Yield Of DNA Double-strand Breaks Research Articles

A fast Monte Carlo cell-by-cell simulation for radiobiological effects in targeted radionuclide therapy using pre-calculated single-particle track standard DNA damage data.

We developed a new method that drastically speeds up radiobiological Monte Carlo radiation-track-structure (MC-RTS) calculations on a cell-by-cell basis. The technique is based on random sampling and superposition of single-particle track (SPT) standard DNA damage (SDD) files from a "pre-calculated" data library, constructed using the RTS code TOPAS-nBio, with "time stamps" manually added to incorporate dose-rate effects. This time-stamped SDD file can then be input into MEDRAS, a mechanistic kinetic model that calculates various radiation-induced biological endpoints, such as DNA double-strand breaks (DSBs), misrepairs and chromosomal aberrations, and cell death. As a benchmark validation of the approach, we calculated the predicted energy-dependent DSB yield and the ratio of direct-to-total DNA damage, both of which agreed with published in vitro experimental data. We subsequently applied the method to perform a superfast cell-by-cell simulation of an experimental in vitro system consisting of neuroendocrine tumor cells uniformly incubated with 177Lu. The results for residual DSBs, both at 24 and 48 h post-irradiation, are in line with the published literature values. Our work serves as a proof-of-concept demonstration of the feasibility of a cost-effective "in silico clonogenic cell survival assay" for the computational design and development of radiopharmaceuticals and novel radiotherapy treatments more generally.

Effects of Differing Underlying Assumptions in In Silico Models on Predictions of DNA Damage and Repair.

The induction and repair of DNA double-strand breaks (DSBs) are critical factors in the treatment of cancer by radiotherapy. To investigate the relationship between incident radiation and cell death through DSB induction many in silico models have been developed. These models produce and use custom formats of data, specific to the investigative aims of the researchers, and often focus on particular pairings of damage and repair models. In this work we use a standard format for reporting DNA damage to evaluate combinations of different, independently developed, models. We demonstrate the capacity of such inter-comparison to determine the sensitivity of models to both known and implicit assumptions. Specifically, we report on the impact of differences in assumptions regarding patterns of DNA damage induction on predicted initial DSB yield, and the subsequent effects this has on derived DNA repair models. The observed differences highlight the importance of considering initial DNA damage on the scale of nanometres rather than micrometres. We show that the differences in DNA damage models result in subsequent repair models assuming significantly different rates of random DSB end diffusion to compensate. This in turn leads to disagreement on the mechanisms responsible for different biological endpoints, particularly when different damage and repair models are combined, demonstrating the importance of inter-model comparisons to explore underlying model assumptions.

Cellular lethal damage of 64Cu incorporated in mammalian genome evaluated with Monte Carlo methods.

Targeted Radionuclide Therapy (TRT) with Auger Emitters (AE) is a technique that allows targeting specific sites on tumor cells using radionuclides. The toxicity of AE is critically dependent on its proximity to the DNA. The aim of this study is to quantify the DNA damage and radiotherapeutic potential of the promising AE radionuclide copper-64 (64Cu) incorporated into the DNA of mammalian cells using Monte Carlo track-structure simulations. A mammalian cell nucleus model with a diameter of 9.3 μm available in TOPAS-nBio was used. The cellular nucleus consisted of double-helix DNA geometrical model of 2.3 nm diameter surrounded by a hydration shell with a thickness of 0.16 nm, organized in 46 chromosomes giving a total of 6.08 giga base-pairs (DNA density of 14.4 Mbp/μm3). The cellular nucleus was irradiated with monoenergetic electrons and radiation emissions from several radionuclides including 111In, 125I, 123I, and 99mTc in addition to 64Cu. For monoenergetic electrons, isotropic point sources randomly distributed within the nucleus were modeled. The radionuclides were incorporated in randomly chosen DNA base pairs at two positions near to the central axis of the double-helix DNA model at (1) 0.25 nm off the central axis and (2) at the periphery of the DNA (1.15 nm off the central axis). For all the radionuclides except for 99mTc, the complete physical decay process was explicitly simulated. For 99mTc only total electron spectrum from published data was used. The DNA Double Strand Breaks (DSB) yield per decay from direct and indirect actions were quantified. Results obtained for monoenergetic electrons and radionuclides 111In, 125I, 123I, and 99mTc were compared with measured and calculated data from the literature for verification purposes. The DSB yields per decay incorporated in DNA for 64Cu are first reported in this work. The therapeutic effect of 64Cu (activity that led 37% cell survival after two cell divisions) was determined in terms of the number of atoms incorporated into the nucleus that would lead to the same DSBs that 100 decays of 125I. Simulations were run until a 2% statistical uncertainty (1 standard deviation) was achieved. The behavior of DSBs as a function of the energy for monoenergetic electrons was consistent with published data, the DSBs increased with the energy until it reached a maximum value near 500 eV followed by a continuous decrement. For 64Cu, when incorporated in the genome at evaluated positions (1) and (2), the DSB were 0.171 ± 0.003 and 0.190 ± 0.003 DSB/decay, respectively. The number of initial atoms incorporated into the genome (per cell) for 64Cu that would cause a therapeutic effect was estimated as 3,107 ± 28, that corresponds to an initial activity of 47.1 ± 0.4 × 10-3 Bq. Our results showed that TRT with 64Cu has comparable therapeutic effects in cells as that of TRT with radionuclides currently used in clinical practice.

