DSB Yield Research Articles

Modelling DNA damage induced by different energy photons and tritium beta-particles

Purpose: To model the production of single- and double-strand breaks (ssb and dsb) in DNA by ionizing radiations. To compare the predicted effectiveness of different energy photon radiations and tritium beta-particles. Materials and methods : Modelling is carried out by Monte Carlo and includes consideration of direct energy depositions in DNA molecules, the production of species, their diffusion and interactions with each other and DNA. Computer-generated electron tracks in liquid water are used to model energy deposition and to derive the initial positions of chemical species. Atomistic representation of the DNA in B form with a first hydration shell is used. Photon radiations in the energy range 70keV-1 MeV and tritium beta-particles are considered. Results: A tentative increase for dsb yield has been predicted for 70keV photons and tritium compared with 137Cs. This increase is more pronounced for complex dsb. Double-strand breaks are much more prone compared with ssb to combine with additional strand breaks and base damage, which contributes to break complexity. At least half of DNA breaks are hydroxyl radical mediated. Conclusions: The developed model makes predictions compatible with features of available experimental data. Break complexity has to be addressed in biophysical modelling when the relative effectiveness of radiations in DNA damage is studied. Obtained data strongly argue against the dominance of direct radiation action in DNA damage in the cellular environment predicted by some theoretical studies.

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 e ffectiveness) 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 10 9 dsb/Gy/bp for X-rays and 7.8 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 10 9 and 3.4 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.

Rejoining of DNA double-strand breaks induced by accelerated nitrogen ions.

Rejoining of radiation-induced DNA double-strand breaks (dsb) was measured in cultured cells with pulsed-field gel electrophoresis after radiation doses in the range of 5-30 Gy. Human glioma, U-343MG and Chinese hamster, V79, cells were irradiated with either accelerated nitrogen ions of high linear energy transfer, LET approximately 125 keV/ microns, or photons from 60Co. The induction frequencies of dsb were similar for the two radiation qualities with a relative biological effectiveness, RBE, of 0.90 and 0.89 for the human and hamster cell lines respectively. The biphasic rejoining kinetics differed significantly between the two radiation qualities when studied in the human glioma cells. The difference was seen within the first hour after irradiation and after 6 h there were considerable differences in both the total amount of unrejoined dsb and the fraction of dsb rejoined during the slow phase. When rejoining was analysed 20-22 h after irradiation, the nitrogen ions gave 2.5-2.9 times more residual dsb than the gamma photons. The results for the hamster V79 cells were, up to 2h after irradiation, similar, but the difference between the two radiation qualities was less accentuated. In summary, similar initial yields of dsb after exposure of cells to high or low LET resulted in both radiation quality and cell type-dependent differences when the rejoining of these breaks were compared.

Two types of double-strand breaks in electron and photon tracks and their relation to exchange-type chromosome aberrations.

Yields of DNA double-strand breaks (dsb), i.e. the average number of dsb, N, per relative molar mass, M(r), and dose, D, produced by electrons and photons in the energy range 50 eV-1 MeV were calculated. The experimental data of dsb induction by ultrasoft x-rays and by photons agree well with the calculated yields of dsb as a function of photon energy. The dsb are classified into simple and complex ones. Energy transfers of less than about 200 eV producing at least two ionizations generate mainly simple dsb, while low-energy electrons with an initial energy between 200 and 500 eV induce preferentially complex dsb. Assuming that dsb is the main DNA lesion leading to exchange-type chromosome aberrations (etca), three different mechanisms have to be considered: 1) complex dsb on its own; 2) interaction between two dsb induced by the same primary particle; and 3) interaction between two dsb induced by different primary particles. Mechanisms 1) and 2) produce a linear term, whereas mechanism 3) leads to a quadratic term for the yield of etca. The sum of contributions 1) and 2) to the yield of dicentrics describes fairly well the non-trivial structure of the experimental data. The results suggest that interaction between complex dsb does not contribute significantly to the formation of dicentrics via mechanism 3).

