ACCELERATED PAPER: Relationship between the intracellular reactive oxygen species and the induction of oxidative DNA damage in human neutrophil-like cells

To clarify the mechanisms of intracellular induction of oxidativeDNA damage, we have investigated the concentrations of intracellularreactive oxygen species and the amounts of 8-hydroxydeoxyguanosine(8OHdG), a mutagenic oxidative DNA damage, in human neutrophil-likecells, dimethylsulfoxide-differentiated HL60 (DMSO-HL60). Wedetermined intracellular concentrations of hydrogen peroxideand superoxide by flow cytometry with dichlorofluorescein diacetateand hydroethidine, respectively. We determined the 8OHdG amountswith an electrochemical detector connected to HPLC after anaerobicsample processing. DMSO-HL60 releases superoxide upon stimulationwith phorbol myristate acetate, and the released superoxidedismutates to hydrogen peroxide. Stimulation of DMSO-HL60 with100 nM phorbol myristate acetate increased intracellular hydrogenperoxide, superoxide and 8OHdG (control). Addition of 1000 U/mlcatalase decreased hydrogen peroxide (313% of control) and 8OHdG(20.3%). Addition of 100 U/ml SOD decreased superoxide (18.7%)and 8OHdG (41.6%). Addition of 1 mM deferoxamine decreased 8OHdG(30.4%), but increased hydrogen peroxide (129.6%). Additionof 200 µM 4-acetamido-4-isothiocyanostilbene-2, 2'-disulfonicacid decreased superoxide (59.9%) and 8OHdG (42.0%). Additionof 0.4% ethanol had no effect on superoxide concentration (102.2%),but tended to decrease hydrogen peroxide (83.5%) and 8OHdG (84.3%).Pretreatment of DMSO-HL60 with 0.1 mM FeSO₄ increased 8OHdG(1173%), but decreased hydrogen peroxide(75.8%). These findingsindicate that the extracellularly released superoxide and hydrogenperoxide diffuse into the cell, but that such reactive oxygenspecies are not the direct molecules to induce 8OHdG. Our resultssuggest that 8OHdG is induced by the hydroxyl radical whichis generated from intracellular hydrogen peroxide and superoxide-reducedFe.