Modelling oxygen effects on the in- and out-of-field radiosensitivity of cells exposed to intensity-modulated radiation fields

Objective. The delivery of intensity-modulated radiation fields has improved the conformity of dose to tumour targets during radiotherapy (RT). Previously, it has been shown that intercellular communication between cells positioned in- and outside of the radiation field impacts cellular radiosensitivity under hypoxic and normoxic conditions. However, the mechanism of intercellular communication in hypoxia remains to be fully understood. In this study, the cell-killing effects of intercellular communication in hypoxia were modelled in an effort to better understand the underlying mechanisms of response. Approach. By irradiating a 50% area of the culture dish (half-field exposure), experimental dose-response curves for cell survival and residual DNA double-strand breaks (DSBs) were generated in prostate (DU145) and non-small cell lung cancer (H1299) cells. The oxygen enhancement ratio (OER) was determined from early DSB yields (corresponding to relative direct damage) and used to model the in- and out-of-field radiosensitivity. Main results. The developed integrated microdosimetric-kinetic (IMK) model successfully predicted the experimental dose responses for survival and lethal lesions, and provides a mechanistic interpretation that the probability of hits for releasing cell-killing signals is dependent on oxygen. This experimental and modelling study also suggests that residual DSBs correspond to logarithmic survival fraction (meaning lethal lesions) for in- and out-of-field cells. Our data suggest that the OER value determined using uniform-field exposure can be applied to predict the in- and out-of-field radiosensitivity of cells following exposure to intensity modulated beams. Significance. The developed IMK model facilitates a more precise understanding of intercellular signalling following exposure to intensity-modulated radiation fields.

Evaluation of the Yield of DNA Double-Strand Breaks for Carbon Ions Using Monte Carlo Simulation and DNA Fragment Distribution

The aim of this work was to provide a method to evaluate the yield of DNA double-strand breaks (DSBs) for carbon ions, overcoming the bias in existing methods due to the nonrandom distribution of DSBs. A previously established biophysical program based on the radiation track structure and a multilevel chromosome model was used to simulate DNA damage induced by x-rays and carbon ions. The fraction of activity retained (FAR) as a function of absorbed dose or particle fluence was obtained by counting the fraction of DNA fragments larger than 6 Mbp. Simulated FAR curves for the 250 kV x-rays and carbon ions at various energies were compared with measurements using constant-field gel electrophoresis. The doses or fluences at the FAR of 0.7 based on linear interpolation were used to estimate the simulation error for the production of DSBs. The relative difference of doses at the FAR of 0.7 between simulation and experiment was -8.5% for the 250 kV x-rays. The relative differences of fluences at the FAR of 0.7 between simulations and experiments were -17.5%, -42.2%, -18.2%, -3.1%, 10.8%, and -14.5% for the 34, 65, 130, 217, 2232, and 3132 MeV carbon ions, respectively. In comparison, the measurement uncertainty was about 20%. Carbon ions produced remarkably more DSBs and DSB clusters per unit dose than x-rays. The yield of DSBs for carbon ions, ranging from 10 to 16 Gbp-1Gy-1, increased with linear energy transfer (LET) but plateaued in the high-LET end. The yield of DSB clusters first increased and then decreased with LET. This pattern was similar to the relative biological effectiveness for cell survival for heavy ions. The estimated yields of DSBs for carbon ions increased from 10 Gbp-1Gy-1 in the low-LET end to 16 Gbp-1Gy-1 in the high-LET end with 20% uncertainty.