DNA double strand break production and rejoining in V79 cells irradiated with light ions

Low energy protons and other densely ionizing light ions are known to have RBE>1 for cellular end points relevant for stochastic and deterministic effects. The occurrence of a close relationship between them and induction of DNA dsb is still a matter of debate. We studied the production of DNA dsb in V79 cells irradiated with low energy protons having LET values ranging from 11 to 31 keV/μm, i.e. in the energy range characteristic of the Bragg peak, using the sedimentation technique. We found that the initial yield of dsb is quite insensitive to proton LET and not significantly higher than that observed with X-rays, in agreement with recent data on V79 cells irradiated with alpha particles of various LET up to 120 keV/μm. By contrast, RBE for cell inactivation and for mutation induction rises with the proton LET. In experiments aimed at evaluating the rejoining of dsb after proton irradiation we found that the amount of dsb left unrepaired after 120 min incubation is higher for protons than for sparsely ionizing radiation. These results indicate that dsb are not homogeneuos with respect to repair and give support to the hypothesis that increasing LET leads to an increase in the complexity of DNA lesions with a consequent decrease in their reparability.

Repair of DNA double-strand breaks induced in Saccharomyces cerevisiae using different gamma-ray dose-rates: a pulsed-field gel electrophoresis analysis.

We investigated the effects of gamma-ray exposures at high dose-rate (HDR, 23.2 Gy/min) and low dose-rate (LDR, 0.47 Gy/min) on survival and the induction of DNA double-strand breaks (dsb) in a diploid wild-type (D7) and the repair-deficient mutant strain rad52/rad52 of Saccharomyces cerevisiae. Analysis by pulsed-field gel electrophoresis (PFGE) using a contour homogeneous electric field apparatus revealed that, at HDR, in the range 0-400 Gy, dsb are induced as a linear function of gamma-ray dose. Liquid holding recovery in non-nutrient medium (LHR) for 48 h of wild-type cells treated at HDR, significantly increased survival and reduced the yield of dsb. Such changes did not occur in rad52/rad52 cells defective in the repair of dsb. Thus, in gamma-irradiated wild-type cells, an efficient repair of dsb is taking place during LHR. Treatments of wild-type cells at LDR resulted in higher survival and an approximately two-fold lower yield of dsb than at HDR. Such a dose-rate effect was absent in rad52/rad52 cells suggesting that, in wild-type cells during LDR exposures, significant amounts of dsb can be repaired. This repair could be very much accentuated by 48-h LHR of wild-type cells treated at LDR. The relationship observed between gamma-ray survival and dsb repair clearly indicates that increases in survival of wild-type cells, during LDR as compared with HDR exposures and after LHR, are strongly related to the repair of dsb.

Induction of DNA damage in gamma-irradiated nuclei stripped of nuclear protein classes: differential modulation of double-strand break and DNA-protein crosslink formation.

The influence of chromatin proteins on the induction of DNA double-strand breaks (dsb) and DNA-protein crosslinks (dpc) by gamma-radiation was investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaCl, whereas nuclear matrix proteins were not extractable even by 2.0 M NaCl. The yield of dsb increased with progressive removal of proteins from chromatin. Whilst removal of low molecular weight non-histone proteins and histone H1 resulted in small increases in the production of dsb, removal of histones H2A/H2B, all histones, or all proteins led to 18.4, 46.4 and 55.5-fold increases in the yield of dsb, respectively, relative to irradiated cells. Therefore, both histones and non-histone proteins contribute to the radioprotection of DNA, core histones being the major radio-protectors. In contrast, depletion of chromatin proteins caused little or no effect on the induction of dpc until the chromatin was extracted with > or = 1.4 M NaCl. However, our studies indicated no direct, quantitative correlation between the removal of histones and the induction of dpc. The data support our previous conclusion that nuclear matrix protein rather than the majority of the histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions.

Effect of radiation quality on lesion complexity in cellular DNA.

Understanding the critical lesions induced by ionizing radiation in DNA and their relationship to cellular effects is an important challenge in radiation biology. Much evidence has suggested that DNA double-strand breaks (dsb) are important lesions. Establishing a cause and effect relationship between initial levels of DNA dsb, their repair rate or the level of residual unrepaired breaks, and cellular effects has proved difficult in mammalian cells. Several studies have measured yields of DNA dsb after irradiation with radiations of differing linear energy transfer (LET). In general the RBEs for dsb induction (20-100 keV/microns) have been lower than the RBEs measured for cell survival and in many cases are around 1.0. Several studies have shown differences in the rejoining of dsb with less dsb rejoined after high-LET irradiation in comparison with low-LET radiation. These results suggest that there may be differences in the types of lesions induced by different radiations and scored as DNA dsb using current techniques. Track structure modelling studies have suggested that some lesions induced will be clustered at the sites of energy depositions and that uniquely large energy deposition events are produced by high-LET radiations. Assays need to be developed to measure complex lesions in both model DNA and cellular systems. Different levels of complexity need to be considered such as clustering of radicals close to DNA, localized areas of DNA damage (1-20 bp) and lesions which may be induced over larger distances. Studies using new and existing assays of DNA damage, coupled with irradiation at various LETs, are directed at understanding the role of lesion complexity in relation to cellular effects.

