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To determine whether the half-times (T1/2) of the DNA repair processes measured by alkaline elution increased in a dose-dependent manner, exponentially growing 9L/Ro rat brain tumor cells were irradiated with doses of 15-50 Gy, and their DNA repair kinetics was measured by alkaline elution. At 15 Gy, the DNA repair kinetics was biphasic with the fast phase having a T1/2 approximately 6 min and the slow phase having a T1/2 approximately 42 min. As the dose was increased to 50 Gy, the fast-phase T1/2 remained at approximately 6 min, but the slow-phase T1/2 increased to approximately 87 min. Although a dose-dependent increase in the T1/2 of the slow phase of DNA repair (saturation) was measured by alkaline elution, both the absolute value of the slow-phase T1/2 and the dependency of the slow-phase T1/2 on dose were less than those measured by alkaline sucrose gradient sedimentation in zonal rotors with slow reorienting gradient capability. Thus these two techniques appear either to depend on different hydrodynamic properties of the DNA or to have different coefficients of dependency for the same hydrodynamic properties of the DNA. The lower sensitivity for detection of the dose dependency of DNA repair makes it unlikely that the alkaline elution technique will be useful for quantitatively relating the shape of mammalian cell survival curves to the doses at which saturation of a DNA repair process occurs.

Previous work from our laboratory suggested that neuronal chromatin structure may be altered immediately after exposure to ionizing radiation. In the present study, whole brains of 4-month-old male Fisher 344 rats were irradiated with a dose of 25 Gy. The kinetics of restoring the chromatin structure to its unirradiated state was investigated in rat cerebellar neurons using three different approaches: (1) measurement of changes in the DNA superhelical structure by the fluorescent halo assay, (2) measurement of changes in chromatin accessibility to digestion by micrococcal nuclease, and (3) measurement of changes in the accessibility of the nuclear-matrix-associated DNA to digestion by DNase I. Immediately after irradiation, the topological constraints on the DNA loops were altered, the chromatin was more accessible to m. nuclease digestion, and the DNA associated with the nuclear matrix was more resistant to digestion by DNase I. Return of the chromatin structure to its unirradiated state as measured by each of the three methods followed biphasic kinetics with the fast phase having a half-time of several minutes and the slow phase having a half-time of several hours. The kinetics are similar to that previously reported for repair of radiation-induced DNA damage in mammalian cells. Although the independent assays used in this study seemed to follow the same kinetics, their relationship at the molecular level remains to be determined.

We have reported that the half-time (T1/2) for the slow phase of repair of radiation-induced DNA lesions increased with dose from 12.5 to 50.0 Gy in both 9L rat brain tumor cells and cerebellar neurons. In this manuscript these studies have been extended to lower doses to determine at what dose this DNA repair process becomes unsaturated. Our alkaline sucrose sedimentation technique in zonal rotors limits determination of the slow phase T1/2 to doses of greater than or equal to 6 Gy for 9L tumor cells and greater than or equal to 4 Gy for cerebellar neurons. The slow phase T1/2 in cerebellar neurons appears constant at doses less than or equal to 6 Gy and then increases exponentially at higher doses; survival of whole brain irradiated rats does not begin to decrease until doses greater than 6 Gy. The slow phase T1/2 in 9L tumor cells is either saturated or just becoming saturated at 6 Gy; a dose just before the final slope of the 9L survival curve is reached. These data support the hypothesis that the shape of mammalian cell survival curves is related to the saturation of a DNA repair process.