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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.

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.

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 importance of research to reduce uncertainties in risk assessment of low and protracted exposures is now recognised globally. In Europe a new initiative, called 'Multidisciplinary European LOw Dose Initiative' (MELODI), has been proposed by a 'European High Level and Expert Group on low-dose risk research' (www.hleg.de), aimed at integrating national and EC (Euratom) efforts. Five national organisations: BfS (DE), CEA (FR), IRSN (FR), ISS (IT) and STUK (FI), with the support of the EC, have initiated the creation of MELODI by signing a letter of intent. In the forthcoming years, MELODI will integrate in a step-by-step approach EU institutions with significant programmes in the field and will be open to other scientific organisations and stakeholders. A key role of MELODI is to develop and maintain over time a strategic research agenda (SRA) and a road map of scientific priorities within a multidisciplinary approach, and to transfer the results for the radiation protection system. Under the coordination of STUK a network has been proposed in the 2009 Euratom Programme, called DoReMi (Low-Dose Research towards Mutidisciplinary Integration), which can help the integration process within the MELODI platform. DoReMi and the First MELODI Open Workshop, organised by BfS in September 2009, are now important inputs for the European SRA.

We simulated the irradiation of human fibroblasts with gamma rays, protons and helium, carbon and iron ions at a fixed dose of 5 Gy. The simulations were performed with the biophysical Monte Carlo code PARTRAC. From the output of the code, containing in particular the genomic positions of the radiation-induced DNA double-strand breaks (DSBs), we obtained the DNA fragmentation spectra. Very small fragments, in particular those related to "complex lesions" (few tens of base pairs), are probably very important for the late cellular consequences, but their detection is not possible with the common experimental techniques. We paid special attention to the differences among the various ions in the production of these very small fragments; in particular, we compared the fragmentation spectra for ions of the same specific energy and for ions of the same LET (linear energy transfer). As found previously for iron ions, we found that the RBE (relative biological effectiveness) for DSB production was considerably higher than 1 for all high-LET radiations considered. This is at variance with the results obtainable from experimental data, and it is due to the ability to count the contribution of small fragments. It should be noted that for a given LET this RBE decreases with increasing ion charge, due mainly to the increasing mean energy of secondary electrons. A precise quantification of the DNA initial damage can be of great importance for both radiation protection, particularly in open-space long-term manned missions, and hadrontherapy.

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.

We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1-5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1-23 kbp and 23-5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1-5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since gamma rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

In this work we have compared the pattern of DNA fragmentation (in the size range 1–5700 kbp) induced in human fibroblasts by γ-rays with that induced by Fe ions with an energy of 115 MeV/u; Fe ions are considered of biological significance for radiation protection issues during long term astronauts’ travels. The study has taken into account the comparison of the experimental fragmentation spectra, their analysis performed through the implementation of a phenomenological model, and Monte Carlo simulations performed with the PARTRAC code. The phenomenological method characterizes in an approximate but simple way the nonrandom nature of the experimental fragment distribution caused by high LET radiation; it has the advantage to take into account in a detailed way the background fragmentation. The PARTRAC simulations, on the other hand, thanks to the accurate representation of the chromatin geometry and of the physical and physico-chemical processes associated with the energy deposition by radiation, offer the possibility to compute the spectra and to compare them with the experimental data. We found a satisfactory agreement between the experimental data and the results obtained with PARTRAC, recently upgraded with the implementation of ions heavier than alpha particles; this agreement represents a first validation of the code. A relevant result is represented by the very high number of fragments that, according to the Monte Carlo simulations, are produced by iron ions in the size range

