Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase

X-ray-induced DNA base damage can be detected using endonuclease III and fo rmamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sen sitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method t hat detects DNA damage in individual cells, In using this approach to measu re the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiat ion-induced strand breaks, base damage was detected more easily after highe r doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen p eroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently w hen few direct strand breaks were present, Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as di rect strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximate to 1.0, and (2) the presence of direct DNA strand breaks (>2000-1000 per cell) prevents acc urate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method. (C) 1999 by Radiation Research Society.