Effectiveness of Flattening-Filter-Free versus Flattened Beams in V79 and Glioblastoma Patient-Derived Stem-like Cells.

Literature data on the administration of conventional high-dose beams with (FF) or without flattening filters (FFF) show conflicting results on biological effects at the cellular level. To contribute to this field, we irradiated V79 Chinese hamster lung fibroblasts and two patient-derived glioblastoma stem-like cell lines (GSCs-named #1 and #83) using a clinical 10 MV accelerator with FF (at 4 Gy/min) and FFF (at two dose rates 4 and 24 Gy/min). Cell killing and DNA damage induction, determined using the γ-H2AX assay, and gene expression were studied. No significant differences in the early survival of V79 cells were observed as a function of dose rates and FF or FFF beams, while a trend of reduction in late survival was observed at the highest dose rate with the FFF beam. GSCs showed similar survival levels as a function of dose rates, both delivered in the FFF regimen. The amount of DNA damage measured for both dose rates after 2 h was much higher in line #1 than in line #83, with statistically significant differences between the two dose rates only in line #83. The gene expression analysis of the two GSC lines indicates gene signatures mimicking the prognosis of glioblastoma (GBM) patients derived from a public database. Overall, the results support the current use of FFF and highlight the possibility of identifying patients with candidate gene signatures that could benefit from irradiation with FFF beams at a high dose rate.

Radiation dose rate effects: what is new and what is needed?

Despite decades of research to understand the biological effects of ionising radiation, there is still much uncertainty over the role of dose rate. Motivated by a virtual workshop on the "Effects of spatial and temporal variation in dose delivery" organised in November 2020 by the Multidisciplinary Low Dose Initiative (MELODI), here, we review studies to date exploring dose rate effects, highlighting significant findings, recent advances and to provide perspective and recommendations for requirements and direction of future work. A comprehensive range of studies is considered, including molecular, cellular, animal, and human studies, with a focus on low linear-energy-transfer radiation exposure. Limits and advantages of each type of study are discussed, and a focus is made on future research needs.

Reduced Environmental Dose Rates Are Responsible for the Increased Susceptibility to Radiation-Induced DNA Damage in Larval Neuroblasts of Drosophila Grown inside the LNGS Underground Laboratory.

A large amount of evidence from radiobiology studies carried out in Deep Underground Laboratories support the view that environmental radiation may trigger biological mechanisms that enable both simple and complex organisms to cope with genotoxic stress. In line with this, here we show that the reduced radiation background of the LNGS underground laboratory renders Drosophila neuroblasts more sensitive to ionizing radiation-induced (but not to spontaneous) DNA breaks compared to fruit flies kept at the external reference laboratory. Interestingly, we demonstrate that the ionizing radiation sensitivity of flies kept at the LNGS underground laboratory is rescued by increasing the underground gamma dose rate to levels comparable to the low-LET reference one. This finding provides the first direct evidence that the modulation of the DNA damage response in a complex multicellular organism is indeed dependent on the environmental dose rate.

A forty-year journey from "classical" biophysics and radiobiology to hadrontherapy, space radiation and low dose rate underground radiobiology.

PURPOSE: As a biologist who, since the beginning of her involvement in science, has collaborated closely with physicists, I want to share my forty years of experience describing the events that introduced me to the world of charged particle radiation biology as well as that of low doses/dose rates, with related implications in medicine and radiation protection. CONCLUSION: The main features of my experience can be summarized in the development of an interdisciplinary culture and in the interest in technological advances for the study of biological responses to radiation in different scenarios, relevant for public health. Mine was a journey that began by chance, but which led me to a world that proved to be of great interest to me. With the current advances in science, the new generations of scientists have new opportunities that I wish them to face with the same interest and enthusiasm that I felt for such an interdisciplinary field as that of radiation biology.

The role of dosimetry and biological effects in metastatic castration-resistant prostate cancer (mCRPC) patients treated with 223Ra: first in human study.

