Measurement of DNA Double-strand Breaks in CHO Cells at Various Stages of the Cell Cycle Using Pulsed Field Gel Electrophoresis: Calibration by Means of125I Decay

Abstract

Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of ¹²⁵I decay accumulation, under conditions preventing repair, and is based on the observation that each ¹²⁵I decay in the DNA produces approximately one dsb. Iodine was incorporated into DNA in the form of 5′-iododeoxyuridine and decay accumulation was allowed in cells kept frozen at − 80°C. Since widely different DNA damage dose-response curves were obtained in cells exposed to X-rays in various phases of the cell cycle, calibration was performed using synchronized populations of cells that were allowed to accumulate DNA damage in G₁, G₁/S, mid-S, and G₂ + M. For this purpose the fraction of activity (in DNA) released from the plug (FAR) was measured and correlated to the number of ¹²⁵I decays accumulated during the elapsed period of time. Fluctuations in the FAR per ¹²⁵I decay were observed throughout the cell cycle that were similar to those previously reported for the FAR per Gy of X-rays. Comparison of the FAr per ¹²⁵I decay with the FAR per Gy gave an induction of 21 ± 3, 31 ± 3, 21 ± 3 and 26 ± 8 dsb per Gy per diploid DNA complement for G₁, G₁/S, S, and G₂ + M cells, respectively. The results suggest that the observed fluctuations in the FAR per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. We propose that similar phenomena underlie the observed fluctuations throughout the cell cycle in the fraction of activity eluted per Gy in the non-unwinding filter elution assay. ¹²⁵I decays accumulated at 4°C in partly purified DNA from cells embedded in agarose plugs and lysed immediately, gave FAR identical to those obtained with cells kept frozen. This finding suggests that indirect effects do not significantly contribute to DNA damage induction by ¹²⁵I decay, and indicate that calibration of electrophoresis techniques for dsb measurements can be carried out using this simplified protocol.