Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

Summary

The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O ^⋅₂ ⁻ radical itself, generated byγ-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O ^⋅₂ ⁻. Addition of H₂0₂ to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O ^⋅₂ ⁻ becomes comparable to that initiated by^⋅OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O ^⋅₂ ⁻ into^⋅OH radicals directly at the target molecule,

$$\begin{gathered} DNA - Cu^{2 + } + O_2 ^{ \cdot - } \to DNA - Cu^ + + O_2 \hfill \\ DNA - Cu^ + + H_2 O_2 \to DNA \cdots OH + Cu^2 + OH^ - \hfill \\ \end{gathered} $$

in a “site-specific” mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs withoutγ-irradiation by autocatalysis of DNA/Cu(II)/H₂0₂ systems. In this context we observed that Cu(II) in the DNA-Cu²⁺ complex (unlike free Cu²⁺) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu²⁺/Cu⁺ couple appears to be higher than that of the Fe³⁺/Fe²⁺ couple when the ions are complexed with DNA.

Metal-catalysed DNA damage by O ^⋅-₂ also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II)) efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.