BACKGROUND: 223Ra is currently used for treatment of metastatic castration resistant prostate cancer patients (mCRPC) bone metastases with fixed standard activity. Individualized treatments, based on adsorbed dose (AD) in target and non-target tissue, are absolutely needed to optimize efficacy while reducing toxicity of α-emitter targeted therapy. This is a pilot first in human clinical trial aimed to correlate dosimetry, clinical response and biological side effects to personalize 223Ra treatment. METHODS: Out of 20 mCRPC patients who underwent standard 223Ra treatment and dosimetry, in a subset of 5 patients the AD to target and non-target tissues was correlated with clinical effects and radiation-induced chromosome damages. Before each 223Ra administrations, haematological parameters, PSA and ALP values were evaluated. Additional blood samples were obtained baseline (T0), at 7 days (T7), 30 days (T30) and 180 days (T180) to evaluate chromosome damage. After administration WB planar 223Ra images were obtained at 2-4 and 18-24 h. Treatment response and toxicity were monitored with clinical evaluation, bone scan, 18F-choline-PET/CT, PSA value and ALP while haematological parameters were evaluated weekly after 223Ra injection and 2 months after last cycle. RESULTS: 1. a correlation between AD to target and clinical response was evidenced with threshold of 20 Gy as a cut-off to obtain tumor control; 2. the AD to red marrow was lower than 2 Gy in all the patients with no apparently correlation between dosimetry and clinical toxicity. 3. a high dose dependent increase of the number of dicentrics and micronuclei during the course of 223Ra therapy was observed and a linear correlation has been found between blood AD (BAD) and number of dicentrics. CONCLUSIONS: This study provides some interesting preliminary evidence to be further investigated: dosimetry may be useful to identify a more appropriate 223Ra administered activity predicting AD to target tissue; a dose dependent complex chromosome damage occurs during 223Ra administration and this injury is more evident in heavily pre-treated patients; dosimetry could be used for radioprotection purpose. TRIAL REGISTRATION: The pilot study has been approved from the Ethics Committee of Regina Elena National Cancer Institute (N:RS1083/18-2111).

Underground Radiobiology: A Perspective at Gran Sasso National Laboratory.

Scientific community and institutions (e. g., ICRP) consider that the Linear No-Threshold (LNT) model, which extrapolates stochastic risk at low dose/low dose rate from the risk at moderate/high doses, provides a prudent basis for practical purposes of radiological protection. However, biological low dose/dose rate responses that challenge the LNT model have been highlighted and important dowels came from radiobiology studies conducted in Deep Underground Laboratories (DULs). These extreme ultra-low radiation environments are ideal locations to conduct below-background radiobiology experiments, interesting from basic and applied science. The INFN Gran Sasso National Laboratory (LNGS) (Italy) is the site where most of the underground radiobiological data has been collected so far and where the first in vivo underground experiment was carried out using Drosophila melanogaster as model organism. Presently, many DULs around the world have implemented dedicated programs, meetings and proposals. The general message coming from studies conducted in DULs using protozoan, bacteria, mammalian cells and organisms (flies, worms, fishes) is that environmental radiation may trigger biological mechanisms that can increase the capability to cope against stress. However, several issues are still open, among them: the role of the quality of the radiation spectrum in modulating the biological response, the dependence on the biological endpoint and on the model system considered, the overall effect at organism level (detrimental or beneficial). At LNGS, we recently launched the RENOIR experiment aimed at improving knowledge on the environmental radiation spectrum and to investigate the specific role of the gamma component on the biological response of Drosophila melanogaster.

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

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

Low Radiation Environment Switches the Overgrowth-Induced Cell Apoptosis Toward Autophagy.