Impact of the Lorentz force on electron track structure and early DNA damage yields in magnetic resonance-guided radiotherapy

Magnetic resonance-guided radiotherapy (MRgRT) has been developed and installed in recent decades for external radiotherapy in several clinical facilities. Lorentz forces modulate dose distribution by charged particles in MRgRT; however, the impact of Lorentz forces on low-energy electron track structure and early DNA damage induction remain unclear. In this study, we estimated features of electron track structure and biological effects in a static magnetic field (SMF) using a general-purpose Monte Carlo code, particle and heavy ion transport code system (PHITS) that enables us to simulate low-energy electrons down to 1 meV by track-structure mode. The macroscopic dose distributions by electrons above approximately 300 keV initial energy in liquid water are changed by both perpendicular and parallel SMFs against the incident direction, indicating that the Lorentz force plays an important role in calculating dose within tumours. Meanwhile, DNA damage estimation based on the spatial patterns of atomic interactions indicates that the initial yield of DNA double-strand breaks (DSBs) is independent of the SMF intensity. The DSB induction is predominantly attributed to the secondary electrons below a few tens of eV, of which energy deposition patterns are not considerably affected by the Lorentz force. Our simulation study suggests that treatment planning for MRgRT can be made with consideration of only changed dose distribution.

Development of modified microdosimetric kinetic model for relative biological effectiveness in proton therapy.

To predict the biological effects of ionising radiation, the quantity of biological dose is introduced instead of the physical absorbed dose. In proton therapy, a constant relative biological effectiveness (RBE) of 1.1 is usually applied clinically as recommended by the International Commission of Radiation Units and Measurements. This study presents a new model, based on the modified microdosimetric kinetic model (MMKM), for calculating variable RBE values based on experimental data on the induction of DNA double-strand breaks (DSBs) within cells. The MMKM was proposed based on experimental data for the yield of DSBs in mammalian cells, which allows modification of the yield of primary lesions in the MKM. In this approach, a unique function named f(LET), which describes the relation between RBE and linear energy transfer (LET), was considered for charged particles. In the presented model (DMMKM), the MMKM approach was developed further by considering different f(LET)s for different relevant ions involved in energy deposition events in proton therapy. Although experimental data represent the dependence of the yield of primary lesions on the ion species, the DSB yield (assumed as the main primary lesion) is assumed independent of the ion species in the MMKM. In the DMMKM, by considering the yield of primary lesions as a function of the ion species, the α and β values are in better agreement with the experimental data as compared to those of the MKM and MMKM approaches. The biological dose in the DMMKM is predicted to be lower than that in the MMKM. Further, in the proposed model, the variation of the β parameter is higher than the constant value assumed in the MKM, at the distal end of the spread-out Bragg peak (SOBP). Moreover, the level of cell death estimated by the MMKM at the SOBP region is higher than that obtained based on the DMMKM. It is concluded that considering modified f(LET)s in the model developed here is more consistent with experimental results than when MMKM and MKM approaches are considered. The DMMKM examines the biological effects with full detail and will, therefore, be effective in improving proton therapy.

Monte Carlo Simulation of Double-Strand Break Induction and Conversion after Ultrasoft X-rays Irradiation.

