Suppression by cobalt (II) and enhancement by iron (II) ions of DNA damage induced by 2,2'-azobis (2-amidinopropane) dihydrochloride
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Plasmid DNA damage induced by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and the effects of Co(II) and Fe(II) ions on this damage were investigated under aerobic conditions by agarose gel electrophoresis. DNA singlestrand breaks were observed depending on AAPH concentration and treatment time. DNA damage induced by treatment with 10 mM AAPH for 2 hours at 37°C was not inhibited by superoxide dismutase (<200 U), catalase (<200 U), or mannitol (<200 mM), although the damage slightly tended to be decreased by 200 U superoxide dismutase. Damage was inhibited by EDTA or deferoxamine in a concentration-dependent manner. These results suggested the involvement of superoxide anion and metal ions in damage formation. When DNA was incubated with 1mM AAPH, DNA damage was enhanced by 0.001-0.1 mM Fe(II) ion, whereas damage induced by AAPH and Fe(II) ion was totally resolved by 0.1 mM deferoxamine. In contrast, ≥1 mM concentration of Co(II) ion had a suppressive effect on AAPH-induced damage. Co(II) ion in the concentration range of 0.01-0.1 mM suppressed DNA damage induced by both 5 mM AAPH and 0.1 mM Fe(II) ion ; however, a higher concentration (≥5 mM) of Co(II) ion enhanced DNA damage and caused severe DNA breaks, making the DNA bands invisible even in the absence of AAPH. Spectrophotometric analysis suggested the direct interaction of AAPH and Co(II) ion. The above results suggested that Co(II) ion has biphasic effects on DNA damage induced by AAPH depending on its concentration, although Fe(II) ion simply enhances the AAPH-induced DNA damages.
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