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

Abstract

To clarify the mechanisms of intracellular induction of oxidative DNA damage, we have investigated the concentrations of intracellular reactive oxygen species and the amounts of 8-hydroxydeoxyguanosine (80HdG), a mutagenic oxidative DNA damage, in human neutrophil-like cells, dimethylsulfoxide-differentiated HL60 (DMSO-HL60). We determined intracellular concentrations of hydrogen peroxide and superoxide by flow cytometry with dichlorofluorescein diacetate and hydroethidine, respectively. We determined the 80HdG amounts with an electrochemical detector connected to HPLC after anaerobic sample processing. DMSO-HL60 releases superoxide upon stimulation with phorbol myristate acetate, and the released superoxide dismutates to hydrogen peroxide. Stimulation of DMSO-HL60 with 100 nM phorbol myristate acetate increased intracellular hydrogen peroxide, superoxide and 80HdG (control). Addition of 1000 U/ml catalase decreased hydrogen peroxide (31.3% of control) and 8OHCLG (20.3%). Addition of 100 U/ml SOD decreased superoxide (18.7%) and 80HdG (41.6%). Addition of 1 mM deferoxamine decreased 80HdG (30.4%), but increased hydrogen peroxide (129.6%). Addition of 200 μM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid decreased superoxide (59.9%) and 80HdG (42.0%). Addition of 0.4% ethanol had no effect on superoxide concentration (102.2%), but tended to decrease hydrogen peroxide (83.5%) and 80HdG (84.3%). Pretreatment of DMSO-HL60 with 0.1 mM FeSO4 increased 80HdG (117.3%), but decreased hydrogen peroxide (75.8%). These findings indicate that the extracellularly released superoxide and hydrogen peroxide diffuse into the cell, but that such reactive oxygen species are not the direct molecules to induce 80HdG. Our results suggest that 80HdG is induced by the hydroxyl radical which is generated from intracellular hydrogen peroxide and superoxide-reduced Fe.