Evidence suggesting that iron and calcium are interrelated in oxidant-induced DNA damage

The effect of iron chelators and agents that buffer cytosolic-free calcium ([Ca²⁺]_i) on hydrogen peroxide-induced DNA strand breaks in LLC-PK₁ cells has not been previously examined. In addition, the interrelationship between iron and calcium in the pathogenesis of DNA damage has not been studied in any model of tissue injury. Exposure of LLC-PK₁ cells to 1 mM hydrogen peroxide resulted in marked DNA damage, as measured by the alkaline unwinding assay (residual intact double stranded DNA at 10 min, control: 88 ± 1%; hydrogen peroxide-treated cells: 17 ± 3%, N = 8). The iron chelators, 1,10-phenanthroline and deferoxamine, and agents which buffer [Ca²⁺]_i, BAPTA and quin-2, provided highly significant protection against hydrogen peroxide-induced DNA strand breaks. We then examined the effect of iron chelators on hydrogen peroxide-induced rise in [Ca²⁺]_i in LLC-PK₁ cells. Both 1,10-phenanthroline and deferoxamine prevented the marked and sustained rise in [Ca²⁺]; induced by exposure of LLC-PK₁ cells to 1 mM hydrogen peroxide ([Ca²⁺]_i at 15 min, control 100 ± 3 nM; hydrogen peroxide 195 ± 14 nM; 1,10-phenanthroline + hydrogen peroxide 100 ± 4 nM; deferoxamine + hydrogen peroxide 106 ± 4 nM; N = 4). We excluded the possibility that the iron chelators were directly chelating calcium by performing experiments using a cell free system. We also confirmed that BAPTA and quin-2, in concentrations used in our study, chelate calcium but not iron or copper. Our data thus indicate a role for both iron and calcium in hydrogen peroxide-induced DNA strand breaks, and suggest that the effect of iron chelators in preventing DNA damage may be related at least in part to their ability to prevent oxidant-induced rise in [Ca²⁺J_i.