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)-O-center dot), 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)-O-center dot and biologically effective targets. Furthermore, an assessment of plasmid DNA breakage showed that the Fenton reaction system did not effectively induce DNA breakage. The impact of the site of the Fenton reaction, i.e., hydroxyl radical ((OH)-O-center dot) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2), or pre-incubated with FeSO4 and then H2O2 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 FeSO4 exerted protective effects against H2O2-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO4 enhanced cell lethality induced by H2O2, whereas a pre-incubation with a high concentration of FeSO4 exerted protective effects. FeSO4 distributed extracellularly or on the surface of cells neutralized H2O2 outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of (OH)-O-center dot, occurred inside cells. An assessment of plasmid DNA breakage showed that (OH)-O-center dot induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated (OH)-O-center dot does not appear to be DNA.