EVIDENCE FOR INVOLVEMENT OF MULTIPLE IRON SPECIES IN DNA SINGLE-STRAND SCISSION BY H2O2 IN HL-60 CELLS

Some of the properties of cellular iron species which react with H2O2 to cause DNA single-strand breaks in HL-60 cells were characterized in control cells and in cells made deficient of iron using 4,7-phenylsul fonyl-1,10-phenanthroline (bathophenanthroline disulfonic acid or BPS) and ascorbate. Single-strand breaks were measured using alkaline elut ion of DNA of cells treated at 4 degrees to minimize repair during tre atment. Strand breakage in the presence of 10% serum was only 40% of t hat in the absence of serum. This effect was traced to reaction of H2O 2 with metals, most likely iron, in serum. Dimethyl sulfoxide (Me(2)SO ) inhibited a maximum of 65% of breaks in control cells. The diffusion distance from the site of generation of hydroxyl radicals to the site of reaction with DNA for the Me(2)SO-inhibitable fraction was 6.9 nm. There was no significant alteration in the fraction of Me(2)SO-inhibi table strand breaks or in diffusion distance in iron-deficient cells, though total strand breaks decreased by 70%. When the effect of extrac ellular iron in serum was taken into account, 60 mu M orthophenanthrol ine (OP) inhibited a maximum of 85% of strand breaks. In cells pretrea ted with 60 mu M OP, the Me(2)SO-inhibitable fraction of the remaining strand breaks decreased to 32%, while the diffusion distance decrease d to 4.1 nm. These data indicate the existence of a number of differen t iron species, as characterized by overlapping but not coincidental i nhibition by OP and Me(2)SO, and by differing diffusion distances.