The effects of ionising radiation on deoxyribonucleic acid. Part 4. The role of hydrogen peroxide

Abstract

The direct and indirect mechanisms for ionising-radiation-induced damage to deoxyribonucleic acid (DNA) have been probed by e.s.r. spectroscopy and by analysis for strand breaks. Irradiation of frozen aqueous DNA was shown by e.s.r. spectroscopy to give guanine and thymine ion radicals (G+˙ and T–˙) together with hydroxyl radicals. The latter are trapped in the ice crystallites and do not interact with DNA even on annealing. Addition of H2O2 has been shown to affect the e.s.r. spectrum of the DNA phase. H2O2 competes with T for electron capture as indicated by reduction to TH˙. The production of ˙OH close to DNA is inferred by the appearance of e.s.r. features assigned to sugar radicals. On annealing, the sugar radicals were lost at temperatures significantly below those at which the DNA radicals centred on G and T normally react; this provides an explanation for the fact that sugar radical intermediates have not hitherto been detected in the decay of these primary DNA radicals. At high H2O2 concentrations HO2˙ was detected. Two pathways for its formation are proposed: (i) direct reaction of ˙OH with H2O2(ii) reaction with G+˙. In support of the latter, we observed reduction of G+˙ in the presence of H2O2. Concurrent strand-break analyses using pasmid DNA (pBR 322) under conditions similar to those of the e.s.r. experiments showed that the presence of H2O2 leads to a radiation-dependent enhancement of single- and double-strand breaks. The significance of these results to experiments in which the ionising radiation is fractionated as part of a freeze–thaw cycle is discussed.