DNA double-strand breaks induced by low energy protons in V79 cells.

The initial production of DNA double-strand breaks (dsb) was determined in V79 Chinese hamster cells irradiated with proton beams of 3.24, 1.50 and 0.88 MeV, corresponding to values of unrestricted LET evaluated at the cell midplane of 10.9, 20.0 and 30.5 keV/micron, respectively. X-rays were used for comparison. Dsb were measured with the low speed sedimentation technique in neutral sucrose gradients. The initial yield of dsb rose linearly with the dose and did not significantly depend on the proton LET, in contrast with the results obtained in previous studies for cell inactivation and mutation induction. Also, no significant differences for dsb induction were found between protons and X-rays. Two possible explanations, not necessarily mutually exclusive, are proposed: (1) dsb are not the only lesions involved in cellular effects; and (2) the initial number of dsb is not the only important parameter since a fundamental role is played by the degree of clustering, i.e. the association of dsb with other dsb or other types of damage.

Lethality of heavy ion-induced DNA double-strand breaks in mammalian cells.

Cell killing and the production of DNA double-strand breaks (dsbs) were measured in parallel for V79 cells and a human tumour cell line (Caski) following exposure to radiations of differing linear energy transfer (LET) including accelerated heavy ions. Dsb induction was assessed by neutral filter elution (pH 7.4) and elution from agarose plugs in pulsed-field electrophoresis. The elution data were consistent with linear lesion induction and allowed to derive dsb yields or production cross-sections (sigma dsb), respectively. Both cell lines as well as the two elution approaches gave comparable results. sigma dsb was found to rise up to the heaviest ions used (LET = 11,500 keV/microns) but relative biological effectiveness was always smaller than unity. Slopes of the exponential survival curves (final slope with 60Co-radiation) were normalized to the respective dsb yields and allowed to calculate the relative lethality per induced dsb. This parameter increased with LET up to about 300 keV/microns followed by a steep decline for the very heavy ions. The extent of dsb rejoining was concomitantly reduced with ionization density.

Constraints on energy deposition and target size of multiply damaged sites associated with DNA double-strand breaks.

We suggest that the observed experimental data on relative double-strand break (dsb) yield as a function of radiation quality can act as valuable constraints in defining the type of energy deposition which causes this basic lesion in radiation biology. Both heavy-ion and alpha-particle data show sufficient trends for quantitative comparisons with calculation to be made. We use the technique of track-structure simulation and search for energy-deposition clusters (containing at least a given number of ionizations in a given diameter) whose relative frequencies (compared to sparsely ionizing radiation) correlate with the relative biological effects (RBEs) for dsb induction. We conclude that locally multiply damaged sites (LMDS) which cause dsb are probably energy depositions of at least two to five ionizations localized, respectively, in sites of diameters of 1-4 nm. Although our derived cluster sizes should be viewed in light of the quality of the experimental data and uncertainties in the computer simulations at the nanometre level, it is unlikely that these estimates of cluster sizes would change greatly.

Metal ions protect DNA against strand breakage induced by fast neutrons.

Single and double strand breaks (SSB and DSB) are induced by fast neutrons in plasmid (pBR322) DNA in 1 mM potassium phosphate buffer (pH 7.25). Increasing the concentration of monovalent (Na+, Cs+, Li+), divalent (Mg2+, Ca2+) and trivalent (Al3+, Co3+ (NH3)6) metal cations strongly decreases the yield of DSB. The extent of the observed protection depends on the valence of the cation. The production of SSB is only slightly decreased, except for Al3+ and Co3+ (NH3)6, whose effects are particularly large (complete protection at 1 and 0.1 mM respectively). Circular dichroism spectra show that Al3+ induces an important structural change of DNA at the ion concentration where the protection becomes total. This change is probably a condensation (collapse), as in the well-known case of Co3+ (NH3)6. Our results suggest two mechanisms of protection by metal ions: (i) the induction of structural changes of DNA, that render less accessible the critical sites of attack by OH. radicals; and (ii) the stabilization of the double helical regions between two close-set nicks on opposite strands, that hinders the effective double strand breakage of DNA.

