Abstract
DNA damage induction dose response curves were measured in synchronous and plateauphase CHO cells following exposure to sparsely ionizing radiation using pulsed-field gel electrophoresis and non-unwinding filter elution; assays that have been shown to respond specifically to the presence of double strand (dsb) breaks in the DNA. The fraction of DNA-associated activity released (FAR) from the agarose plug, where cells were embedded, and the fraction of activity eluted (FAE) from the filter were measured and used as parameters for the quantitation of the DNA damage present after pulsed-field gel electrophoresis and non-unwinding filter elution, respectively. Dose response curves for FAR and FAE were, as expected in general sigmoid, but under certain lysis conditions, the data obtained in the low dose region could be fitted by straight lines. FAR and FAE per Gy fluctuated throughout the cell cycle in a quantitatively similar manner suggesting that both techniques respond to similar cell cycle associated effects. High values were obtained for these parameters in cells irradiated in G1 or G2+M; the values decreased as cells entered S, and reached a minimum in the middle of this phase. Calibration of pulsed field gel electrophoresis by means of 125I-decay demonstrated that the fluctuations in the FAR per unit dose do not reflect variations in the production of DNA dsb. It is, therefore, proposed that the observed phenomena reflect alterations in the physicochemical properties of the DNA induced by the inception of replication, and caused by the presence of forks and eyes in the molecules. In both assays, the resulting DNA damage induction dose response curves could be altered by modifying the lysis conditions employed. In general, the sensitivity of the assay increased when NLS (sodium-N-laurylsarcosine) was substituted for SDS (sodium dodecyl sulphate) and when high temperatures (50–60°C) were used during lysis. These findings indicate that correlations established between the magnitude of the generated signal, by either technique, and DNA dsb, hold only for the particular cell population used and the specific lysis conditions employed. Extrapolation of such correlations for the interpretation of results obtained with different cell populations treated under different conditions may lead to erroneous conclusions.
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Iliakis, G., Metzger, L., Okayasu, R., Pantelias, G., Cicilioni, O. (1991). Measurement of DNA Double Strand Breaks in Mammalian Cells: Comparison Between Pulsed Field Gel Electrophoresis and Non-Unwinding Filter Elution. In: Fielden, E.M., O’Neill, P. (eds) The Early Effects of Radiation on DNA. NATO ASI Series, vol 54. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-75148-6_6
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DOI: https://doi.org/10.1007/978-3-642-75148-6_6
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