Jp. Banath et al., Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase, RADIAT RES, 151(5), 1999, pp. 550-558
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.