Low radiation doses can affect and modulate cell responses to various stress stimuli, resulting in perturbations leading to resistance or sensitivity to damage. To explore possible mechanisms taking place at an environmental radiation exposure, we set-up twin biological models, one growing in a low radiation environment (LRE) laboratory at the Gran Sasso National Laboratory, and one growing in a reference radiation environment (RRE) laboratory at the Italian National Health Institute (Istituto Superiore di Sanità, ISS). Studies were performed on pKZ1 A11 mouse hybridoma cells, which are derived from the pKZ1 transgenic mouse model used to study the effects of low dose radiation, and focused on the analysis of cellular/molecular end-points, such as proliferation and expression of key proteins involved in stress response, apoptosis, and autophagy. Cells cultured up to 4 weeks in LRE showed no significant differences in proliferation rate compared to cells cultured in RRE. However, caspase-3 activation and PARP1 cleavage were observed in cells entering to an overgrowth state in RRE, indicating a triggering of apoptosis due to growth-stress conditions. Notably, in LRE conditions, cells responded to growth stress by switching toward autophagy. Interestingly, autophagic signaling induced by overgrowth in LRE correlated with activation of p53. Finally, the gamma component of environmental radiation did not significantly influence these biological responses since cells grown in LRE either in incubators with or without an iron shield did not modify their responses. Overall, in vitro data presented here suggest the hypothesis that environmental radiation contributes to the development and maintenance of balance and defense response in organisms.

Targeting cancer stem cells: protons versus photons.

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

Fruit Flies Provide New Insights in Low-Radiation Background Biology at the INFN Underground Gran Sasso National Laboratory (LNGS).

Deep underground laboratories (DULs) were originally created to host particle, astroparticle or nuclear physics experiments requiring a low-background environment with vastly reduced levels of cosmic-ray particle interference. More recently, the range of science projects requiring an underground experiment site has greatly expanded, thus leading to the recognition of DULs as truly multidisciplinary science sites that host important studies in several fields, including geology, geophysics, climate and environmental sciences, technology/instrumentation development and biology. So far, underground biology experiments are ongoing or planned in a few of the currently operating DULs. Among these DULs is the Gran Sasso National Laboratory (LNGS), where the majority of radiobiological data have been collected. Here we provide a summary of the current scenario of DULs around the world, as well as the specific features of the LNGS and a summary of the results we obtained so far, together with other findings collected in different underground laboratories. In particular, we focus on the recent results from our studies of Drosophila melanogaster, which provide the first evidence of the influence of the radiation environment on life span, fertility and response to genotoxic stress at the organism level. Given the increasing interest in this field and the establishment of new projects, it is possible that in the near future more DULs will serve as sites of radiobiology experiments, thus providing further relevant biological information at extremely low-dose-rate radiation. Underground experiments can be nicely complemented with above-ground studies at increasing dose rate. A systematic study performed in different exposure scenarios provides a potential opportunity to address important radiation protection questions, such as the dose/dose-rate relationship for cancer and non-cancer risk, the possible existence of dose/dose-rate threshold(s) for different biological systems and/or end points and the possible role of radiation quality in triggering the biological response.

Effects of reduced natural background radiation on Drosophila melanogaster growth and development as revealed by the FLYINGLOW program.

Natural background radiation of Earth and cosmic rays played a relevant role during the evolution of living organisms. However, how chronic low doses of radiation can affect biological processes is still unclear. Previous data have indicated that cells grown at the Gran Sasso Underground Laboratory (LNGS, L'Aquila) of National Institute of Nuclear Physics (INFN) of Italy, where the dose rate of cosmic rays and neutrons is significantly reduced with respect to the external environment, elicited an impaired response against endogenous damage as compared to cells grown outside LNGS. This suggests that environmental radiation contributes to the development of defense mechanisms at cellular level. To further understand how environmental radiation affects metabolism of living organisms, we have recently launched the FLYINGLOW program that aims at exploiting Drosophila melanogaster as a model for evaluating the effects of low doses/dose rates of radiation at the organismal level. Here, we will present a comparative data set on lifespan, motility and fertility from different Drosophila strains grown in parallel at LNGS and in a reference laboratory at the University of L'Aquila. Our data suggest the reduced radiation environment can influence Drosophila development and, depending on the genetic background, may affect viability for several generations even when flies are moved back to normal background radiation. As flies are considered a valuable model for human biology, our results might shed some light on understanding the effect of low dose radiation also in humans.

Irradiation of Mesenchymal Stromal Cells With Low and High Doses of Alpha Particles Induces Senescence and/or Apoptosis.

