Prediction of DNA rejoining kinetics and cell survival after proton irradiation for V79 cells using Geant4-DNA.

PURPOSE: Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology. METHODS: We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs. RESULTS: By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction. CONCLUSION: This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints.

Changes in Radiosensitivity to Gamma-Rays of Lymphocytes from Hyperthyroid Patients Treated with I-131.

This study investigated the peripheral blood lymphocytes (PBL) response to a dose of γ-rays in patients treated with radioiodine (I-131) for hyperthyroidism vs. healthy controls, to gain information about the individual lymphocytes' radio-sensitivity. Blood samples were taken from 18 patients and 10 healthy donors. Phosphorylated histone variant H2AX (γ-H2AX) and micronuclei (MN) induction were used to determine the change in PBL radio-sensitivity and the correlations between the two types of damage. The two assays showed large inter-individual variability in PBL background damage and in radio-sensitivity (patients vs. healthy donors). In particular, they showed an increased radio-sensitivity in 36% and 33% of patients, decrease in 36% and 44%, respectively. There was a scarce correlation between the two assays and no dependence on age or gender. A significant association was found between high radio-sensitivity conditions and induced hypothyroidism. PBL radio-sensitivity in the patient group was not significantly affected by treatment with I-131, whereas there were significant changes inter-individually. The association found between clinical response and PBL radio-sensitivity suggests that the latter could be used in view of the development of personalized treatments.

Ionizing Radiation-Induced Epigenetic Modifications and Their Relevance to Radiation Protection.

The present system of radiation protection assumes that exposure at low doses and/or low dose-rates leads to health risks linearly related to the dose. They are evaluated by a combination of epidemiological data and radiobiological models. The latter imply that radiation induces deleterious effects via genetic mutation caused by DNA damage with a linear dose-dependence. This picture is challenged by the observation of radiation-induced epigenetic effects (changes in gene expression without altering the DNA sequence) and of non-linear responses, such as non-targeted and adaptive responses, that in turn can be controlled by gene expression networks. Here, we review important aspects of the biological response to ionizing radiation in which epigenetic mechanisms are, or could be, involved, focusing on the possible implications to the low dose issue in radiation protection. We examine in particular radiation-induced cancer, non-cancer diseases and transgenerational (hereditary) effects. We conclude that more realistic models of radiation-induced cancer should include epigenetic contribution, particularly in the initiation and progression phases, while the impact on hereditary risk evaluation is expected to be low. Epigenetic effects are also relevant in the dispute about possible "beneficial" effects at low dose and/or low dose-rate exposures, including those given by the natural background radiation.

The Response of Living Organisms to Low Radiation Environment and Its Implications in Radiation Protection.

Life has evolved on Earth for about 4 billion years in the presence of the natural background of ionizing radiation. It is extremely likely that it contributed, and still contributes, to shaping present form of life. Today the natural background radiation is extremely small (few mSv/y), however it may be significant enough for living organisms to respond to it, perhaps keeping memory of this exposure. A better understanding of this response is relevant not only for improving our knowledge on life evolution, but also for assessing the robustness of the present radiation protection system at low doses, such as those typically encountered in everyday life. Given the large uncertainties in epidemiological data below 100 mSv, quantitative evaluation of these health risk is currently obtained with the aid of radiobiological models. These predict a health detriment, caused by radiation-induced genetic mutations, linearly related to the dose. However a number of studies challenged this paradigm by demonstrating the occurrence of non-linear responses at low doses, and of radioinduced epigenetic effects, i.e., heritable changes in genes expression not related to changes in DNA sequence. This review is focused on the role that epigenetic mechanisms, besides the genetic ones, can have in the responses to low dose and protracted exposures, particularly to natural background radiation. Many lines of evidence show that epigenetic modifications are involved in non-linear responses relevant to low doses, such as non-targeted effects and adaptive response, and that genetic and epigenetic effects share, in part, a common origin: the reactive oxygen species generated by ionizing radiation. Cell response to low doses of ionizing radiation appears more complex than that assumed for radiation protection purposes and that it is not always detrimental. Experiments conducted in underground laboratories with very low background radiation have even suggested positive effects of this background. Studying the changes occurring in various living organisms at reduced radiation background, besides giving information on the life evolution, have opened a new avenue to answer whether low doses are detrimental or beneficial, and to understand the relevance of radiobiological results to radiation protection.

Targeting cancer stem cells: protons versus photons.

