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) was developed to determine radiation-induced DNA double-strand breaks (DSBs) in CHO-K1 cells after exposure to X-rays or heavy charged particles. The fraction of DNA eluted into the gel matrix depends on the quantity of DSBs introduced. In agreement with a recent report, SFGE and pulsed-field electrophoresis were found to be equally sensitive in DSB detection. With radiolabeled DNA from cell cultures, the absolute amount of DNA migrating out of agarose plugs into the gel was quantified by determining the radioactivity in the gel lane. Alternatively, relative measurements of the amount of DNA released into the gel were achieved with a standardized protocol for both SFGE and a subsequent densitometric scanning of photographic negatives from gels stained with ethidium bromide. After calibration with the radioactive method, the fractions of DNA retained could be calculated directly from the data obtained with the densitometric assay to set up classical dose-effect curves. This procedure was validated for its application with heavy ions using an 500 MeV/u lead beam.