Metallothionein protects DNA from copper-induced but not iron-induced cleavage in vitro

Abstract

Iron and copper ions mediate generation of reactive oxygen radicals from O2 and H2O2 by the Fenton reaction: these radicals are capable of damaging DNA. We studied (a) the ability of these metals to induce double-strand breaks in DNA in vitro in the presence of H2O2 and ascorbic acid as donors of reactive oxygen, and (b) the ability of the metal-binding protein metallothionein (MT) to protect DNA from damage. Strand cleavage was measured by loss of fluorescence after binding to ethidium bromide and by increased mobility of DNA in agarose. The results show that Cu(II), Fe(II) and Fe(III) all can induce damage to calf thymus DNA under our experimental conditions. Cu(II)-induced DNA damage was dose-dependent and the degree of damage was proportional to the concentration of H2O2. On the other hand, DNA fragmentation was significant only in the presence of high concentrations of Fe(II) or Fe(III). Addition of Zn-MT to the reaction mixture prior to addition of Cu(II) inhibited fragmentation of DNA in a dose-dependent manner but had little effect on iron induced damage. Other proteins (histone or albumin) were not effective in protecting DNA from Cu-induced damage, as compared to Zn-MT. The formation of Cu(I) from Cu(II) in the presence of hydrogen peroxide and ascorbate was also inhibited by addition of Zn-MT. Thus, MT may protect DNA from damage by free radicals by sequestering copper and preventing its participation in redox reactions.