Barry D. Michael

A review of dsb induction data for varying quality radiations

PURPOSE This short review summarizes the data obtained with various techniques for measuring the yields of double strand breaks (dsb) produced by particle radiations of differing linear energy transfer (LET) in order to obtain relative biological effectiveness (RBE) values. RESULTS AND CONCLUSIONS Studies aimed at understanding the interactions of different types of radiation with cellular DNA have monitored the yields of DNA dsb versus radiation quality. Several techniques have been used to measure dsb yields in mammalian cells, and these include: neutral sedimentation gradients, filter elution and more recently pulsed field gel electrophoresis techniques (PFGE). Recent developments in PFGE have allowed the measurement of both the yields and the distribution of breaks within the genome, which go part of the way to explaining the RBE values close to 1.0 previously measured using other approaches with various radiation qualities. It is clear that future studies to determine the effectiveness of radiations of differing LET must use techniques that determine both yields and distributions of dsb, and assays need to be developed to allow these measurements at biologically relevant doses.

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/micron 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 alpha-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-5.7 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 repeating structures found in intact cells.

Low-Dose Binary Behavior of Bystander Cell Killing after Microbeam Irradiation of a Single Cell with Focused CK X Rays

Abstract Schettino, G., Folkard, M., Michael, B. D. and Prise, K. M. Low-Dose Binary Behavior of Bystander Cell Killing after Microbeam Irradiation of a Single Cell with Focused CK X Rays. Radiat. Res. 163, 332–336 (2005). Although conclusive evidence has been obtained for the presence of radiation-induced bystander effects, the mechanisms that trigger and regulate these processes are still largely unknown. The bystander effect may play a critical role in determining the biological effectiveness of low-dose exposures, but questions on how to incorporate it into current models and extrapolate the risks of radiation-induced carcinogenesis are still open. The Gray Cancer Institute soft X-ray microbeam has been used to investigate the dose–response relationship of the bystander effect below 0.5 Gy. The survival response of V79 cells was assessed after the irradiation of a single cell within a population with a submicrometer-size beam of carbon K X rays (278 eV). Above 0.3 Gy, the measured bystander cell killing was in agreement with previously published data; however, a significant increase in the scatter of the data was observed in the low-dose region (<0.3 Gy). The data distribution observed indicates a binary behavior for triggering of the bystander response. According to our hypothesis, the probability of triggering a bystander response increases approximately linearly with the dose delivered to the single selected cell, reaching 100% above about 0.3 Gy. The magnitude of the bystander effect, when triggered, is approximately constant with the dose and results in an overall ∼10% reduction in survival in our system. This suggests that the event that triggers the emission of the bystander signal by the hit cell is an all-or-nothing process. Extrapolation of the data indicates that when a single fast electron traverses a V79 cell, there is a probability of ∼0.3% that the cell will emit the bystander signal. The data presented in this paper have also been analyzed statistically to test the possibility that complex DNA double-strand breaks may be the initial critical event.

The relationship between radiation-induced DNA double-strand breaks and cell kill in hamster V79 fibroblasts irradiated with 250 kVp X-rays, 2.3 MeV neutrons or 238Pu alpha-particles.

Using the neutral filter elution technique, the induction of DNA double-strand breaks (dsb) has been measured in 250 kVp X-irradiated V79-379A Chinese hamster cells irradiated under air or nitrogen. The dose-effect curves for induced dsb were curvilinear, mirroring cell survival curves, such that there was an approximately linear relationship between induced dsb and lethal lesions (-In (cell survival)) which was independent of oxygen. With cells irradiated with 2.3 MeV neutrons or 238Pu alpha-particles the correlations between lethal events and dsb, although also approximately linear, do not match those for X-rays. With neutrons there is approximately a 2.5-fold reduction in the level of dsb induction per lethal event. Thus either the apparently linear relationships found are spurious, and there is no general correlation between induced dsb and lethal effect, or there are qualitative differences between neutron, alpha-particle and X-ray induced dsb that give them differing probabilities of cell kill.

Electron Affinic Sensitization: Part II: Para-Nitroacetophenone: A Radiosensitizer for Anoxic Bacterial and Mammalian Cells

SummaryThe radiosensitizing properties of the highly electron-affinic nitroketone, para-nitroacetophenone (PNAP) have been investigated in resistant and sensitive bacteria, fern spores, mammalian cells in tissue culture and in suspensions of bacteriophage. Radiosensitization occurs in anoxic suspensions of Serratia marcescens Micrococcus radiodurans and in anoxic preparations of Chinese hamster cells line V79–379A. The sensitization of the mammalian cells (enhancement ratio 1·7 for 0·4 mM PNAP) is not inhibited in the presence of serum protein. No sensitization was observed for the spore of the fern Osmunda regalis.PNAP resembles oxygen in conferring some radioprotection on suspensions of bacteriophage T7 irradiated in broth.

