Mauro Belli

The aim of this position statement is to bring to the forefront the necessity for maintaining and enhancing high competence in assessing the impact of low dose and low dose rate exposure on human health and the urge for funding to achieve this within Europe. Exposure to low dose/dose rates of radiation can arise from multiple scenarios or events, including natural radiation exposure, the use of radiation in medicine, industry and energy production, terrorist actions and following a nuclear incident or war. Technological developments involving radiation are progressing rapidly and have the potential to benefit mankind and societal issues. The benefit of high dose exposure during radiotherapy is a well-funded area. However, the health consequences of exposure to low doses is not well understood and the area of radiation protection research (RPR) is poorly funded. High quality RPR is essential to allow updating of radiation safety regulations for optimal protection from natural, medical and occupational exposure and for assessment of radiation incidents. Continuous evaluation of risks is essential as technological developments result in new types of radiation exposure. We will overview the technologies and situations which can potentially lead to low dose exposure, evaluate what has been gained from RPR and the questions that still need addressing, discuss the current state of RPR in Europe and highlight the consequences of a failure to adequately fund this area. We conclude that increased funding for RPR is essential to maintain high competence and to allow adequate protection of the public to inevitable low dose radiation exposure.

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

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 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.

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.

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 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.

Adaptive response (AR) is a term that has been generally accepted to describe the ability of a low ‘priming’ radiation dose to decrease the cell response to a subsequent higher ‘challenging’ dose. The main proposed mechanisms to explain AR are: increased efficiency of DNA repair and induction of antioxidant enzymes. A model that considers a modulation of the efficiency of DNA repair activity and of the level of antioxidant enzymes, starting from the framework of a lethal–potentially lethal (LPL) model is proposed. The LPL model has been extended with the inclusion of the dynamic variables representing the efficiency of repair, the levels of radiation induced radicals and of antioxidant enzymes. The model used here is able to describe the protective effect of a priming dose. Moreover, in agreement with the data in the literature, the simulations show that the AR happens in given priming dose and priming dose-rate ranges only, and requires at least 4 h to develop. In order to get more insights into the role of cell–cell communication as factors affecting the AR, experimental studies were planned using sparse or confluent AG1522 cell monolayer. The results obtained after gamma irradiation suggest that cell density is a crucial factor for observing an AR.