Ischemia-induced brain iron delocalization: effect of iron chelators.

Tissue damage in cerebral ischemia may be produced by acidosis-induced delocalization of intracellular iron which acts as a catalyst in oxidative reactions. Acidosis was induced either by homogenization and incubation of rat cortical homogenates in acidified buffers or by submitting hyperglycemic rats to complete ischemia, a procedure that leads to intracellular lactic acidosis. The level of low molecular weight species (LMWS) iron was measured after filtration of tissue homogenates through a 10,000 Mr ultrafiltration membrane. When cortical tissue was homogenized in buffer pH 7, the level of LMWS iron was equal to 0.21 microgram/g. It was significantly enhanced by acidification of the homogenization medium, reaching 0.34 microgram/g at pH 6 and 0.75 microgram/g at pH 5. When the tissue was homogenized in water, the LMWS iron level reached 0.17 microgram/g in normoglycemic rats and 0.38 microgram/g (p < .05) in hyperglycemic rats. Both aerobic incubation of homogenates for 1 h at 37 degrees C and inclusion of EDTA in the homogenization medium led to further increases in the iron level. In order to demonstrate the deleterious role of iron in brain ischemia, the effect of treatment with bipyridyl, an iron-chelating agent, was assessed by measuring regional brain edema by the specific gravity method, 24 h following induction of thrombotic brain infarction. The treatment significantly attenuated the development of brain edema, reducing the water content of the infarcted area by about 2.5%. Taken together, these results support the hypothesis that a significant component of brain ischemic injury involves an iron-dependent mechanism.

Effect of intracellular iron loading on lipid peroxidation of brain slices.

The effect of artificially elevated cell iron content on oxygen-derived free radical production was assessed in brain slices by use of an iron ligand, 8-hydroxyquinoline (HQ). The iron complex Fe(3+)-HQ exhibited a high lipid solubility evidenced by n-octanol/water partition coefficient and was avidely taken up by brain slices. The catalytically active form of Fe3+ within the complex was evidenced by measuring the rate of ascorbate oxidation. Lipid peroxidation was assessed by measuring the thiobarbituric acid-reactive substances (TBARS) in brain homogenates or slices exposed to two doses of Fe(3+)-HQ (10 microM/20 microM, 100 microM/200 microM) or Fe(3+)-citrate (10 microM, 100 microM). Addition of the iron complexes to homogenates or slices resulted in a dose-dependent increase in lipid peroxidation. In homogenates, the effects were grossly similar with both complexes, whereas in slices the effects of Fe-HQ were significantly higher than those of Fe-citrate. Lipid peroxidation persisted in washed slices preexposed to Fe-HQ, but not in slices preexposed to the hydrophilic iron complex Fe-citrate. Fe-HQ-induced lipid peroxidation in slices was enhanced in the presence of H2O2, an effect that was not seen using Fe-citrate. Addition of Fe-HQ to brain homogenates in the presence of salicylic acid resulted in the production of 2,3-dihydroxybenzoic acid and the effect was potentiated in the presence of H2O2. This model of iron cell loading may be useful for evaluating the efficacy of antioxidant drugs.

Effects of increasing intracellular reactive iron level on cardiac function and oxidative injury in the isolated rat heart.

Elevation of cell iron content was produced by use of a lipophilic iron ligand, 8-hydroxyquinoline (HQ), capable of transferring catalytically active iron into cells. The Fe(3+)-HQ complex labeled with 59Fe was avidly taken up by isolated perfused hearts contrary to the hydrophilic complex Fe(3+)-citrate. Hearts perfused in aerobic conditions with Krebs-Henseleit buffer were exposed for 15 min to the iron complexes, Fe(3+)-HQ (5 microM/10 microM and 10 microM/20 microM), or Fe(3+)-citrate (10 microM), and then perfused for 30 min with normal buffer. Exposure to the high dose of Fe(3+)-HQ (10 microM/20 microM) resulted in early and irreversible decreases in coronary flow and heart rate (-48% and -33%, respectively), initial increases followed by decreases in left ventricular systolic pressure and +dP/dt, and increase in left ventricular end-diastolic pressure (+80%). The low dose of Fe(3+)-HQ (5 microM/10 microM) mimicked with a lower magnitude the effects of the high dose, whereas Fe(3+)-citrate had no effects on cardiac parameters. Only hearts exposed to the high dose of Fe(3+)-HQ exhibited a significant increase (+60%) in thiobarbituric acid-reactive substance level, an index of lipid peroxidation. The production of hydroxyl radicals was investigated by measuring 2,3-dihydroxybenzoic acid level in the coronary effluent after addition of salicylic acid (1 mM) in the perfusate. An immediate and high increase (x6) was seen during heart exposure to Fe(3+)-HQ (10 microM/20 microM) and to Fe(3+)-citrate (10 microM). Considering Fe(3+)-citrate had no effect on cardiac function and lipid peroxidation it was concluded that this hydroxyl radical formation occurring in the extracellular space was not implicated in Fe(3+)-HQ-induced cardiac dysfunction. These results demonstrate the deleterious effect of increasing intracellular reactive iron level in non-ischemic hearts.