Hydrogen peroxide-induced cell damage: The role of free radicals and iron complexes

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.This thesis presents data exploiting one of the important chemico-biological characteristics of the transition metal iron: its ability to exist in two oxidation states. Manipulation of the reactivity of iron by ligands has been reflected by the oxygen utilisation or the amount of ferrozine-detectable Fe2+. Ligand-dependent cell associated iron has been demonstrated by prussian blue staining and ferrozinedetectable iron. Iron/8-hydroxyquinoline enters the cell by diffusion, whereas iron dextran is endocytosed as is evident from extensive vesicular staining. The involvement of iron in the cytotoxicity of H202 has been examined (a) indirectly by introduction of reducing agents which restore the cytotoxicity of H202 at 4°C (an effect which is abolished by desferrioxamine) and (b) directly by extracellularly added iron, which enhances the cytotoxicity if the iron is internalised but protects the cell if the iron remains extracellular. Cytotoxicity has been estimated by plating efficiency and by a modified tritiated thymidine incorporation assay permitting a 24 hour delay before exposure to the label. Direct free radical interaction with a ligand itself is exemplified by the production of the nitroxide free radical in desferrioxamine. This causes extensive damage to yeast alcohol dehydrogenase but can be protected by ascorbate, methionine and iron/EDTA but not iron dextran. The findings lend further support for the suggestion that free radical generation by the Fenton reaction enhances the potential cytotoxic effect of H202 provided the reactions occur at critical sites within the cell.Financial support obtaind from Fisons Pharmaceuticals Plc