This paper estimates the yields of DNA double-strand breaks (DSBs) induced by ultrasoft X-rays and uses the DSB yields and the repair outcomes to evaluate the relative biological effectiveness (RBE) of ultrasoft X-rays. We simulated the yields of DSB induction and predicted them in the presence and absence of oxygen, using a Monte Carlo damage simulation (MCDS) software, to calculate the RBE. Monte Carlo excision repair (MCER) simulations were also performed to calculate the repair outcomes (correct repairs, mutations, and DSB conversions). Compared to 60Co γ-rays, the RBE values for ultrasoft X-rays (titanium K-shell, aluminum K-shell, copper L-shell, and carbon K-shell) for DSB induction were respectively 1.3, 1.9, 2.3, and 2.6 under aerobic conditions and 1.3, 2.1, 2.5, and 2.9 under a hypoxic condition (2% O2). The RBE values for enzymatic DSBs were 1.6, 2.1, 2.3, and 2.4, respectively, indicating that the enzymatic DSB yields are comparable to the yields of DSB induction. The synergistic effects of DSB induction and enzymatic DSB formation further facilitate cell killing and the advantage in cancer treatment.

Development of a DNA damage model that accommodates different cellular oxygen concentrations and radiation qualities

Research regarding cellular responses at different oxygen concentrations (OCs) is of immense interest within the field of radiobiology. Therefore, this study aimed to develop a mechanistic model to analyze cellular responses at different OCs. A DNA damage model (the different cell oxygen level DNA damage [DICOLDD] model) that examines the oxygen effect was developed based on the oxygen fixation hypothesis, which states that dissolved oxygen can modify the reaction kinetics of DNA-derived radicals generated by ionizing radiation. The generation of DNA-derived radicals was simulated using the Monte Carlo method. The decay of DNA-derived radicals due to the competing processes of chemical repair, oxygen fixation, and intrinsic damaging was described using differential equations. The DICOLDD model was fitted to the previous experimental data obtained under different irradiation configurations and validated by calculating the yields of DNA double-strand breaks (DSBs) after exposure to 137 Cs as well as cell survival fractions (SFs) using a mechanistic model of cellular survival. Moreover, we used the DICOLDD model to calculate DNA DSB damage yields after irradiation with 0.5-50MeV protons. Generally, DSB yields calculated after exposure to 137 Cs at different OCs correspond to statistical uncertainties of previous experimental results. Calculated SFs of CHO and V79 cells exposed to photons, protons, and alpha particles at different OCs generally concur with those obtained in previous studies. Our results demonstrated that the variation in DSB yields was less than 10% when the cellular OC decreased from 21% to 5%. Additionally, DSB yields changed drastically when OC dropped below 1%. We developed a DNA damage model to evaluate the oxygen effect and provide evidence that a reaction-kinetic model of DNA-derived radicals induced by ionizing radiation suffices to explain the observed oxygen effects. Therefore, the DICOLDD model is a powerful tool for the analysis of cellular responses at different OCs after exposure to different types of radiation.

Effects of combined exposure to modeled radiation and gravitation factors of the interplanetary flight: Monkeys' cognitive functions and the content of monoamines and their metabolites; cytogenetic changes in peripheral blood lymphocytes

In a study on primates (Macaca mulatta), neurobiological and radiobiological effects have been studied of the synchronous combined action of 7-day antiorthostatic hypokinesia and exposure of the monkeys' head first to γ-rays during 24 h and then to accelerated 12C ions. The neurobiological effects were evaluated by the cognitive functions which model the basic elements of operator activity and the concentration of monoamines and their metabolites in peripheral blood. The radiobiological effects were evaluated by the chromosomal aberration and DNA double-strand break (DSB) yield in peripheral blood lymphocytes. The results of the cognitive function research show that the typological features of the animals' higher nervous activity are the prevailing factor that determines changes in these functions. The monkey of the strong balanced type effectively retained its cognitive functions after the exposures, while in the weak unbalanced type animals these functions were impaired. These changes went along with a decrease in the concentration of monoamines and their metabolites and an increase in the DNA DSB and chromosomal aberration yield in lymphocytes.

