Purpose The purpose of this study is to correlate the physical dose to non-target tissues (blood) to the radiation-induced chromosome damage in terms of the dicentrics and micronuclei (MN) in peripheral blood lymphocytes (PBL) after radium-223 (223RaCl2) therapy in patients with bone metastasis derived from prostate cancer [1] . Methods Four patients undergoing 223RaCl2 therapy for skeletal diseases have been enrolled in this prospective clinical study. The effective dose to blood per injected activity was calculated considering the alpha, beta and gamma emission of Ra-223 and considering a standard man weight of 70 kg. PBL cultures for dicentric assay and MN were performed before treatment (T0), 6 days (T1) and 30 days (T2) after the first cycle of treatment and after the end of the therapy (T3). Haematological toxicity parameters (blood cell count) have been monitored during therapy and analysed along with doses to blood. Results The administration of 223RaCl2 produces a high dose dependent increase of radiation-induced chromosome damage in terms of dicentrics and MN induction observed in the circulating lymphocytes. Surprisingly, the increase of chromosome damage observed between T1 and T2 is not due to an 223RaCl2 addition dose, suggesting that circulating lymphocytes were exposed to an extra dose by the emissions from the target areas. In addition, clinical monitoring during the treatment showed a progressive increasing fatigue, leucopenia and anemia with a partial recovery after some months after the end of therapy. Conclusions The cytogenetic data suggest a persistence of the radiation emission from the target tissue to non-target ones and seem to be correlated to the observed haematological toxicity, highlighting possible adverse effects related to this therapy. These data need further investigation in order to evaluate the potential side effects to normal, non-target tissue in patients treated with alpha emitters.

Purpose The use of ionising radiation in medicine has been steadily increasing, and this trend is set to continue, with obvious health benefits for the population thanks to improved diagnostic and therapy technologies.However, this increase in radiation exposure levels also raises a number of safety concerns:the potential health effects among patients and medical workers need to be evaluated, dose evaluation tools for clinical practice need to be developed, and practices need to be optimised in order to reduce exposure doses and ensure adequate radiation protection.A new EC-funded project will bring together medical and radiation scientists, physicists and clinicians to enhance the radiation protection of patients and medical professionals.The four-year MEDIRAD project (2017–2021) is led by the European Institute for Biomedical Imaging Research (EIBIR) and comprises a consortium of 33 partners from 14 European countries. Methods The MEDIRAD Project consists of six interdependent and complimentary work packages (WP), listed in Table. Work Packages Description WP1: Project management and dissemination Scientific and clinical coordination, ethics management, knowledge management and exploitation, internal and external communication WP2: Dose evaluation and optimisation in medical imaging Optimisation of chest CT, interventional procedures and multimodality imaging, and development of imaging and radiation dose biobank WP3: Impact of low dose radiation exposure Standardisation, biokinetic modelling and treatment planning, dosimetry, biomarkers of absorbed doses, protocol for epidemiological study WP4: Possible health impact of paediatric scanning Epidemiological study on cardiovascular changes after radiotherapy, measuring markers of exposure and risk modelling WP5: Breast radiotherapy and secondary cardiovascular risk Epidemiological study of paediatric CTs and cancer, including (epi) genetic biomarkers of sensitivity, dosimetry and statistical analyses WP6: Bringing together medical & nuclear scientific communities Formulation of science-based policy recommendations, consultation of stakeholders, organisation of dissemination seminars Results MEDIRAD has three major operational objectives:to improve organ dose estimation and registration; to evaluate and understand the mechanisms of the effects of medical exposures, focusing on two outcomes of public health relevance:cardiovascular effects of radiotherapy in breast cancer treatment, and cancer risks following CT scanning in children and adolescents; and to develop science-based consensus policy recommendations for the effective protection of patients, workers and the general public.The ISS, due to its long and consolidated experience in the field of Quality Assurance in Radiological Sciences, has been invited to contribute to the latter issue. Conclusions MEDIRAD’s overall goal is to address these needs by enhancing the scientific bases and practice of radiation protection in medicine. MEDIRAD has received funding from the Euratom research and training programme 2014–2018 (grant agreement N°755523). More information can be found at http://www.medirad-project.eu/ .

The EC-funded project MEDIRAD addresses numerous low-dose exposure situations for patients and workers in the medical context, to further develop risk models and draw operational recommendations for improving radiation protection. The four-year project (2017-2021) relies on a 33-partner consortium from 14 European countries and is coordinated by the European Institute for Biomedical Imaging Research (EIBIR, AT). Prof. Elisabeth Cardis (ISGlobal, ES) and Prof. Guy Frija (Universite Paris Descartes, FR) are the scientific coordinator and the clinical coordinator, respectively. The MEDIRAD Project consists of six interdependent and complimentary Work Packages (WPs). WP1: project management and dissemination; WP2: dose evaluation and optimisation in medical imaging; WP3: impact of low-dose radiation exposure from I-131 radioiodine in thyroid cancer treatment; WP4: breast radiotherapy and secondary cardiovascular risks; WF5: possible health impact of paediatric scanning; WP6: bringing together medical and nuclear scientific communities for radiation protection purposes. The Italian National Institute of Health (ISS), thanks to its consolidated experience in the field of quality assurance in radiological sciences, will contribute to the latter issue coordinating a Working Group to develop recommendations on patient radiological protection, directed to the medical communities, considering the scientific outcomes of the MEDIRAD WPs and the stakeholders'comments.