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 spatial distribution of radiation-induced DNA breaks within the cell nucleus depends on radiation quality in terms of energy deposition pattern. It is generally assumed that the higher the radiation linear energy transfer (LET), the greater the DNA damage complexity. Using a combined experimental and theoretical approach, we examined the phosphorylation-dephosphorylation kinetics of radiation-induced γ-H2AX foci, size distribution and 3D focus morphology, and the relationship between DNA damage and cellular end points (i.e., cell killing and lethal mutations) after exposure to gamma rays, protons, carbon ions and alpha particles. Our results showed that the maximum number of foci are reached 30 min postirradiation for all radiation types. However, the number of foci after 0.5 Gy of each radiation type was different with gamma rays, protons, carbon ions and alpha particles inducing 12.64 ± 0.25, 10.11 ± 0.40, 8.84 ± 0.56 and 4.80 ± 0.35 foci, respectively, which indicated a clear influence of the track structure and fluence on the numbers of foci induced after a dose of 0.5 Gy for each radiation type. The γ-H2AX foci persistence was also dependent on radiation quality, i.e., the higher the LET, the longer the foci persisted in the cell nucleus. The γ-H2AX time course was compared with cell killing and lethal mutation and the results highlighted a correlation between cellular end points and the duration of γ-H2AX foci persistence. A model was developed to evaluate the probability that multiple DSBs reside in the same gamma-ray focus and such probability was found to be negligible for doses lower than 1 Gy. Our model provides evidence that the DSBs inside complex foci, such as those induced by alpha particles, are not processed independently or with the same time constant. The combination of experimental, theoretical and simulation data supports the hypothesis of an interdependent processing of closely associated DSBs, possibly associated with a diminished correct repair capability, which affects cell killing and lethal mutation.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour, with very poor prognosis. The high recurrence rate and failure of conventional treatments are expected to be related to the presence of radio-resistant cancer stem cells (CSCs) inside the tumour mass. CSCs can both self-renew and differentiate into the heterogeneous lineages of cancer cells. Recent evidence showed a higher effectiveness of C-ions and protons in inactivating CSCs, suggesting a potential advantage of Hadrontherapy compared with conventional radiotherapy for GBM treatment. To investigate the mechanisms involved in the molecular and cellular responses of CSCs to ionising radiations, two GBM stem cell (GSC) lines, named lines 1 and 83, which were derived from patients with different clinical outcomes and having different metabolic profiles (as shown by NMR spectroscopy), were irradiated with 137Cs photons and with protons or C-ions of 62 MeV u-1 in the dose range of 5-40 Gy. The biological effects investigated were: cell death, cell cycle progression, and DNA damage induction and repair. Preliminary results show a different response to ionising radiation between the two GSC lines for the different end points investigated. Further experiments are in progress to consolidate the data and to get more insights on the influence of radiation quality. © The Author 2015.

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.

To quantify the role played by radiation track structure and background fragments in modulating DNA fragmentation in human cells exposed to gamma-rays and light ions.Human fibroblasts were exposed in vitro to different doses (in the range from 40 - 200 Gy) of (60)Co gamma-rays and 0.84 MeV protons (Linear Energy Transfer, LET, in tissue 28.5 keV/microm). The resulting DNA fragments were scored under two electrophoretic conditions, in order to optimize separation in the size ranges 0.023 - 1.0 Mbp and 1.0 - 5.7 Mbp. In parallel, DNA fragmentation was simulated both with a phenomenological approach based on the "generalized broken-stick" model, and with a mechanistic approach based on the PARTRAC (acronym of PARticle TRACk) Monte Carlo code (1.32 MeV photons were used for the simulation of (60)Co gamma-rays).For both gamma-rays and protons, the experimental dose response in the range 0.023 - 5.7 Mbp could be approximated as a straight line, the slope of which provided a yield of (5.3 +/- 0.4) x 10(-9) Gy(-1) bp(-1) for gamma-rays and (7.1 +/- 0.6) x 10(-9) Gy(-1) bp(-1) for protons, leading to a Relative Biological Effectiveness (RBE) of 1.3 +/- 0.2. From both theoretical analyses it appeared that, while gamma-ray data were consistent with double-strand breaks (DSB) random induction, protons at low doses showed significant deviation from randomness, implying enhanced production of small fragments in the low molecular weight part of the experimental range. The theoretical analysis of fragment production was then extended to ranges where data were not available, i.e. to fragments larger than 5.7 Mbp and smaller than 23 kbp. The main outcome was that small fragments (23 kbp) are produced almost exclusively via non-random processes, since their number is considerably higher than that produced by a random insertion of DSB. Furthermore, for protons the number of these small fragments is a significant fraction (about 20%) of the total number of fragments; these fragments remain undetected in these experiments. Calculations for 3.3 MeV alpha particle irradiation (for which no experimental data were available) were performed to further investigate the role of fragments smaller than 23 kbp; in this case, besides the non-random character of their production, their number resulted to be at least as much as half of the total number of fragments.Comparison between experimental data and two different theoretical approaches provided further support to the hypothesis of an important role of track structure in modulating DNA damage. According to the theoretical approaches, non-randomness of fragment production was found for proton irradiation for the smaller fragments in the experimental size range and, in a significantly larger extent, for fragments of size less than 23 kbp, both for protons and alpha particles.