Microdosimetric investigation of the radiation quality of low-medium energy electrons using Geant4-DNA

The increasing clinical use of low-energy photon and electron sources (below few tens of keV) has raised concerns on the adequacy of the existing approximation of an energy-independent radiobiological effectiveness. In this work, the variation of the quality factor (Q) and relative biological effectiveness (RBE) of electrons over the low-medium energy range (0.1 keV–1 MeV) is examined using several microdosimetry-based Monte Carlo methodologies with input data obtained from Geant4-DNA track-structure simulations. The sensitivity of the results to the different methodologies, Geant4-DNA physics models, and target sizes is examined. Calculations of Q and RBE are based on the ICRU Report 40 recommendations, the Kellerer-Hahn approximation, the site version of the theory of dual radiation action (TDRA), the microdosimetric kinetic model (MKM) of cell survival, and the calculated yield of DNA double strand breaks (DSB). The stochastic energy deposition spectra needed as input in the above approaches have been calculated for nanometer spherical volumes using the different electron physics models of Geant4-DNA. Results are normalized at 100 keV electrons which is here considered the reference radiation. It is shown that in the energy range ~50 keV-1 MeV, the calculated Q and RBE are approximately unity (to within 1–2%) irrespective of the methodology, Geant4-DNA physics model, and target size. At lower energies, Q and RBE become energy-dependent reaching a maximum value of ~1.5–2.5 between ~200 and 700 eV. The detailed variation of Q and RBE at low energies depends mostly upon the adopted methodology and target size, and less so upon the Geant4-DNA physics model. Overall, the DSB yield predicts the highest RBE values (with RBEmax≈2.5) whereas the MKM the lowest RBE values (with RBEmax≈1.5). The ICRU Report 40, Kellerer-Hahn, and TDRA methods are in excellent agreement (to within 1–2%) over the whole energy range predicting a Qmax≈2. In conclusion, the approximation Q=RBE=1 was found to be valid only above ~50 keV whereas at lower energies both Q and RBE become strongly energy-dependent. It is envisioned that the present work will contribute towards establishing robust methodologies to determine theoretically the energy-dependence of radiation quality of individual electrons which may then be used in subsequent calculations involving practical electron and photon radiation sources.

Calculated relative biological effectiveness (RBE) for initial DNA double-strand breaks (DSB) from flattening filter and flattening filter-free 6 MV X-ray fields.

Objectives:We evaluated the radiobiological effectiveness based on the yields of DNA double-strand breaks (DSBs) of field induction with flattening filter (FF) and FF-free (FFF) photon beams.Methods:We used the particle and heavy ion transport system (PHITS) and a water equivalent phantom (30 × 30 × 30 cm3) to calculate the physical qualities of the dose-mean lineal energy (yD) with 6 MV FF and FFF. The relative biological effectiveness based on the yields of DNA-DSBs (RBEDSB) was calculated for standard radiation such as 220 kVp X-rays by using the estimating yields of SSBs and DSBs. The measurement points used to calculate the in-field yD and RBEDSB were located at a depth of 3, 5, and 10 cm in the water equivalent phantom on the central axis. Measurement points at 6, 8, and 10 cm in the lateral direction of each of the three depths from the central axis were set to calculate the out-of-field yD and RBEDSB.Results:The RBEDSB of FFF in-field was 1.7% higher than FF at each measurement depth. The RBEDSB of FFF out-of-field was 1.9 to 6.4% higher than FF at each depth measurement point. As the distance to out-of-field increased, the RBEDSB of FFF rose higher than those of FF. FFF has a larger RBEDSB than FF based on the yields of DNA-DSBs as the distance to out-of-field increased.Conclusions:The out-of-field radiobiological effect of FFF could thus be greater than that of FF since the spreading of the radiation dose out-of-field with FFF could be a concern compared to the FF.Advances in knowledge:The RBEDSB of FFF of out-of-field might be larger than FF.