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 (137)Cs 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 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.

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 gamma-rays and C-ions having E similar to 45 MeV/u at the cell entrance, corresponding to LET (in water) similar to 49 keV/mu m. 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 gamma-rays than for C-ions. However, in the first 12 h the fraction of residual gamma -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 gamma-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 gamma-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.

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.

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 paper we present the main outcomes of a wide collaborative effort (carried out, within the INFN project, "EPICA" and in part within the European projects "RISC-RAD" and "NOTE" and the ASI project MoMa-COUNT), both experimental and theoretical, devoted to the characterization and quantification of the induction of DNA-targeted and non-DNA-targeted molecular and cellular biological endpoints, following irradiation of human cells with different Charged particles The work was mainly aimed at reaching a better understanding of the mechanisms governing the physical and biophysical pathways leading from the initial energy deposition by radiation in matter to the induction of observable radiobiological damage, with particular focus on the role played by radiation quality. More, specifically we characterized the induction of DNA DSB within different fragment-size ranges outlining the effectiveness of high-LET radiation at inducing small fragments and thus clustered DNA breaks, which can evolve in terms of endpoints like chromosome aberrations (CAs). This was confirmed by the development and application of a model of CA induction based oil the assumption that only DNA breaks can lead to aberrations. Concerning non-DNA-targeted damage, we quantified the time-dependent induction of medium-mediated DNA damage in bystander cells and we characterized the time and dose dependence of cytokine concentration in the medium of sham-irradiated and irradiated cells, since medium-mediated bystander damage is thought to arise from molecular signalling between irradiated and unirradiated cells. The mechanisms governing such Signalling were investigated developing a model and a MC code simulating cytokine release, diffusion mid internalization, showing good agreement with experimental data. Non-DNA-targeted effects were further characterized by MRS investigation of the radiation effects on lipids and oxidative metabolism, which are particularly relevant also considering that, they may differently expressed in different tumors and ill normal tissues.

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.

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.

This paper reports on DNA DSB induction in human fibroblasts by iron ions of different energies, namely 5, 1 GeV/u, 414 and 115 MeV/u, in absence or presence of different shields (PMMA, Al and Pb). Measure of DNA DSB was performed by calibrated Pulsed Field Gel Electrophoresis using the fragment counting method. The RBE-LET relationships for unshielded and shielded beams were obtained both in terms of dose average LET and of track average LET. Weak dependence on these parameters was observed for DSB induction. The shielding efficiency, evaluated by the ratio between the cross sections for unshielded and shielded beams, depends not only on the shield type and thickness, but also on the beam energy. Protection is only observed at high iron ions energy, especially at 5 GeV/u, where PMMA shield gives higher protection compared to Al or Pb shields of the same thickness expressed in g/cm2.

Abstract Antonelli, F., Belli, M., Cuttone, G., Dini, V., Esposito, G., Simone, G., Sorrentino, E. and Tabocchini, M. A. Induction and Repair of DNA Double-Strand Breaks in Human Cells: Dephosphorylation of Histone H2AX and its Inhibition by Calyculin A. Radiat. Res. 164, 514–517 (2005). Phosphorylation of histone H2AX at serine 139 (γ-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 γ rays in MRC-5 human fibroblasts was studied. The number of γ-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 γ-H2AX foci occurred. This indicates that the number of γ-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 γ-H2AX dephosphorylation and DSB rejoining are independent phenomena. We found that the maintenance of the phosphate group at Ser 139 in γ-H2AX does not represent an obstacle for DSB rejoining. Preliminary experiments performed with 62 MeV/nucleon carbon ions have shown a longer persistence of γ-H2AX foci with respect to γ rays, consistent with the induction of damage that is more severe and difficult to repair.

Purpose:To quantify the role played by radiation track structure and background fragments in modulating DNA fragmentation in human cells exposed to γ-rays and light ions.Materials and methods: Human fibroblasts were exposed in vitro to different doses (in the range from 40 – 200 Gy) of 60Co γ-rays and 0.84 MeV protons (Linear Energy Transfer, LET, in tissue 28.5 keV/μm). 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 60Co γ-rays).Results: For both γ-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) • 10−9 Gy−1 bp−1 for γ-rays and (7.1 ± 0.6) • 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 γ-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.Conclusion: 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.

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 γ-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 γ-rays in the size range 1–23 kbp; (3) a non-random DNA DSB induction by Fe ions.

PURPOSE: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.MATERIALS AND METHODS: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.RESULTS: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.CONCLUSION: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.