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 (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.