Utilization of Spin-Trapping Reactions for Effective Detection of DNA Damage

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Biological oxidative stress is triggered by the reactions of oxygen radicals with DNA.Oxygen radicals first induced DNA radicals. The DNA radicals then react with oxygens to form peroxy radicals which proceed to turn out to be many kinds of DNA damage. Therefore, several methods have been developed as tools to qualitatively and quantitatively measure DNA final products. Since the kinds of DNA radicals are fewer than those of final products in mumber, it seems better to detect and identify DNA damage at the stage of radicals. In the present study the spin-trapping method was applied for this purpose. The spin trapping is a method to trap unstable, short-lived free radicals by spin trapping reagent. The spin-trapped raticals, the spin adducts, are subsequently analyzed by electron spin resonance spectrometry (ESR). i-Methyl-2-nitrosopropane (MNP) was used as a spin trapping reagent. X irradiation was employed to produce oxygen radicals which reacted with DNA.An effort to detect and identify the precursor radicals of both DNA strand breaks and 8-hydroxydeoxyadenosinn (oh^8dG) was made with spcial emphasis on the detection of precursors of chromosomal aberrations and point mutations, respectively. As a result, the formation of radicals at the C4' site of the sugar moiety and at the N7 site of the guanine base moiety were observed as precursor radicals of strand breaks and oh^8dG, respectively. Evidence for the C4' sugar radical as a precursor of strand breaks was further confirmed by experiments using two 10mers of oligonucleotides, oligo(dC)_<10> and oligo(dT)_<10>, as DNA models. The oligomers were ^<32>P-labeled at the 5' terminus and the numbers of oxygen-radical-induced strand breaks in the presence or absence of MNP were measured by bio-image analyzer BAS 2000. It was observed that the trapping of the C4' sugar radical by MNP surely suppressed the numbers of strand breaks in both oligomers.