To characterize the differences among the experimental DNA fragmentation spectra induced in Chinese hamster V79 cells by gamma-rays, low-energy protons and alpha-particles through the use of a phenomenological model.A model of DNA fragmentation was developed as a generalization of the broken-stick model, in which the double-strand breaks induced by radiation were considered randomly placed, but in which the manifestly non-random fragmentation of the control sample was fully taken into account and considered as the initial fragment distribution. Further, an analytical method was introduced that allowed an evaluation of the deviation from randomness of the fragmentation induced by radiation.The analysis of the experimental distribution of DNA fragments showed that there was a progressive departure from randomness in radiation-induced fragmentation going from gamma-rays to protons and then to alpha-particles. This deviation was characterized by an enhanced induction of fragments, and therefore by a larger correlation of double-strand breaks, in the experimental range of lower molecular weights.The analysis shows that low-energy light ions induce DNA fragmentation, at the loop level of the chromatin organization, that can be significantly non-random. The same analysis can readily be applied at different length scales, and thus it could offer a basis for the study of the link between DNA damage, correlated at various spatial scales and biological end-points.

Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used gamma rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by gamma rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Very little is known about the influence of environmental radiation on living matter. In principle, important information can be acquired by analysing possible differences between parallel biological systems, one in a reference-radiation environment (RRE) and the other in a low-radiation environment (LRE). We took advantage of the unique opportunity represented by the cell culture facilities at the Gran Sasso National Laboratories of the Istituto Nazionale di Fisica Nucleare, where environment dose rate reduction factors in the underground (LRE), with respect to the external laboratory (RRE), are as follows: 10(3) for neutrons, 10(7) for directly ionizing cosmic rays and 10 for total γ-rays. Chinese hamster V79 cells were cultured for 10 months in both RRE and LRE. At the end of this period, all the cultures were kept in RRE for another 6 months. Changes in the activities of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX) and spontaneous mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus were investigated. The results obtained suggest that environmental radiation might act as a trigger of defence mechanisms in V79 cells, specifically those in reference conditions, showing a higher degree of defence against endogenous damage as compared to cells grown in a very low-radiation environment. Our findings corroborate the hypothesis that environmental radiation contributes to the development of defence mechanisms in today living organisms/systems.

Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/micrometer protons, 123 keV/micrometer helium-4 ions and gamma rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respect to that induced by comparable doses of gamma rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for gamma rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage repairability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by gamma rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.

To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET).V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE).The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2 h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0.The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.

An experimental set-up is described for irradiating cultured mammalian cells with ultrasoft X-rays produced by a laser–plasma source developed at the ENEA-CRE in Frascati (Italy), using a non-monochromatic beam of energies (0.90÷1.56) keV. Dosimetry methods have been developed for such a beam, taking into account the different X-ray attenuation for the various energy components. The dependence on cell depth of the dose (expressed in terms of kerma) in V79 cell nucleus has been evaluated, assuming various geometrical models that can be generalized to other types of cells.

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.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.

Purpose RBE-LET relationships for cell inactivation and hprt mutation in V79 cells have been studied with mono-energetic low-energy proton beams at the radiobiological facility of the INFN-Laboratori Nazionali di Legnaro (LNL), Padova, Italy. Materials and methods V79 cells were irradiated in mono-layer on mylar coated stainless steel petri dishes, in air. Inactivation data were obtained at 7.7, 34.6 and 37.8 keV/microm and hprt mutation was studied at 7 7 and 37.8 keV/microm. Additional data were also collected for both the end points with the proton LET already considered in our previous publications, namely 11.0, 20.0 and 30.5 keV/microm. Results A maximum in the RBE-LET relationship for cell inactivation was found at around 31 keV/microm, while the RBE for mutation induction increased continuously with LET. Conclusions The proton RBE-LET relationship for cell inactivation is shifted to lower LET values compared with that for heavier ions. For mutation induction, protons of LET equal to 7.7keV/microm gave an RBE value comparable with that obtained by helium ions of about 20 keV/microm. Mutagenicity and lethality caused by protons at low doses in the LET range 7.7-31 keV/microm were proportional, while the data at 37.8 keV/microm suggest that this may not hold at higher LET values.

