DNA Double Strand Breaks in Ehrlich Ascites Tumour Cells at Low Doses of X-rays. I. Determination of Induced Breaks by Centrifugation at Reduced Speed

Summary

DNA double strand breaks (dsb) were determined in Ehrlich ascites tumour cells at doses down to 5 Gy. The method is based on the separation of DNA from other components by heating in a solution of pronase and detergents held in wide-mouth syringes, which were also used to facilitate the application of the released high molecular weight DNA to sucrose gradients. Purified DNA was sedimented in neutral sucrose gradients at low speed to reduce speed artifacts. The sedimentation profiles were analysed using a computer program and the number of dsb was determined by simulation of random breaks in the mass distribution of the control sample and by comparison of this simulated profile with that of the irradiated one. The number of dsb formed was proportional to X-ray dose in the range of 5 to 2000 Gy. The induction per dose was found to be nm^−1r D⁻¹ = (11·7 ± 2) × 10⁻¹² Gy⁻¹.