Inhibition of Hydroperoxide-Induced DNA Single-Strand Breakage by 1,10-Phenanthroline in HL-60 Cells: Implications for Iron Speciation

The speciation of intracellular iron which reacts with hydroperoxides to cause DNA damage was investigated in HL-60 cells using 1,10-phenanthroline (OP) to compete with ligands for iron. HL-60 cells were treated with various concentrations of OP for 30 min at 37°C followed by 30 min treatment with H2O2ort-butylhydroperoxide (tert-BuOOH) at 4°C. Single-strand breaks (SSBs) were measured by alkaline elution. OP (5 μM) completely inhibited production of SSBs bytert-BuOOH. This contrasts with results for H2O2, for which it was not possible to inhibit all SSBs even at OP concentrations up to 60 μM. Induction of SSBs bytert-BuOOH decreased according to a SSB:OP ratio of 1:3.28 ± 0.23 (± 1 SE). This is consistent with inhibition achieved by occupation of all coordination sites on iron by OP, such as by formation of Fe(OP)3. In contrast, after subtraction of the noninhibitable fraction, SSBs induced by H2O2decreased according to a 1:1.53 ± 0.26 ratio. This suggests that inhibition of H2O2-induced SSBs is partially or wholly achieved by formation of different structures than Fe(OP)3.Tert-BuOOH did not cause SSBs in nuclei isolated from HL-60 cells. However, H2O2induced SSBs which were inhibited 45 ± 8% (±1 SD) by dimethyl sulfoxide. Analysis of the concentration dependence of inhibition by dimethyl sulfoxide indicated that the H2O2-reactive iron in nuclei possessed an average distance of 4.8 nm from DNA. This compares with a previously obtained distance in whole cells of 6.9 nm, for which 5 μMOP pretreatments decreased to 4.8 nm. These data indicate thattert-BuOOH generates DNA-damaging radicals through reaction with a subset of the different cell iron species which react with H2O2.