Recent studies on cancer stem cells revealed they are tumorigenic and able to recapitulate the characteristics of the tumour from which they derive, so that it was suggested that elimination of this population is essential to prevent recurrences after any treatment. However, there is evidence that cancer stem cells are inherently resistant to conventional (photon) radiotherapy. Since the use of proton beam therapy in cancer treatment is growing rapidly worldwide, mainly because of their excellent dosimetric properties, the possibility could be considered that they also have biological advantages through preferential elimination of cancer stem cells.Indeed, a review of preclinical data suggest that protons and photons differ in their biological effects on cancer stem cells, with protons offering potential advantages, although the heterogeneity of cancer stem cells and the different proton irradiation modalities make the comparison of the results not so easy. Further research to understand the mechanisms underlying such effects is important for their possible exploitation in clinics and to perform proton beam therapy optimization.

The European initiative on low-dose risk research: from the HLEG to MELODI.

The importance of low-dose risk research for radiation protection is now widely recognised. The European Commission (EC) and five European Union (EU) Member States involved in the Euratom Programme set up in 2008 a 'High Level and Expert Group on European Low Dose Risk Research' (HLEG) aimed at identifying research needs and proposing a better integration of European efforts in the field. The HLEG revised the research challenges and proposed a European research strategy based on a 'Multidisciplinary European LOw Dose Initiative' (MELODI). In April 2009, five national organisations, with the support of the EC, created the initial core of MELODI (http://www.melodi-online.eu) with a view to integrate the EU institutions with significant programmes in the field, while being open to other scientific organisations and stakeholders, and to develop an agreed strategic research agenda (SRA) and roadmap. Since then, open workshops have been organised yearly, exploring ideas for SRA implementation. As of October 2014, 31 institutions have been included as members of MELODI. HLEG recommendations and MELODI SRA have become important reference points in the radiation protection part of the Euratom Research Programme. MELODI has established close interactions through Memorandum of Understanding with other European platforms involved in radiation protection (Alliance, NERIS and EURADOS) and, together with EURADOS, with the relevant medical European Associations. The role of Joint Programming in priority setting, foreseen in the forthcoming EU Horizon 2020, calls for keeping MELODI an open, inclusive and transparent initiative, able to avoid redundancies and possible conflicts of interest, while promoting common initiatives in radiation protection research. An important issue is the establishment of a proper methodology for managing these initiatives, and this includes the set-up of an independent MELODI Scientific Committee recently extended to Alliance, NERIS and EURADOS, with the aim of identifying research priorities to suggest for the forthcoming Euratom research calls.

The European strategy on low dose risk research and the role of radiation quality according to the recommendations of the "ad hoc" High Level and Expert Group (HLEG).

Health effects of exposures at low doses and/or low dose rates are recognized as requiring intensive research activity to answer several questions. To address these issues at a strategic level in Europe, with the perspective of integrating national and EC efforts (in particular those within the Euratom research programmes), a "European High Level and Expert Group (HLEG) on low dose risk research" was formed and carried out its work during 2008. The Group produced a report published by the European Commission in 2009 and available on the website http://www.hleg.de . The more important research issues identified by the HLEG were as follows: (a) the shape of dose-response for cancer; (b) the tissue sensitivities for cancer induction; (c) the individual variability in cancer risk; (d) the effects of radiation quality (type); (e) the risks from internal radiation exposure; and (f) the risks of, and dose response relationships for, non-cancer diseases. In this paper, the radiation quality issues are especially considered, since they are closely linked to health problems and related radioprotection in space and in emerging radiotherapeutic techniques (i.e., hadrontherapy). The peculiar features of low-fluence, high-LET radiation exposures can question in particular the validity of the radiation-weighting factor (w ( R )) approach. Specific strategies are therefore needed to assess such risks. A multi-scale/systems biology approach, based on mechanistic studies coordinated with molecular-epidemiological studies, is considered essential to elucidate differences and similarities between specific effects of low- and high-LET radiation.

An alpha-particle irradiator for radiobiological research and its implementation for bystander effect studies.

An experimental system based on an improved version of an existing alpha-particle irradiator has been developed for radiobiological studies, in particular those investigating bystander effects. It consists of a 20-mm-diameter stainless steel chamber that can be equipped alternatively with 244Cm or 241Am sources of different activities. Mylar-based petri dishes 56 mm in diameter were specially designed to house adaptors for permeable membrane inserts that reproduce the geometry of commercial cell culture insert companion plates. Characterization of the radiation field at the cell level was performed by experimental measurements and calculations. The average incident LET was about 122 keV/microm for 244Cm and about 125 keV/microm for 241Am. Dose rates at the chosen source-sample distance were 2.8 and 88.6 mGy/min, respectively. These low dose rates are suitable for our planned experiments on low-dose effects. For both sources, the uniformity of the alpha-particle dose was better than +/-7%, and the photon dose calculated at the cell entrance was negligible compared to the alpha-particle dose. The irradiator is small enough to be inserted into a cell incubator for irradiation under physiological conditions or into a refrigerator to prevent metabolic processes during irradiation. Benchmark experiments using the 241Am source to examine DNA double-strand breaks in directly hit and bystander primary human fibroblasts have shown that the irradiator can be used successfully for bystander effect studies.