Investigation of the mechanisms underlying the biological effects induced by densely ionizing radiation has relevant implications in both radiation protection and therapy. In particular, the possible advantages of hadrontherapy with respect to conventional radiotherapy in terms of high conformal tumor treatment and sparing of healthy tissues are well known. Further improvements are limited by lack of radiobiological knowledge, particularly about the specific cellular response to the damage induced by particles of potential interest for tumor treatment. This study compares early and late effects induced in AG01522 normal human primary fibroblasts by γ-rays and C-ions having E ∼ 45 MeV/u at the cell entrance, corresponding to LET (in water) ∼ 49 keV/μm. Different end points have been investigated, namely: cell killing and lethal mutation, evaluated as early and delayed reproductive cell death, respectively; chromosome damage, as measured by micronuclei induction (MN); DNA damage, in terms of DSB induction and repair, as measured by the H2AX phosphorylation/dephosphorylation kinetics. Linear dose-response relationships were found for cell killing and induction of lethal mutations, with RBEs of about 1.3 and 1.6 respectively, indicating that the presence of genomic instability is greater in the progeny of C-ions irradiated cells. H2AX phosphorylation/dephosphorylation kinetics have shown a maximum foci number at 30 min after irradiation, higher for γ-rays than for C-ions. However, in the first 12 h the fraction of residual γ-H2AX foci was higher for C-ions irradiated cells, indicating a lower removal rate, possibly related to multiple/more complex damage along the particle track, with respect to the sparse lesions produced by γ-rays. MN induction, observed after 72 h from irradiation, was also greater for C-ions. Overall, these data indicate a more severe DNA damage induced by 45 MeV/u C-ions with respect to γ-rays, likely responsible of an increased cellular misrepair, leading to the greater observed levels of chromosome damage and, eventually, of genomic instability. They give strong support to the idea that higher damage severity at molecular level, determined by the typical deposition pattern of densely ionizing radiation, is the earliest relevant factor for the more severe late effects at cellular level. © 2013 AIP Publishing LLC.

A large-aperture, long-pulse XeCl excimer laser has been used to generate a soft-X-ray plasma source. Several laser optical configurations have been employed to optimise X-ray emission, including positive-branch unstable resonators and injection by seeding the gain region of the laser with a small commercial excimer laser, resulting in power densities in the range 1012–1014 W cm−2. The characteristics of the plasma source for each different laser configuration and for different target materials are investigated. The most suitable source conditions (spectral energy distribution, time duration, etc.) for specific applications are discussed.

The results for inactivation of V79 cells irradiated by monoenergetic deuterons with LET of 18, 26 and 31 keV.µm-1 and their direct comparison with protons of comparable LET are presented. The experimental results show that at 31 keV.µm-1 cell inactivation is similar for protons and deuterons while, at lower LET, deuterons appear to be less lethal than protons. The use of parameters other than L(, such as L100 or Z2 eff/ß2, does not completely reconcile the differences in biological effectiveness among the particles pointing out their inadequacy for describing the radiation quality of light ions. The experimental results have been compared with the Monte Carlo track structure simulations, using the code MOCA 14, carried out for monoenergetic track segments of protons, deuterons and alpha particles of selected LET in the range 20-40 keV.µm-1. Existing track structure codes, and indeed essentially all other physical descriptions of radiations used to date, are unable to explain the observed differences in biological effectiveness between deuterons and protons.

We report the characterization of a soft x-ray plasma source generated by a long-pulse XeCl excimer laser system. The output energy is 4 J at a wavelength of 308 nm in a 100-ns pulse. The intensity of radiation on target is estimated to be 4 x 1012 W cm-2. X-ray emission spectra of the plasma have been recorded using a double focusing spatial resolution spectrometer with a spherical mica crystal. From these measurements, the plasma temperature and electron density have been estimated. Various applications of such a plasma source have been investigated. First images of whole intact living cells from our system, imaged using the technique of soft x-ray contact microscopy, utilizing x rays in the water window” region (280-530 eV), are shown. The suitability of the source for other applications, for example, x-ray lithography and radiation damage studies, to living cells are discussed. Possible improvements to the x-ray source for the various applications are proposed. © 1995 IOS Press. All rights reserved.