Сравнительные количественные исследования фокусов H2AX, образующихся в фибробластах лёгкого человека, инкубированных в средах, содержащих меченный тритием тимидин или аминокислоты

We compared the formation of γH2AX foci (marker of DNA double-strand breaks) in human lung fibroblasts (MRC-5 line) during their 24-h incubation in a medium containing 3H-labeled thymidine or amino acids (glycine, alanine, and proline) with specific radioactivity from 100 to 400 MBq/liter. A linear dependence of changes in the number of γH2AX foci on the specific radioactivity of the medium was revealed. The quantitative yield of DNA double-strand breaks under the influence of 3H-thymidine was more than 2-fold higher than under the influence of 3H-labeled amino acids. Comparative analysis of the yields of DNA double-strand breaks during cell incubation with 3H-labeled amino acids showed that 3H-alanine produced more pronounced effect that 3H-proline, which is consistent with the data on the content of their non-radioactive analogs in chromatin proteins.

Stochastic multicellular modeling of x-ray irradiation, DNA damage induction, DNA free-end misrejoining and cell death

The repair or misrepair of DNA double-strand breaks (DSBs) largely determines whether a cell will survive radiation insult or die. A new computational model of multicellular, track structure-based and pO2-dependent radiation-induced cell death was developed and used to investigate the contribution to cell killing by the mechanism of DNA free-end misrejoining for low-LET radiation. A simulated tumor of 1224 squamous cells was irradiated with 6 MV x-rays using the Monte Carlo toolkit Geant4 with low-energy Geant4-DNA physics and chemistry modules up to a uniform dose of 1 Gy. DNA damage including DSBs were simulated from ionizations, excitations and hydroxyl radical interactions along track segments through cell nuclei, with a higher cellular pO2 enhancing the conversion of DNA radicals to strand breaks. DNA free-ends produced by complex DSBs (cDSBs) were able to misrejoin and produce exchange-type chromosome aberrations, some of which were asymmetric and lethal. A sensitivity analysis was performed and conditions of full oxia and anoxia were simulated. The linear component of cell killing from misrejoining was consistently small compared to values in the literature for the linear component of cell killing for head and neck squamous cell carcinoma (HNSCC). This indicated that misrejoinings involving DSBs from the same x-ray (including all associated secondary electrons) were rare and that other mechanisms (e.g. unrejoined ends) may be important. Ignoring the contribution by the indirect effect toward DNA damage caused the DSB yield to drop to a third of its original value and the cDSB yield to drop to a tenth of its original value. Track structure-based cell killing was simulated in all 135306 viable cells of a 1 mm3 hypoxic HNSCC tumor for a uniform dose of 1 Gy.

Modeling gold nanoparticle radiosensitization using a clustering algorithm to quantitate DNA double-strand breaks with mixed-physics Monte Carlo simulation.

The radiosensitization properties of gold nanoparticles (GNPs) are investigated using a simple Geant4 cell model considering a realistic cell geometry and a clustering algorithm to characterize the number of DNA double-strand breaks (DSBs). A mixed-physics approach is taken for accurate modeling of low-energy photon interactions in the different regions of the model using Geant4-DNA physics within the cell, and Livermore physics within gold. Density-based spatial clustering of applications with noise (DBSCAN), a clustering algorithm, is used to directly quantitate DNA DSBs after irradiation. The simulation was run using different sizes of GNPs, different distances of GNPs from the cell nucleus, and several combinations of these two conditions. Four types of radiation were simulated in the work: 80-keV monoenergetic photons, 100-keV monoenergetic photons, a 250-kVp photon spectrum, and a 6-MV flattening filter free (FFF) photon spectrum. A variable enhancement in DSB yield, nucleus dose, and cell dose was observed when there are GNPs in the cell cytoplasm, and increases with larger GNPs and proximity to the nucleus. The distance of the GNPs from the nucleus has a large impact on the DSB yield and nucleus dose, but little to no effect on the cell dose. The cell dose enhancement factor of 80keV photons varies from 1.037-1.125 at 0.2µm for 30-100nm GNPs to 1.040-1.127 at 4μm. The DSB enhancement factor varies from 1.050 to 1.174 at 0.2µm to a marginal effect of <1.01 at 4μm. For 100keV, the dose enhancement factor is from 1.142-1.470 at 0.2µm to 1.106-1.371 at 4μm. The DSB enhancement factor varies from 1.249-1.813 at 0.2µm to almost no effect at 4μm. For 250kVp, the dose enhancement factor is from 1.117-1.393 at 0.2μm to 1.110-1.342 at 4μm. The DSB enhancement factor varies from 1.183-1.600 at 0.2μm to a marginal effect of ~1.03 at 4μm. A 6-MV FFF shows a dose enhancement factor of 1.061-1.103 at 0.2μm and 1.053-1.107 at 4μm. The DSB yield varies from 1.070-1.143 at 0.2μm to a marginal effect at 4μm. The stark difference in behavior for DSB yield when compared to cell dose highlights the importance of evaluating more complex radiobiological quantities rather than dose alone when evaluating the radiosensitization properties from metallic nanomaterials. The nucleus dose showed similar characteristics to the DSB yield demonstrating the ability of the method to predict DNA damage and its relationship with nuclear dose. The proposed method provides a way to explore the radiobiological mechanisms of radiation-induced DNA damages, and it aids to evaluate the physical radiosensitization properties of GNP-aided radiotherapy, which can be easily combined with radiochemical DSB quantitation in order to better understand the intricate DNA damage induction mechanisms that are involved in GNP-aided radiotherapy.

