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-phenant hroline (OF) to compete with ligands for iron. HL-60 cells were treate d with various concentrations of OP for 30 min at 37 degrees C followe d by 30 min treatment with H2O2 or t-butylhydroperoxide (tert-BuOOH) a t 4 degrees C. Single-strand breaks (SSBs) were measured by alkaline e lution. OP (5 mu M) completely inhibited production of SSBs by tert-Bu OOH This contrasts with results for H2O2, for which it was not possibl e to inhibit all SSBs even at OP concentrations up to 60 mu M. Inducti on of SSBs by tert-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 OF, such as by format ion of Fe(OP)(3). In contrast, after subtraction of the noninhibitable fraction, SSBs induced by H2O2 decreased according to a 1:1.53 +/- 0. 26 ratio. This suggests that inhibition of H2O2-induced SSBs is partia lly or wholly achieved by formation of different structures than Fe(OP )(3). Tert-BuOOH did not cause SSBs in nuclei isolated from HL-60 cell s. However, H2O2 induced SSBs which were inhibited 45 +/- 8% (+/- 1 SD ) by dimethyl sulfoxide. Analysis of the concentration dependence of i nhibition by dimethyl sulfoxide indicated that the H2O2-reactive iron in nuclei possessed an average distance of 4.8 nm from DNA This compar es with a previously obtained distance in whole cells of 6.9 nm, for w hich 5 mu M OP pretreatments decreased to 4.8 ma, These data indicate that tert-BuOOH generates DNA-damaging radicals through reaction with a subset of the different cell iron species which react with H2O2. (C) 1996 Academic Press, Inc.