Calcium Indicator Dye Quin2 Inhibits Hydrogen Peroxide-Induced DNA Strand Break Formation via Chelation of Iron

Exposure of HeLa cells to H2O2 at 4°C caused DNA strand breakage which was prevented by the metal chelator 1,10-phenanthroline, but not by neocuproine. This is believed to indicate the participation of iron, rather than copper, in the formation of reactive hydroxyl radicals (· OH) from H2O2. The calcium indicator dye Quin2 also prevented H2O2-induced DNA fragmentation. The inhibition of oxidant-induced DNA fragmentation at 37°C by Quin2 is often presented as evidence for the involvement of Ca2+-dependent endonucleases in damage. However, our finding that Quin2 inhibits DNA fragmentation at 4°C led us to investigate the possibility that Quin2 may also inhibit DNA damage via its effects on metal-dependent · OH formation. Using ESR spin trapping techniques and in vitro DNA oxidation measurements, we found that the binding of Fe by Ouin2 does not prevent · OH formation, but inhibits DNA damage. This is believed to reflect the prevention of iron ion binding to DNA by Quin2, directing · OH generation to the bulk solution, thereby preventing damage. The Cu-Quina complex was found to be a poor catalyst of both · OH formation and in vitro DNA damage. These findings suggest that the frequently reported protective effect of Quina toward DNA in cells exposed to oxidants may be due to the chelation of metal ions rather than the prevention of Ca2+-dependent endonuclease activation.