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

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 (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, 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.