DNA double-strand break distributions in X-ray and alpha-particle irradiated V79 cells: Evidence for non-random breakage

Many studies have shown that with increasing LET of ionizing radiation the RBE (relative biological effectiveness) for dsb (double strand breaks) induction remains around 1.0 despite the increase in the RBE for cell killing. This has been attributed to an increase in the complexity of lesions, classified as dsb with current techniques, at multiply damaged sites. This study determines the molecular weight distributions of DNA from Chinese hamster V79 cells irradiated with X-rays or 110 keV/mu m alpha-particles. Two running conditions for pulsed-field gel-electrophoresis were chosen to give optimal separation of fragments either in the 225 kbp-5.7 Mbp range or the 0.3 kbp to 225 kbp range. Taking the total fraction of DNA migrating into the gel as a measure of fragmentation, the RBE for dsb induction was less than 1.0 for both molecular weight regions studied. The total yields of dsb were 8.2 x 10(-9) dsb/Gy/bp for X-rays and 7.8 x 10(-9) dsb/Gy/bp for a-particles, measured using a random breakage model. Analysis of the RBE of alpha-particles versus molecular weight gave a different response. In the 0.4 Mbp-57 Mbp region the RBE was less than 1.0; however, below 0.4 Mbp the RBE increased above 1.0. The frequency distributions of fragment sizes were found to differ from those predicted by a model assuming random breakage along the length of the DNA and the differences were greater for alpha-particles than for X-rays. An excess of fragments induced by a single-hit mechanism was found in the 8-300 kbp region and for X-rays and alpha-particles these corresponded to an extra 0.8 x 10(-9) and 3.4 x 10(-9) dsb/bp/Gy, respectively. Thus for every alpha-particle track that induces a dsb there is a 44% probability of inducing a second break within 300 kbp and for electron tracks the probability is 10%. This study shows that the distribution of damage from a high LET alpha-particle track is significantly different from that observed with low LET X-rays. In particular, it suggests that the fragmentation patterns of irradiated DNA may be related to the higher-order chromatin repealing structures found in intact cells.

A charged-particle microbeam .2. A single-particle micro-collimation and detection system.

The use of a charged-particle microbeam provides a unique opportunity to control precisely, the number of particles traversing individual cells and the localization of dose within the cell. The accuracy of 'aiming' and of delivering a precise number of particles crucially depends on the design and implementation of the collimation and detection system. This report describes the methods available for collimating and detecting energetic particles in the context of a radiobiological microbeam. The arrangement developed at the Gray Laboratory uses either a 'V'-groove or a thick-walled glass capillary to achieve 2-5 mu m spatial resolution. The particle detection system uses an 18 mu m thick transmission scintillator and photomultiplier tube to detect particles with >99% efficiency.

A charged-particle microbeam .1. Development of an experimental system for targeting cells individually with counted particles

Charged-particle microbeams provide a unique opportunity to control precisely, the dose to individual cells and the localization of dose within the cell. The Gray Laboratory is now routinely operating a charged-particle microbeam capable of delivering targeted and counted particles to individual cells, at a dose-rate sufficient to permit a number of single-cell assays of radiation damage to be implemented. By this means, it is possible to study a number of important radiobiological processes in ways that cannot be achieved using conventional methods. This report describes the rationale, development and current capabilities of the Gray Laboratory microbeam.