COMBINATION OF STATIC-FIELD GEL-ELECTROPHORESIS AND DENSITOMETRIC SCANNING FOR THE DETERMINATION OF RADIATION-INDUCED DNA DOUBLE-STRAND BREAKS IN CHO CELLS

An experimental setup using static-field gel electrophoresis (SFGE) wa s developed to determine radiation-induced DNA double-strand breaks (D SBs) in CHO-K1 cells after exposure to X-rays or heavy charged particl es. The fraction of DNA eluted into the gel matrix depends on the quan tity of DSBs introduced. In agreement with a recent report, SFGE and p ulsed-field electrophoresis were found to be equally sensitive in DSB detection. With radiolabeled DNA from cell cultures, the absolute amou nt of DNA migrating out of agarose plugs into the gel was quantified b y determining the radioactivity in the gel lane. Alternatively, relati ve measurements of the amount of DNA released into the gel were achiev ed with a standardized protocol for both SFGE and a subsequent densito metric scanning of photographic negatives from gels stained with ethid ium bromide. After calibration with the radioactive method, the fracti ons of DNA retained could be calculated directly from the data obtaine d with the densitometric assay to set up classical dose-effect curves. This procedure was validated for its application with heavy ions usin g an 500 MeV/u lead beam.