We have studied the Cu binding site in Cu(II)–ATP, Cu(II)–tripolyphosphate, and Cu(II)–D‐ribose complexes in aqueous solution at several pH values ranging from 3 to 12 by means of high‐resolution x‐ray spectroscopy. We have measured the spectra in the x‐ray absorption near edge structure (XANES) region to probe the tetragonal distortion of the Cu site in these compounds, as indicated by optical d–d spectroscopy. The spectrophotometric studies proposed that Cu(II) bind to the oxygen atoms of the phosphate groups of ATP with a distorted octahedral symmetry at low pH, while at high pH, the metal atom would be bound to the hydroxyl groups of the ribose moiety with an almost planar symmetry. We have compared the measured XANES spectra with multiple scattering calculations, by using as a structural model a simple tetragonal CuO6 cluster with fixed equatorial distance (1.95 A) and variable axial distance (1.95–2.7 A). The multiple scattering calculations fit well the experimental data of Cu–ATP in solution, and ...

The results reported form part of a series of experiments to substantiate and extend the findings by Belli et al. (1989) that protons are more biologically effective at cell killing than alpha-particles of the same LET. The irradiations were carried out using the Variable Energy Cyclotron (VEC) at the Harwell Laboratories. V79-4 Chinese hamster cells were exposed to alpha-particles and protons with LETs of 20 and 23 keV microns-1 in the dose range 40-150 Gy. X-rays were also used for comparison. Two methods were used for measurement of initial DNA double-strand breaks: sedimentation and DNA precipitation assays. The dose-response relationships were found to be well fitted by straight lines in all cases. With the sedimentation assay a slightly lower yield of dsb was found from protons than from alpha-particles of the same LET. The yield from X-rays was not significantly different from either. The precipitation assay showed similar yields of DNA damage from both particle types but significantly higher yields from X-rays. This may reflect a difference in the type of lesions scored by the two methods. Since the initial amount of dsb does not account for the observed differences in cellular response to radiations of different qualities, it is likely that these are related to the nature of the dsb (affecting reparability) or to the occurrence of other types of molecular damage.

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.

The mutation induction at the HGPRT locus has been studied in V79-753B Chinese hamster cells irradiated with proton beams with energies of 3.36, 1.70 and 1.16 MeV, corresponding to average LET values of 10.6, 17.8 and 23.9 keV/microns, respectively. The mutation curve obtained with 200 kV X-rays was used for comparison. The mutation frequency induced by all the proton beams is considerably higher than that induced at the same dose by X-rays and it is linearly related to the dose. Moreover, the proton effectiveness increases with the LET. The RBEs (evaluated as the initial slope ratios) are 5.0 +/- 0.8, 5.4 +/- 0.8 and 7.7 +/- 1.2 for protons with average LETs of 10.6, 17.8 and 23.9 keV/microns, respectively. These values are higher than those reported in the literature for other ions of comparable LET. This finding parallels what we have already found for cell inactivation (for which RBEs of 3.0, 4.6 and 7.3 were obtained at the same LETs), and indicates that for mutation induction, also, the RBE-LET relationship may depend on the type of radiation.

The relationships between cell killing, mutation induction and DNA double (dsb) and single (ssb) strand breaks have been studied in V79 cells irradiated with X-rays under oxic and anoxic conditions in the presence and in the absence of dimethylsulphoxide (DMSO). Curvilinear relationships were found between all pairs of endpoints, except for dsb versus ssb. Statistical analysis of experimental data has shown that in the absence of DMSO there is evidence of good correlations between cell killing, mutation induction and dsb in oxic and anoxic conditions. However, when DMSO was present, no significant correlation was found. In the presence of oxygen DMSO always exerts a protective effect while in anoxia it is generally much less protective and induces a strong sensitization with respect to mutation induction. Possibly DMSO acts not only as a radical scavenger but also as an agent inducing chromatin relaxation and/or under anoxia, forming highly mutagenic short-term radicals. The present data suggest that lethal and mutational events are at least partially independent and not proportional to the initial number of DNA breaks. This may imply that either other kinds of lesions are involved in cell lethality and mutability, or dose-dependent repair mechanisms of dsb have to be considered.

Human cell line/Carbon-ion beams/Cell inactivation/Relative Biological Effectiveness (RBE)/Linear Energy Transfer (LET). 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 γ-rays following exposure to monoenergetic carbon-ion beams (incident LET 13–303 keV/μm) and at various positions along the ionization curve of a therapeutic carbon-ion beam, corresponding to three doseaveraged LET (LETd) values (40, 50 and 75 keV/μm). 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 γ-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 tum our cells radioresistant to X- or γ-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.

