EVIDENCE FOR A HYPOXIC FIXATION REACTION LEADING TO THE INDUCTION OF SSB AND DSB IN IRRADIATED DNA

Purpose: To measure hypoxic chemical fixation processes of radiation d amage in both isolated plasmid DNA and in GSH-depleted E. coli cells. Materials and methods: Plasmid pBR322 DNA was irradiated with a single 5 ns pulse of 400 keV electrons under hypoxic conditions. At pre-set times, immediately before or after the electron pulse, the chamber con taining the DNA was exposed to a high-pressure shot of hydrogen sulphi de (H2S) gas. Results: DNA irradiated before contact with the H2S puls e was more sensitive to the production of both single strand breaks (s sb) and double strand breaks (dsb) than DNA irradiated after the addit ion of H2S. The post-irradiation protection of DNA by H2S was time-dep endent, having first-order rate constants of 21 s(-1) for ssb and 10 s (-1) for dsb. Conclusions: This is the first direct kinetic evidence f or the involvement of a hypoxic fixation reaction in the production of DNA damage by ionizing radiation. It indicates that long-lived radica l damage is induced in DNA which, even at times of 20-50 ms after irra diation, can be chemically repaired, or rescued, by the addition of a thiol agent. This reaction may partially explain the predicted decreas e in oxygen enhancement ratio (OER) with linear energy transfer (LET) on the basis of the increased clustering of radicals produced on the D NA by tracks of ionizing radiation. As radical multiplicity increases with LET there is a greater chance that some of the radicals will beco me fixed in the absence of oxygen leading to an increased probability of damage under hypoxia and a reduction in the OER.