Impact of dimethyl sulfoxide on irradiation-related DNA double-strand-break induction, -repair and cell survival.

Dimethyl sulfoxide (DMSO) is an effective radical scavenger and, when added to cells, reduces the initial number of radiation-induced DNA double-strand breaks (DSB). The aim of this study was to investigate modification by DMSO of both DSB induction and DSB repair by means of pulsed-field gel electrophoresis (PFGE) as well as gamma-H2AX immunofluorescence staining. WiDr cells (human colon carcinoma provided by DKFZ) were incubated with 2% DMSO for 2h (or mock-treated) prior to irradiation with varying X-ray doses and subsequent incubation for repair. Sample processing for PFGE analysis or counting of γ-H2AX foci was performed according to standard protocols. Effects on apoptosis induction and cell survival were investigated additionally by standard protocols. DMSO reduced DSB yield after 20-80Gy measured by PFGE. A qualitatively similar result was found after low-dose irradiation (1Gy) using γ-H2AX immunofluorescence staining. During incubation for repair, both DNA fragment rejoining (PFGE) as well as γ-H2AX foci removal occurred at a reduced rate when cells had been pre-treated with DMSO. But this effect was clearly more pronounced for the PFGE-analyzed double-strand breakage, particularly at early repair times. WiDr cells treated with DMSO (2%) showed a significantly increased clonogenic survival after irradiation doses above 8Gy. Apoptosis rates were not changed by DMSO. The radio-protective effect of DMSO, well known from other PFGE studies, could be confirmed for the formation of γ-H2AX foci. DSB generated in the presence of DMSO were less rapidly repaired. DMSO showed radio-protective effects on clonogenic survival but not on apoptosis.

A cell-by-cell Monte Carlo simulation for assessing radiation-induced DNA double strand breaks

This paper presents a cell-by-cell Monte Carlo simulation study that combines charged particle track structure data with an interphase cell nucleus model to quantify DNA double strand breaks (DSBs), spatial distribution of DSBs in a cell nucleus, and resulting potentially lethal or mutagenic events (PLMEs) between DSBs in close proximity. Cell nucleus is simulated according to the chromosome territory-interchromatin compartment (CT-IC) model in that chromatin content is unevenly distributed in chromatin domains (CDs) and IC with a chromatin compaction ratio of 22:1. A particle track structure coordinate (PTSC) library was first generated for each particle type, energy, and dose based on a large number of particle track data obtained by running the Monte Carlo track structure code Geant4-DNA. To assess the DNA DSBs of a cell for a specific particle type, energy, and dose, the corresponding PTSC was selected and “map overlaid” onto 960 unique cell nucleus data sets containing chromatin fiber (CF) locations. Clustering algorithm DBSCAN was next used to identify the clustered energy deposition events occurring inside the CF. These events were then converted to DNA DSBs using a probabilistic approach. The locations of the DSBs thus obtained were, in turn, used to calculate PLMEs within the cell nucleus that can result from DSB proximity and complexity. The results obtained from this simulation study are correctly correlated to the experimental data of DSB yield and the RBE-LET relationships for various types of charged particles and of various energies. The results show agreement with other published radiobiological models.