Previously we reported that yeast and Chinese hamster V79 cells cultured under reduced levels of background environmental ionizing radiation show enhanced susceptibility to damage caused by acute doses of genotoxic agents. Reduction of environmental radiation dose rate was achieved by setting up an underground laboratory at Laboratori Nazionali del Gran Sasso, central Italy. We now report on the extension of our studies to a human cell line. Human lymphoblastoid TK6 cells were maintained under identical in vitro culture conditions for six continuous months, at different environmental ionizing radiation levels. Compared to “reference” environmental radiation conditions, we found that cells cultured in the underground laboratories were more sensitive to acute exposures to radiation, as measured both at the level of DNA damage and oxidative metabolism. Our results are compatible with the hypothesis that ultra-low dose rate ionizing radiation, i.e. environmental radiation, may act as a conditioning agent in the radiation-induced adaptive response.

In the framework of the NOTE Integrated Project (FP6-36465, Euratom), we used for the first time HL60 human promyelocytes for bystander effects studies. This cell line is capable to differentiate in vitro towards either granulocytes or monocytes/macrophages and, therefore, one of the major interest is that it is possible to investigate the nature of signals released from the same cell type in various differentiating conditions and also to evaluate if cell differentiation can be induced through bystander mechanisms. The experiments have been focused (i) to the characterization of the biological system using the appropriate cluster of differentiation (CD) of cell surface molecules for the different cells of interest (i.e., promyelocytes, monocyte-and macrophage-like cells) and (ii) to obtain information about the response to signalling factors from sham/irradiated HL60 promyelocytes in terms of micronuclei (MN) induction and cell killing. Experiments were also performed to characterize the basal levels of ROS and RNS in cells at different stages of differentiation. Monocyte- and macrophage-like differentiation was obtained using vitamin D3 and phorbol diester (12-O-tetradecanoylphorbol-13-acetate, TPA) as inducers, respectively. Irradiation was performed with gamma rays and bystander experiments were carried out using the medium transfer approach. The experiments performed to characterize the differentiation status in the HL60 clone we used show that CD11b and CD11c appear both suitable for identifying macrophage-like and monocyte-like cells. The former are negative while the latter are positive to CD14. Actively proliferating promyelocytes present some positivity to CD95 but not to CD11b and CD11c or CD14. At the present experiments are planned aimed at distinguishing promyelocytes from macrophage-like cells in a mixed population. The results on MN induction in unirradiated promyelocytes by the medium collected from promyelocytes irradiated with 0.5 Gy and incubated for different times at 37°C show an increase after 2 h incubation that disappears at longer incubation times. Measurements of cell killing show that incubation with “conditioned” medium from 1 Gy irradiated promyelocytes leads to an increase in the cloning efficiency of the bystander promyelocytes, giving surviving fractions higher than 1. However, this effect seems more related to factors physiologically released by the cells than to factors induced by irradiation, as indicated by the comparison with medium from sham-irradiated cells. These results suggest that growth stimulating factors, instead of growth inhibiting factors, are released by HL60 promyelocytes.

In low dose radiobiology, there is a strong interest in phenomena that could imply a deviation from the linear-no-threshold (LNT) model, which is currently used to estimate the excess relative cancer risk at low doses of ionizing radiation (IR) via a linear extrapolation from high- and intermediate-dose epidemiology risk data. Such phenomena include the adaptive response as well as bystander effects , occurring in cells which do not experience direct radiation exposure. These phenomena suggest that biological response to harmful agents such as ionising radiation may be more complex than that assumed by the LNT model. In particular, environmental background radiation represents a source of chronic low dose-rate exposure which may condition the response of living systems to acute exposure to genotoxic agents, also including IR itself, via an adaptive response. To clarify this aspect is essential not only to better evaluate the risk of chronic occupational exposure, but also to understand the role of environmental background radiation on biological evolution. Reduction of background environmental IR dose rate exposure is achieved at the National Laboratories at Gran Sasso (LNGS) of the Italian Institute for Nuclear Physics (INFN). Located underneath the Gran Sasso mountain range in central Italy, these laboratories offer an excellent opportunity to continuously maintain cell cultures at extremely reduced levels of environmental background radiation. The Cosmic Silence experiment represents the natural extension, to a human cell culture model (TK6 lymphoblastoid cells), of earlier experimental studies on S. Cerevisiae (L. Satta et. al. Mut Res 1995) and V79 Chinese hamster fibroblasts (L. Satta et. al. Radiat Environ Biophys 2002), which indicated a progressive disappearance of an adaptive response in cells kept under reduced environmental background IR conditions. For the Cosmic Silence experiment, TK6 parallel cultures were set up in the underground LNGS laboratories and in Rome and planned to be maintained and assayed for 12 months. Starting at 6 months of continuous, parallel cultures, measurements of DNA damage (micronuclei) or oxidative metabolism (antioxidant enzymatic activities of SOD, GPx, and Catalase) indicate that significant deprivation of environmental background IR renders cells less tolerant to an acute X-ray exposure of 1-2 Gy. These results are compatible with the concept that environmental background IR is behaving as an adaptive agent.