The repair of DNA damage induced in V79 mammalian cells by the nitroimidazole-aziridine, RSU-1069: Implications for radiosensitization

The induction and repair of single (ssb) and double (dsb) strand breaks in DNA under aerobic or hypoxic conditions have been determined using sucrose sedimentation techniques following incubation of V79 mammalian cells with RSU-1069 or misonidazole, representative of a conventional 2-nitroimidazole radiosensitizer, for 1–1.5 hr at either 293 or 277°K and subsequent irradiation at 277°K. In all cases, the dose dependences for the induction of strand breaks are linear and consistent with an enhancement in the yield of DNA damage induced by the 2-nitroimidazoles under hypoxic conditions. With RSU-1069 at 293°K, the dose dependence of ssb is displaced reflecting DNA damage induced during pre-incubation. From these dependences, it is evident that the enhanced radiosensitization by RSU-1069 may not be accounted for in terms of accumulation of the agent at DNA. From the repair studies, DNA breaks induced by RSU-1069 in the absence of radiation have been shown to persist for at least 3 hr. With a combination of RSU-1069 and radiation under hypoxic conditions, the repair timescale of the induced breaks is significantly longer and an increase in the residual yields of both ssb and dsb (at 2–3 hr) was observed when compared with the observation in the presence of misonidazole or oxygen. From these studies, it is inferred that the enhanced radiosensitization of RSU-1069 at 293°K is a consequence of the formation of non-repairable DNA damage together with a modification of the repairability of the radiation-induced DNA breaks.

ULTRAVIOLET LIGHT INDUCES DOUBLE‐STRAND BREAKS IN DNA OF CULTURED HUMAN P3 CELLS AS MEASURED BY NEUTRAL FILTER ELUTION

Neutral filter elution at pH 7.2 and 9.6 was used to measure the induction of DNA lesions in human P3 teratocarcinoma cells by monochromatic 254-, 270-, 313-, 334-, 365-, and 405-nm radiation and by 60 gamma rays. In this assay DNA double-strand breaks (dsb) increase the rate of elution of DNA from cell lysates on a filter. Yields of dsb as measured by this procedure were determined by using a calibration of the assay that correlates elution parameters with number of dsb caused by disintegration of 125I incorporated into the DNA. Analysis of fluence responses obtained by using the calibrated assay indicated that the number of dsb induced per dalton of DNA as measured by this assay is proportional to the square of the fluence at all the energies of radiation studied, implying that the induction of these lesions may be a two-hit event. Analysis of the relative efficiencies for the induction of dsb by ultraviolet radiation, corrected for quantum efficiency, revealed a spectrum that coincided closely with that for the induction of single-strand breaks (ssb) in the same cells, having a close fit with the spectrum of nucleic acid in the UVC and UVB region below 313 nm, and a shoulder in the UVA region. It was calculated, however, that there may be too few ssb for dsb to result from randomly distributed closely opposed ssb.

The Yield of DNA Double-strand Breaks Produced Intracellularly by Ionizing Radiation: A Review

The mechanisms of radiation damage production are used to examine the following premises: (1) the number of DNA double-strand breaks per unit dose increases with dose; (2) cell type to cell type variations in yield of DNA dsb per dose occur. Two stages of damage production are identified as possible sources of damage yield modulation; numbers of OH. free radicals reacting with the target, and amount of chemical repair occurring on the target radicals. These factors are discussed in the light of the structures within which cellular DNA is packaged and the known rate constants for the reactions involved. It is concluded from our current knowledge that, in the presence of oxygen: (a) the number of DNA dsb is linearly related to dose, and (b) the yields of DNA damage per dose among cell types are constant. There is a caveat to the latter conclusion: the chromatin structure may be different in radiosensitive cell lines. In the absence of such a difference, variations in radiosensitivity with dose or with cell type are assigned to differences in repair speed and/or accuracy.

