Importance of Locations of Iron Ions to Elicit Cytotoxicity Induced by a Fenton-Type Reaction

SIMPLE SUMMARY: The Fenton reaction generates the hydroxyl radical ((•)OH), which is the most reactive and toxic reactive oxygen species and widely recognized as a key player in oxidative stress. To clarify whether this highly reactive molecule travels to its biological target, the effects of the site of the Fenton reaction on cytotoxicity were investigated. Cytotoxicity induced by the Fenton reaction was affected by the distribution of iron ions surrounding and/or being incorporated into cells. Cytotoxicity was enhanced when the Fenton reaction occurred inside cells. Instead of enhancing cytotoxicity, extracellular iron ions exerted protective effects against the cytotoxicity of extracellular hydrogen peroxide in an ion concentration-dependent manner. Distance had a negative impact on the reactivity of extracellular (•)OH and biologically effective targets. Furthermore, an assessment of plasmid DNA breakage showed that the Fenton reaction system did not effectively induce DNA breakage. ABSTRACT: The impact of the site of the Fenton reaction, i.e., hydroxyl radical ((•)OH) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO(4)) and hydrogen peroxide (H(2)O(2)), or pre-incubated with FeSO(4) and then H(2)O(2) was added after medium was replaced to remove iron ions or after the medium was not replaced. Cell lethality in rat thymocytes was estimated by measuring cell sizes using flow cytometry. High extracellular concentrations of FeSO(4) exerted protective effects against H(2)O(2)-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO(4) enhanced cell lethality induced by H(2)O(2), whereas a pre-incubation with a high concentration of FeSO(4) exerted protective effects. FeSO(4) distributed extracellularly or on the surface of cells neutralized H(2)O(2) outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of (•)OH, occurred inside cells. An assessment of plasmid DNA breakage showed that (•)OH induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated (•)OH does not appear to be DNA.