In the framework of the NOTE Integrated Project (FP6-36465, Euratom), we used the alpha particle irradiator set up at the Istituto Superiore di Sanita to investigate DNA damage and cell killing in irradiated and bystander AG1522 primary human fibroblasts. In particular, we studied the timing of medium-mediated bystander effects in order to get insight on the mechanisms involved. To this purpose, both medium transfer and co-culture approaches were used. The experiments were carefully designed in order to produce data useful for modeling. For this reason a great importance was given to standardization of methodologies, and experimental variables such as cell growth phase, cell density, medium thickness/volume, were evaluated and optimized. Cells were seeded in stainless steel Petri dishes with Mylar bottom, especially designed for alpha particle irradiation. They were realized for housing permeable membrane insert(s) and exactly reproduce the geometry of commercial Cell Culture Insert Companion Plates. The alpha particle irradiator was positioned into a CO2 incubator in order to perform irradiation and co-culture incubation in physiological conditions. The data so far obtained show that 30 min of co-culture with 0.5 Gy irradiated cells are sufficient to start a significant induction of DNA damage in bystander cells. This damage increases after 2 h of co-culture to a level twice that of the sham irradiated cells (the percentage of cells showing γ–H2AX foci rising from ~12% to ~25% in bystander cells compared to ~96% in 0.5 Gy irradiated cells). Moreover, the damage induced after 1 h co-culture is further increased by 1 h incubation in the presence of conditioned medium only. Cell killing data, obtained using the medium transfer approach, show a surviving fraction of 0.8 in bystander cells incubated with medium from cells irradiated with 0.1 or 0.5 Gy and kept for 1h at 37°C. It is interesting that the same surviving fraction is obtained in 0.1 Gy irradiated cells, while it drops to 0.2 after the dose of 0.5 Gy. The overall results we obtained for bystander induced effects at molecular and cellular level indicate that crucial signalling from irradiated to bystander cells occurs at relatively short time (0.5-1 h) after irradiation, giving constrains about the possible signalling factors involved.

Experimental data on DNA double strand break (DSB) induction in human fibroblasts (AG1522), following irradiation with several radiation qualities, namely gamma rays, 0.84 MeV protons, 58.9 MeV u -1 carbon ions, iron ions of 115 MeV u -1 , 414 MeV u -1 , 1 GeV u -1 , and 5 GeV u -1 , are presented. DSB yields were measured by calibrated Pulsed Field Gel Electrophoresis in the DNA fragment size range 0.023-5.7 Mbp. The DSB yields show little LET dependence, in spite of the large variation of the latter among the beams, and are slightly higher than that obtained using gamma rays. The highest yield was found for the 5 GeV u -1 iron beam, that gave a value 30% higher than the 1 GeV u -1 iron beam. A phenomenological method is used to parametrise deviation from randomness in fragment size spectra.

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 naive 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 naive bin-by-bin subtraction in all these artificial data sets.

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.

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.

A 2 4 4 Cm alpha-particle irradiator was designed and constructed for radiobiological studies where protracted exposure at a low dose rate of cultured mammalian cells is required. It allows irradiation of a cell monolayerattached to the Mylar bottom of a specially designed Petri dish of 56 mm diameter (∼25 cm 2 area). The irradiator is based on a 20-mm-diameter stainless steel chamber containing a 148 kBq 2 4 4 Cm source. The chamber, flushed with helium gas at a pressure kept slightly above the external pressure, is inserted into a cell incubator where temperature and CO 2 concentration are controlled. Spectrometric and dosimetric characterization of the irradiator was carried out by means of an ion-implanted-silicon charged-particle detector, CR39 detectors, and Monte Carlo simulations with the TRIM code. Average LET of particles incident on the cells at the center of the Petri dish was evaluated to be 120 keV μm - 1 at 59 mm from the source, and the average dose rate was 5.69 x 10 - 5 Gy s - 1 , with +12% and -8% variations at the center and the edge, respectively. The irradiator has been successfully tested and used for several experiments involving 16-d exposure of human fibroblasts monolayers.