Cell killing and DNA damage in Chinese hamster V79 cells treated with hydrogen peroxide.

Hydrogen peroxide induces lesions in cells similar to those from ionizing radiation, by a Fenton-type production of hydroxyl radicals. At 4 degrees C significant levels of DAN single-strand breaks (ssb) could be measured using the alkaline elution technique, after a 20-min incubation with 10(-5) mol dm-3 H2O2. Only at higher concentrations of H2O2 (greater than 10(-4) mol dm-3) where the levels of ssb measured corresponded to that induced by more than 18 Gy of X-rays, was any significant cell killing detected in a clonogenic assay. Cell killing was observed to coincide with the measurement of significant levels of DNA double-strand breaks (dsb) using the filter elution technique at pH 9.6. This suggests that dsb and not ssb are important as regards hydroxyl-radical-induced cell kill, as found for ionizing radiation. The correlation of induced dsb with lethal events showed that the predicted lethal effect of the H2O2-induced dsb was approximately 5 times less than that of X-ray-induced dsb. This is compared with data previously obtained which showed differences in the lethality of dsb with the quality of radiation (Prise et al. 1987).

Time-resolved oxygen effects in irradiated bacteria and mammalian cells: a rapid-mix study

An apparatus combining continuous fast-flow mixing with rapid irradiation has been used to study the time scale of the oxygen effect in irradiated bacteria and mammalian cells. The range of times u...

Late Radiation Damage in the Mouse Bladder as Measured by Increased Urination Frequency

The radiation response of the mouse urinary bladder was assessed by measuring the urination frequency after localized irradiation with 1.8-MeV electrons. The onset of radiation-induced incontinence is late, no changes were observed before 5 months after doses of up to 40 Gy (4000 rad). Beyond this time good dose--response curves were obtained above a threshold dose of 15 Gy. The time of onset of radiation damage was earlier after higher doses, but in all cases the maximum damage was observed by 12 months. There was some recovery of bladder function, especially in the lower dose groups, by 16 months. The increased frequency at 5 to 12 months was associated with epithelial denudation and a loss of the specialized polyploid surface cells. Fibrosis of the deeper layers was not apparent until after 12 months. The increased urination frequency responses are correlated with the delayed compensatory proliferation of the epithelium and with the resistance of the bladder to inflation under applied pressure, which also gave a dose-response relationship at 18 months.

The irradiation of V79 mammalian cells by protons with energies below 2 MeV. Part I: Experimental arrangement and measurements of cell survival.

The relative biological effectiveness (RBE) has been determined for protons with mean energies of 1.9, 1.15 and 0.76 MeV, from measurements of the survival of V79 Chinese hamster cells. The cells are supported as a monolayer and are swept through a beam of scattered protons produced using a 4 MeV Van de Graaff accelerator. An estimation of the dose and unrestricted linear energy transfer (LET) variation within the sensitive volume of the cells is given for the three proton energies. The RBEs for cell survival (relative to 250 kVp X-rays) at the 10 per cent survival level are 1.6, 1.9 and 3.36 for protons with track-average LETs of 17, 24 and 32 keV microns-1 respectively, and the data suggest that protons are most effective at about 40-50 keV microns-1. It is shown that the proton RBEs can be reconciled with those of other light ions if plotted against z*2/beta 2 (where z* is the effective charge and beta is the relative velocity) rather than against LET.

Relative contributions of levels of initial DNA damage and repair of double strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B: Part I. X-rays

In order to investigate the relationships between the induction and rejoining of DNA double-strand breaks (dsb) and their biological consequences it is necessary to measure these lesions uniquely and accurately, especially at relevant low doses of ionizing radiation. Differences in radiosensitivity between cell lines could be due to variations in dsb induction or to differences in the efficiency and/or accuracy of enzymatic repair of these lesions. We have used field-inversion gel electrophoresis to investigate dsb induction and rejoining in V79B parental and XR-V15B ionizing radiation-sensitive mutant cell lines. No difference has been found in the induction of dsb in XR-V15B cells compared with wild type cells; the assay sensitivity permits measurement of dsb induced by doses as low as 1 Gy (p < or = 0.05). The radiosensitivity of the mutant cells is manifested both in a lower fraction of dsb rejoined in the early, fast repair component and longer persistence of unrejoined dsb during post-irradiation incubation. The fraction of dsb remaining unrejoined after prolonged incubations (up to 17 h) correlates well with the higher radiosensitivity of the mutant (as judged by D10 values).