Comparative Analysis of the Formation of γH2AX Foci in Human Mesenchymal Stem Cells Exposed to 3H-Thymidine, Tritium Oxide, and X-Rays Irradiation.

We performed a comparative study of the formation of γН2АХ foci (a marker of DNA doublestrand breaks) in human bone marrow mesenchymal stem cells after 24-h incubation with 3Н-thimidin and tritium oxide with low specific activities (50-800 MBq/liter). The dependence of the number of γH2AX foci on specific activity of 3H-thymidine was described by a linear equation y=2.21+43.45x (R2=0.96), where y is the number of γH2AX foci per nucleus and x is specific activity in 1000 MBq/liter. For tritium oxide, the relationship was described by a linear equation y=2.52+6.70x (R2=0.97). Thus, the yield of DNA double-strand breaks after exposure to 3H-thymidine was 6.5-fold higher than after exposure to tritium oxide. Comparison of the effects of tritium oxide and X-ray radiation on the yield of DNA double-strand breaks showed that the relative biological efficiency of tritium oxide in a dose range of 3.78-60.26 mGy was 1.6-fold higher than that of X-ray radiation. Improvement of the methods of analysis of DNA double-strand breaks repair foci is highly promising in the context of creation of highly sensitive biodosimetry technologies for tritium compounds in humans.

Formation of Direct and Enzymatic DNA Double-Strand Breaks in the Presence of Repair Inhibitors after Exposure to Radiations of Different Quality

With the use of the DNA comet assay and immunocytochemical staining, regularities have been studied in the induction and repair of DNA double-strand breaks(DSBs) in human cells after exposure to 60Co γ-rays and accelerated heavy ions with different linear energy transfer (LET) in the presence of the DNA repair inhibitors cytosine arabinoside and hydroxyurea. It is shown that for heavy ions the agents’ modifying effect decreases with increasing particles’ LET. The approach involving DNA synthesis inhibitors used in this study allows an estimation of the proportion of enzymatic DNA DSBs in total DSB yield after exposure to ionizing radiations of different quality.

A modified microdosimetric kinetic model for relative biological effectiveness calculation

In the heavy ion therapy, not only the distribution of physical absorbed dose, but also the relative biological effectiveness (RBE) weighted dose needs to be taken into account. The microdosimetric kinetic model (MKM) can predict the RBE value of heavy ions with saturation-corrected dose-mean specific energy, which has been used in clinical treatment planning at the National Institute of Radiological Sciences. In the theoretical assumption of the MKM, the yield of the primary lesion is independent of the radiation quality, while the experimental data shows that DNA double strand break (DSB) yield, considered as the main primary lesion, depends on the LET of the particle. Besides, the β parameter of the MKM is constant with LET resulting from this assumption, which also differs from the experimental conclusion. In this study, a modified MKM was developed, named MMKM. Based on the experimental DSB yield of mammalian cells under the irradiation of ions with different LETs, a RBEDSB (RBE for the induction of DSB)-LET curve was fitted as the correction factor to modify the primary lesion yield in the MKM, and the variation of the primary lesion yield with LET is considered in the MMKM. Compared with the present the MKM, not only the α parameter of the MMKM for mono-energetic ions agree with the experimental data, but also the β parameter varies with LET and the variation trend of the experimental result can be reproduced on the whole. Then a spread-out Bragg peaks (SOBP) distribution of physical dose was simulated with Geant4 Monte Carlo code, and the biological and clinical dose distributions were calculated, under the irradiation of carbon ions. The results show that the distribution of clinical dose calculated with the MMKM is closed to the distribution with the MKM in the SOBP, while the discrepancy before and after the SOBP are both within 10%. Moreover, the MKM might overestimate the clinical dose at the distal end of the SOBP more than 5% because of its constant β value, while a minimal value of β is calculated with the MMKM at this position. Besides, the discrepancy of the averaged cell survival fraction in the SOBP calculated with the two models is more than 15% at the high dose level. The MMKM may provide a reference for the accurate calculation of the RBE value in heavy ion therapy.