DNA double strand breaks / DNA repair / Cell cycle checkpoints / Ionizing radiation / Space radiation 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 γ-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.

Use of cellular systems capable of undergoing in vitro differentiation can give useful information on the basic mechanisms of cellular radiation sensitivity. During differentiation the cellular organisation, including the nuclear structure and the intracellular concentration of several compounds and enzymes change drastically. Accordingly, radiation response to ionising radiation is also expected to change. The human proerythroblastoid cell line K562 can be induced to pseudoerythroid differentiation. This process has been characterised and studies have been carried out on DNA single strand break and double strand break induction and repair before and after differentiation commitment. Rejoining studies have been performed for both types of damage and correct double strand break rejoining has been also measured in particular genomic locations. An overview is presented of these results together with preliminary data recently obtained on radiation induced DNA fragmentation as a function of radiation quality.

To compare the results on DNA fragmentation induced in Chinese hamster V79 cells by various doses of gamma-rays and low-energy protons and helium-4 ions.V79 cells were irradiated as monolayers with monoenergetic protons and helium-4 ions; gamma-rays were used as the reference radiation. DNA double-strand breaks were evaluated by calibrated pulsed-field gel electrophoresis using conditions covering the range 5.7 Mbp-23.1 kbp.The fragment-counting method gave double-strand breaks yields and the relative biological effectiveness higher than those obtained by the fraction of activity released method. The frequency distribution of fragments showed that protons and helium ions induced more fragments below the Mbp region than did gamma-rays at the same dose. The distributions for both the irradiated and non-irradiated samples clearly appeared to be non-random.Differences were observed in the yield and spatial correlation, at a molecular size scale characteristic of loop dimensions, of the double-strand breaks induced by gamma-rays and by light ions. These effects may have a role in the observed different cell response to these radiations.

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanita (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.

To analyse the cell inactivation frequencies induced by low energy protons in human cells with different sensitivity to photon radiation.Four human cell lines with various sensitivities to photon irradiation were used: the SCC25 and SQ20B derived from human epithelium tumours of the tongue and larynx, respectively, and the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The cells were irradiated with y-rays and proton beams with linear energy transfer (LET) from 7 to 33 keV/microm. Clonogenic survival was assessed.Survival curves are reported for each cell line following irradiation with gamma-rays and with various proton LETs. The surviving fraction after 2 Gy of gamma-rays was 0.72 for SQ20B cells, and 0.28-0.35 for the other cell lines. The maximum LET proton effectiveness was generally greater than that of gamma-rays. In particular there was a marked increase in beam effectiveness with increasing LET for the most resistant cells (SQ20B) whose 2 Gy-survival varied from 0.72 with gamma-radiation down to 0.37 with 30 keV/microm protons. The relative biological effectiveness (RBE(2 Gy gamma)) with the 30 keV/microm beam, evaluated as the ratio of 2 Gy to the proton dose producing the same inactivation level as that given by 2 Gy of gamma-rays, was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively.RBE for inactivation with high-LET protons increased with the cellular radioresistance to gamma-rays. The cell line with the greatest resistance to gamma-rays was the most responsive to the highest LET proton beam. A similar trend has also been found in studies reported in the literature with He, C, N ions with LET in the range 20-125 keV/microm on human tumour cell lines.

A large volume excimer laser, HERCULES, has been successfully applied as pump for a soft x-ray plasma source. The laser pulse duration has been varied from the natural value of 120 ns down to 10 ns, reaching different emission spectra from the plasma which have resulted to be optimum for different applications of the plasm source itself. Some experimental results on the applications of the source to different fields are presented; the choice of the best laser parameters in relation to the applications of the plasma source are also discussed.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A high brightness x-ray source has been obtained by focusing the large aperture excimer laser HERCULES on a solid target with a laser intensity of more than 10 13 W/cm 2 . The characteristics of the x-ray source for different laser time evolution in the range 6-120 ns are investigated. Experimental result on specific applications of the source, like the soft x-ray contact microscopy and the low photon energy radiobiology will be presented.