The Rejoining of DNA Double-strand Breaks Following Irradiation with238Pu α-particles: Evidence for a Fast Component of Repair as Measured by Neutral Filter Elution

The induction and repair of DNA double-strand breaks (DSB) following exposure to 238Pu alpha-particles was examined in V79-379A cells. The technique of neutral filter elution was used in these investigations at both pH 9.6 and pH 7.2. The initial dsb yield was found to be similar to that seen after 250 kVp X-ray or 2.3 MeV neutron exposure. However, the pattern of dsb rejoining after alpha-particle irradiation did not follow that seen after X-rays or neutrons. A very fast initial component, complete within 2 min of incubation following irradiation, removed 70 per cent of the dsb seen after 40 Gy alpha-particles; very little slow rejoining was seen. This contrasts sharply with the dsb rejoining seen after X-ray or neutron exposure, and presumably reflects the differences in the nature of the dsb induced and the way they are repaired.

An Effect of Elevated Postirradiation pH on the Yield of Double-Strand Breaks in DNA from Irradiated Bacterial Cells

Exposure of DNA isolated from irradiated cells of Escherichia coli to a pH of 9.6 caused a marked increase in the yield of double-strand breaks (dsb). The dsb were measured by sedimentation analysis of E. coli chromosomal DNA using neutral sucrose gradients. After incubation for 4 hr at 37 degrees C and pH 9.6 the dsb yields were 95% and 71% higher than when incubation was at pH 7.0 for irradiation under oxic and anoxic conditions, respectively. This effect was not apparent when dsb were induced enzymatically and it was linearly related to radiation dose. After oxic irradiation, the increase in dsb at pH 9.6 was consistent with first-order kinetics over greater than 2 half-lives (t1/2 = 1.6 hr at 37 degrees C). The effect of elevated pH was largely additive to a previously reported increase in dsb yield caused by ethanol. It is proposed that the effects of elevated pH and of ethanol revealed the presence in intracellularly irradiated DNA of previously unidentified sites where both strands of the DNA were damaged as a result of single radiation events. The possible nature of the proposed sites and the relevance of these findings to the "neutral" elution technique are discussed.

Measurements of DNA Double-Strand Break Yields in E. coli after Rapid Irradiation and Cell Inactivation: The Effects of Inactivation Technique and Anoxic Radiosensitizers

A study has been made of methods for rapidly inactivating cells of E. coli at neutral pH to prevent enzymatic, chemical, or physical modification to DNA damaged by irradiation. The radiation was delivered in a fraction of a second using an electron accelerator. Cell inactivation was with ethanol or a solution (CSE) containing detergent, EDTA, and chloroform. It was found that DNA could be released from irradiated and inactivated cells simply by incubating them with the protease Pronase, and this DNA appeared to be in a form suitable for centrifugational analysis in neutral sucrose gradients. When cells were irradiated in the presence of oxygen, inactivation with ethanol gave a radiation-induced double-strand break (dsb) yield 1.8-fold higher than when inactivation was with CSE. Possible explanations of this are discussed. Using CSE inactivation, an oxygen enhancement ratio for dsb formation of 4.3 was observed and yields of dsb per Gray were in good agreement with results from other laboratories. At concentrations which enhanced cell killing 2.6-fold the "electron-affinic" type anoxic radiosensitizer misonidazole enhanced dsb formation 2.0-fold, whereas the nitroxyl free radical anoxic radiosensitizer norpseudopelletierine-N-oxyl had no significant effect on dsb yield although there was a possible slight enhancement at the higher doses used.

An improved technique of strand break analysis for isodisperse DNA

A technique for determining the double strand break (dsb) yield in irradiated iso-disperse DNA using the molecular length profile of the damaged DNA is described. The technique employs visualisation of the irradiated DNA fragments on the electron microscope followed by length measurement of a randomly selected sample. The range of damage index measurable (0.01–1 dsb/106 Dalton) is substantially greater than that accessible by techniques involving centrifugation. Moreover the use of the whole profile of molecular lengths in the irradiated sample allows errors in the determination to be assessed. Results of experiments with T7 bacteriophage DNA irradiated in dilute aqueous solution indicate that the observed molecular length profiles are in good agreement with those generated theoretically assuming random breakage. The techniques for fitting observed profiles by the method of maximum likelihood to those generated theoretically are described in detail in an appendix. There is no evidence of bias caused by preferential losses in any part of the length profile.