EVIDENCE SUGGESTING THAT IRON AND CALCIUM ARE INTERRELATED IN OXIDANT-INDUCED DNA-DAMAGE

The effect of iron chelators and agents that buffer cytosolic-free cal cium ([Ca2+]i) on hydrogen peroxide-induced DNA strand breaks in LLC-P K1 cells has not been previously examined. In addition, the interrelat ionship between iron and calcium in the pathogenesis of DNA damage has not been studied in any model of tissue injury. Exposure of LLC-PK, c ells to 1 mm hydrogen peroxide resulted in marked DNA damage, as measu red by the alkaline unwinding assay (residual intact double stranded D NA at 10 min, control: 88 +/- 1%; hydrogen peroxide-treated cells: 17 +/- 3%, N = 8). The iron chelators, 1,10-phenanthroline and deferoxami ne, and agents which buffer [Ca2+]i, BAPTA and quin-2, provided highly significant protection against hydrogen peroxide-induced DNA strand b reaks. We then examined the effect of iron chelators on hydrogen perox ide-induced rise in [Ca2+]i in LLC-PK1 cells. Both 1,10-phenanthroline and deferoxamine prevented the marked and sustained rise in [Ca2+]i i nduced by exposure of LLC-PK, cells to 1 mm hydrogen peroxide ([Ca2+]i at 15 min, control 100 +/- 3 nm; hydrogen peroxide 195 +/- 14 nm; 1,1 0-phenanthroline + hydrogen peroxide 100 +/- 4 nm; deferoxamine + hydr ogen peroxide 106 +/- 4 nm; N = 4). We excluded the possibility that t he iron chelators were directly chelating calcium by performing experi ments using a cell free system. We also confirmed that BAPTA and quin- 2, in concentrations used in our study, chelate calcium but not iron o r copper. Our data thus indicate a role for both iron and calcium in h ydrogen peroxide-induced DNA strand breaks, and suggest that the effec t of iron chelators in preventing DNA damage may be related at least i n part to their ability to prevent oxidant-induced rise in [Ca2+]i.