Effectiveness of monoenergetic and spread-out bragg peak carbon-ions for inactivation of various normal and tumour human cell lines.

This work aimed at measuring cell-killing effectiveness of monoenergetic and Spread-Out Bragg Peak (SOBP) carbon-ion beams in normal and tumour cells with different radiation sensitivity. Clonogenic survival was assayed in normal and tumour human cell lines exhibiting different radiosensitivity to X- or gamma-rays following exposure to monoenergetic carbon-ion beams (incident LET 13-303 keV/microm) and at various positions along the ionization curve of a therapeutic carbon-ion beam, corresponding to three dose-averaged LET (LET(d)) values (40, 50 and 75 keV/microm). Chinese hamster V79 cells were also used. Carbon-ion effectiveness for cell inactivation generally increased with LET for monoenergetic beams, with the largest gain in cell-killing obtained in the cells most radioresistant to X- or gamma-rays. Such an increased effectiveness in cells less responsive to low LET radiation was found also for SOBP irradiation, but the latter was less effective compared with monoenergetic ion beams of the same LET. Our data show the superior effectiveness for cell-killing exhibited by carbon-ion beams compared to lower LET radiation, particularly in tumour cells radioresistant to X- or gamma-rays, hence the advantage of using such beams in radiotherapy. The observed lower effectiveness of SOBP irradiation compared to monoenergetic carbon beam irradiation argues against the radiobiological equivalence between dose-averaged LET in a point in the SOBP and the corresponding monoenergetic beams.

Subtraction of background damage in PFGE experiments on DNA fragment-size distributions.

The non-random distribution of DNA breakage in pulsed-field gel electrophoresis (PFGE) experiments poses a problem of proper subtraction of the background damage to obtain a fragment-size distribution due to radiation only. As been pointed out by various authors, a naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram, and may even result in negative values. Previous more systematic subtraction methods have been based mainly on random breakage, appropriate for low-LET radiation but problematic for high LET. Moreover, an investigation is needed whether the background breakage itself is random or non-random. Previously a new generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and any background was proposed, and as now applied it to a set of PFGE data for Fe ions. We developed a Monte Carlo algorithm to compare the naïve subtraction procedure in artificial data sets to the result produced by the new formalism. The simulated data corresponded to various cases, involving non-random (high-LET) or random radiation breakage and random or non-random background breakage. The formalism systematically gives better results than naïve bin-by-bin subtraction in all these artificial data sets.

A robust procedure for removing background damage in assays of radiation-induced DNA fragment distributions.

The non-random distribution of DNA breakage in PFGE (pulsed-field gel electrophoresis) experiments poses a problem of proper subtraction of the background DNA damage to obtain a fragment-size distribution due to radiation only. A naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram. This problem could become more pronounced for high-LET (linear energy transfer) radiation, because the fragment-size distribution manifests a higher frequency of smaller fragments. Previous systematic subtraction methods have been based on random breakage, appropriate for low-LET radiation. Moreover, an investigation is needed to determine whether the background breakage is itself random or non-random. We consider two limiting cases: (1) the background damage is present in all cells, and (2) it is present in only a small subset of cells, while other cells are not contributing to the background DNA fragmentation. We give a generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and apply it to two sets of PFGE data for iron ions.

DNA fragmentation induced in human fibroblasts by accelerated (56)fe ions of differing energies.

