperfusion injury potentiated by iron

Transition metals such as iron and copper potentiate the postischemic reperfusion (I/R) injury induced by oxygen-derived radical and nonradical toxic species (ROS). Various natural and synthetic antioxidants have been previously tested to ameliorate such injury, yet the limitations of the common antioxidants are well known. An alternative strategy for combating oxidative damage is presented wherein cell-permeable, nitroxide stable radicals, which act as SOD-mimics and oxidize reduced metals thus prompting the Fenton-like chemistry, are investigated for utility in ameliorating I/R injury. Our study concentrates on the early effect of nitroxide on the myocardial I/R injury. Isolated rat hearts in the Langendorff configuration were equilibrated with Krebs-Henseleit buffer and then subjected to 18 min of normothermic global ischemia followed by 20 min reperfusion. Iron administered as Fe(III)-citrate (10 microM) did not affect the cardiac function under normoxia but did potentiate I/R injury and decreased the recovery during reperfusion. The iron-induced damage was manifested by further deterioration of the cardiac hemodynamic function and the energy status as reflected by decreased tissue level of phosphorylated nucleotides. Nitroxide at 200 microM protected against the iron-potentiated I/R injury by improving the recovery of the hemodynamic function and the cardiac energy status. Exogenously added iron requires bioreduction to form deleterious Fe(II) bound to critical cellular sites. The nitroxide, which enters the cell and oxidizes the reduced metal instantaneously, provided protection even when administered 2 or 3.5, but not 5 min, after the onset of reperfusion. Thus, its narrow therapeutic time window provides insight into the schedule of the I/R injurious process.     

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron – providing protection to the ischemic heart via preconditioning (PC).PC was accomplished by subjecting isolated rat hearts to three episodes of 2 min ischemia separated by 3 min of reperfusion. Prolonged ischemia followed the PC phase. PC hearts (group I) were compared to hearts subjected to normal perfusion (group II, no ischemia) and to ischemia without PC (group III). Group I showed a marked improvement in the recovery of hemodynamic function vs. group III. Biochemical parameters further substantiated the PC protection provided to group I against prolonged ischemia. Correspondingly, group I presented markedly lower re-distribution and mobilization of iron and copper into the coronary flow, following prolonged ischemia, as evinced from the decrease in total levels, and in the 'free' fraction of iron and copper.During the PC phase no loss of cardiac function was observed. A small wave of re-distribution and mobilization of iron (typically less than 4–8% of the value of 35 min ischemia) was recorded. The cellular content of ferritin (Ft) measured in the heart was significantly higher in group I than in group III (0.90 and 0.54 g/mg, respectively). Also, iron-saturation of Ft was significantly lower for PC hearts, compared to both groups II and III (0.22 vs. 0.32 and 0.31 g/mg, for 35 min ischemia, respectively). These findings are in accord with the proposal that intracellular re-distribution and mobilization of small levels of iron, during PC, cause rapid accumulation of ferritin – the major iron-storage protein.It is proposed that iron play a dual role: (i) It serves as a signaling pathway for the accumulation of Ft following the PC phase. This iron is not involved in cardiac injury, but rather prepares the heart against future high levels of 'free' iron, thus reducing the degree of myocardial damage after prolonged ischemia. (ii) High levels of iron (and copper) are mobilized following prolonged ischemia and cause tissue damage.     

Correlation Between Destruction of Malarial Parasites by Polymorphonuclear Leucocytes and Oxidative Stress

The role of reactive oxygen species (ROS) generated by polymorphonuclear leucocytes (PMNs) in the host response against malaria was investigated. Non-activated human PMNs were added to cultures of P. falciparum in microtitre cells. Parasite viability was evaluated by the incorporation of radioactive hypoxanthine. Using PMN/RBC = 1/150 (starting parasitemia was 1+) the incorporation on the second day in culture was only 61+ of the control cultures. An effect could be observed already after two hours of incubation (30+ reduction at a 1/50 PMN/RBC ratio). A direct contact between the effector and target cells was obligatory for the expression of the damage.

Parasites within G6PD-deficient erythrocytes were more sensitive to the PMNs than normal parasitized erythrocytes. This difference could be attributed to the production of reactive oxygen intermediates in the experimental system, since G6PD-deficient erythrocytes are generally more sensitive to oxidant stress.

Salicylic acid was used as a scavenger and reporter molecule for hydroxyl radical fluxes. It is converted to the corresponding dihydroxybenzoic acid derivatives, which could be detected by HPLC. Uninfected NRBC or parasitized erythrocytes containing young ring forms could trigger the PMNs to produce much less ROS than the mature forms of the parasites. Other factors associated with PMNs may inactivate the parasites, such as phagocytosis, lysosomal enzymes or degradation toxic products of the PMNs. However our results indicate that increased oxidative stress induced by PMNs interfere with the growth of P. falciparum and could play a role in human evolution of abnormal erythrocytes.