Requirement of endogenous iron for cytotoxicity caused by hydrogen peroxide in Euglena gracilis

It is widely thought that redox-active metals in cells such as iron or copper catalyze the reduction of hydrogen peroxide to toxic hydroxyl radicals or their equivalent. However, this has not been directly demonstrated in vivo. To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intracellular iron content can be modulated by the concentration of iron in the defined growth medium without effect on the proliferation rate of the cells. E. gracilis contains two large storage pools of cytosolic iron which can be monitored to assess cellular iron status. The toxicity of H₂O₂ in E. gracilis was inversely related to the amount of iron in the extracellular medium. At high levels of external iron, the metal carried out the Fenton reaction with H₂O₂ outside the cell, producing hydroxyl radicals as detected by electron-spin resonance spin-trapping experiments. This reaction reduced the amount of H₂O₂ that could diffuse into cells to cause toxicity. When cells with different intracellular iron content were placed in iron-deficient media, the the toxicity of H₂O₂, measured by inhibition of proliferation, was directly related to the concentration of internal iron. Using cells deficient in iron, this oxidant did not inhibit proliferation at low concentration but was somewhat effective at higher concentrations. Although the iron chelating agents, 1,10-phenanthroline and desferrioxamine, also depressed cytosolic iron, they were cytotoxic to E. gracilis and so could not be used unambiguously to examine the role of intracellular iron in the toxic effects of hydrogen peroxide.