The use of high-linear energy transfer charged particles is gaining attention as a medical tool because of the emission of radiations with an efficient cell-killing ability. Considerable interest has developed in the use of targeted alpha-particle therapy for the treatment of micrometastases. Moreover, the use of helium beams is gaining momentum, especially for treating pediatric tumors. We analyzed the effects of alpha particles on bone marrow mesenchymal stromal cells (MSCs), which have a subpopulation of stem cells capable of generating adipocytes, chondrocytes, and osteocytes. Further, these cells contribute toward maintenance of homeostasis in the body. MSCs were irradiated with low and high doses of alpha particles or X-rays and a comparative biological analysis was performed. At a low dose (40 mGy), alpha particles exhibited a limited negative effect on the biology of MSCs compared with X-rays. No significant perturbation of cell cycle was observed, and a minimal increase in apoptosis or senescence was detected. Self-renewal was preserved as revealed by the CFU assay. On the contrary, with 2000 mGy alpha particles, we observed adverse effects on the vitality, functionality, and stemness of MSCs. These results are the consequence of different proportion of cells targeted by alpha particles or X-rays and the quality of induced DNA damage. The present study suggests that radiotherapy with alpha particles may spare healthy stem cells more efficaciously than X-ray treatments, an observation that should be taken into consideration by physicians while planning irradiation of tumor areas close to stem cell niches, such as bone marrow. J. Cell. Biochem. 118: 2993-3002, 2017. © 2017 Wiley Periodicals, Inc.

Facility for gamma irradiations of cultured cells at low dose rates: design, physical characteristics and functioning.

We describe a low dose/dose rate gamma irradiation facility (called LIBIS) for in vitro biological systems, for the exposure, inside a CO2 cell culture incubator, of cells at a dose rate ranging from few µGy/h to some tens of mGy/h. Three different (137)Cs sources are used, depending on the desired dose rate. The sample is irradiated with a gamma ray beam with a dose rate uniformity of at least 92% and a percentage of primary 662keV photons greater than 80%. LIBIS complies with high safety standards.

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

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

The complex interactions between radiation induced non-targeted effects and cancer.

Radiation induced non-targeted effects have been widely investigated in the last two decades for their potential impact on low dose radiation risk. In this paper we will give an overview of the most relevant aspects related to these effects, starting from the definition of the low dose scenarios. We will underline the role of radiation quality, both in terms of mechanisms of interaction with the biological matter and for the importance of charged particles as powerful tools for low dose effects investigation. We will focus on cell communication, representing a common feature of non-targeted effects, giving also an overview of cancer models that have explicitly considered such effects.

DNA and cellular effects of charged particles.

Development of new radiotherapy strategies based on the use of hadrons, as well as reduction of uncertainties associated with radiation health risk during long-term space flights, requires increasing knowledge of the mechanisms underlying the biological effects of charged particles. It is well known that charged particles are more effective in damaging biological systems than photons. This capability has been related to the production of spatially correlated and/or clustered DNA damage, in particular two or more double-strand breaks (DSB) in close proximity or DSB associated with other lesions within a localized DNA region. These kinds of complex damages are rarely induced by photons. They are difficult to repair accurately and are therefore expected to produce severe consequences at the cellular level. This paper provides a review of radiation-induced cellular effects and will discuss the dependence of cell death and mutation induction on the linear energy transfer of various light and heavy ions. This paper will show the inadequacy of a single physical parameter for describing radiation quality, underlining the importance of the characteristics of the track structure at the submicrometer level to determine the biological effects. This paper will give a description of the physical properties of the track structure that can explain the differences in the spatial distributions of DNA damage, in particular DSB, induced by radiation of different qualities. In addition, this paper will show how a combined experimental and theoretical approach based on Monte Carlo simulations can be useful for providing information on the damage distribution at the nanoscale level. It will also emphasize the importance, especially for DNA damage evaluation at low doses, of the more recent functional approaches based on the use of fluorescent antibodies against proteins involved in the cellular processing of DNA damage. Advantages and limitations of the different experimental techniques will be discussed with particular emphasis on the still unsolved problem of the clustered DNA damage resolution. Development of biophysical models aimed to describe the kinetics of the DNA repair process is underway, and it is expected to support the experimental investigation of the mechanisms underlying the cellular radiation response.

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

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

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

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

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

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

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

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