DNA fragmentation was studied in the fragment size range 0.023-5.7 Mbp after irradiation of human fibroblasts with iron-ion beams of four different energies, i.e., 200 MeV/nucleon, 500 MeV/nucleon, 1 GeV/nucleon and 5 GeV/nucleon, with gamma rays used as the reference radiation. The double-strand break (DSB) yield (and thus the RBE for DNA DSB induction) of the four iron-ion beams, which have LETs ranging from 135 to 442 keV/mum, does not vary greatly as a function of LET. As a consequence, the variation of the cross section for DSB induction mainly reflects the variation in LET. However, when the fragmentation spectra were analyzed with a simple theoretical tool that we recently introduced, the results showed that spatially correlated DSBs, which are absent after gamma irradiation, increased markedly with LET for the iron-ion beams. This occurred because iron ions produce DNA fragments smaller than 0.75 Mbp with a higher probability than gamma rays (a probability that increases with LET). These sizes include those expected from fragmentation of the chromatin loops with Mbp dimensions. This result does not exclude a correlation at distances smaller than the lower size analyzed here, i.e. 23 kbp. Moreover, the DSB correlation is dependent on dose, decreasing when dose increases; this can be explained with the argument that at increasing dose there is an increasing fraction of fragments produced by DSBs caused by separate, uncorrelated tracks.

Induction and repair of DNA double-strand breaks in human cells: dephosphorylation of histone H2AX and its inhibition by calyculin A.

Phosphorylation of histone H2AX at serine 139 (gamma-H2AX) represents one of the earliest steps in DNA DSB signaling and repair, but the mechanisms of coupling this histone modification to DSB processing remain to be established. In this work, H2AX phosphorylation-dephosphorylation kinetics induced by low doses of gamma rays in MRC-5 human fibroblasts was studied. The number of gamma-H2AX foci increased rapidly, with the maximum reached 20 min after irradiation. Using calyculin A, a protein phosphatase inhibitor, no significant dephosphorylation was found in this time. At longer times, no further induction of gamma-H2AX foci occurred. This indicates that the number of gamma-H2AX foci scored at 20 min can be used as representative of the initial number of DSBs. Pulsed-field gel electrophoresis (PFGE) was also used to determine whether calyculin A-mediated inhibition of gamma-H2AX dephosphorylation and DSB rejoining are independent phenomena. We found that the maintenance of the phosphate group at Ser 139 in gamma-H2AX does not represent an obstacle for DSB rejoining. Preliminary experiments performed with 62 MeV/nucleon carbon ions have shown a longer persistence of gamma-H2AX foci with respect to gamma rays, consistent with the induction of damage that is more severe and difficult to repair.

Influence of PMMA shielding on DNA fragmentation induced in human fibroblasts by iron and titanium ions.

In the framework of a collaborative project on the influence of the shielding on the biological effectiveness of space radiation, we studied DNA fragmentation induced by 1 GeV/nucleon iron ions and titanium ions with and without a 197-mm-thick polymethylmethacrylate (PMMA) shield in AG1522 human fibroblasts. Pulsed- and constant-field gel electrophoresis were used to analyze DNA fragmentation in the size range 1-5700 kbp. The results show that, mainly owing to a higher production of small fragments (1-23 kbp), titanium ions are more effective than iron ions at inducing DNA double-strand breaks (DSBs), their RBE being 2.4 and 1.5, respectively. The insertion of a PMMA shield decreases DNA breakage, with shielding protection factors (ratio of the unshielded/shielded cross sections for DSB production) of about 1.6 for iron ions and 2.1 for titanium ions. However, the DSB yield (no. of DSBs per unit mass per unit dose) is almost unaffected by the presence of the shield, and the relative contributions of the fragments in the different size ranges are almost the same with or without shielding. This indicates that, under our conditions, the effect of shielding is mainly to reduce the dose per unit incident fluence, leaving radiation quality practically unaffected.

Molecular targets in cellular response to ionizing radiation and implications in space radiation protection.

DNA repair systems and cell cycle checkpoints closely co-operate in the attempt of maintaining the genomic integrity of cells damaged by ionizing radiation. DNA double-strand breaks (DSB) are considered as the most biologically important radiation-induced damage. Their spatial distribution and association with other types of damage depend on radiation quality. It is believed these features affect damage reparability, thus explaining the higher efficiency for cellular effects of densely ionizing radiation with respect to gamma-rays. DSB repair systems identified in mammalian cells are homologous recombination (HR), single-strand annealing (SSA) and non-homologous end-joining (NHEJ). Some enzymes may participate in more than one of these repair systems. DNA damage also triggers biochemical signals activating checkpoints responsible for delay in cell cycle progression that allows more time for repair. Those at G1/S and S phases prevent replication of damaged DNA and those at G2/M phase prevent segregation of changed chromosomes. Individuals with lack or alterations of genes involved in DNA DSB repair and cell cycle checkpoints exhibit syndromes characterized by genome instability and predisposition to cancer. Information reviewed in this paper on the basic mechanisms of cellular response to ionizing radiation indicates their importance for a number of issues relevant to protection of astronauts from space radiation.