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DoReMi - Low Dose Research towards

Multidisciplinary Integration

Transitional Research Agenda (TRA) + Annexes

Date of preparation: 30 March 2016

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TRANSITIONAL RESEARCH AGENDA (TRA)

1 Foreword ..................................................................................................................................................................... 4

2 Executive summary ................................................................................................................................................. 4

3 The process and methodology for the development of short-term and long-term research agendas ................................................................................................................................................................................... 6

3.1 The MELODI initiative by the High-Level and Expert Group (HLEG) ....................................... 6

3.2 DoReMi Network of Excellence – an operational tool for setting up the MELODI platform ............................................................................................................................................................................. 8

3.3 Methodology for establishment of the DoReMi Transitional Research Agenda (TRA) .. 11

4 Results obtained during DoReMi .................................................................................................................... 14

4.1 Studies on cancer induction: research on mechanisms and epidemiological approaches, conclusions, perspectives ....................................................................................................................................... 15

4.1.1 Radiation quality specific responses (see question 1): ........................................................... 15

4.1.2 Radiation dose rate specific responses (see question 2): ...................................................... 17

4.1.3 Tissue specific responses (see question 3): ................................................................................. 19

4.1.4 Modifications of risk by genetic and epigenetic factors (see question 4) ...................... 21

4.1.5 Effect of age (see question 5) ............................................................................................................. 25

4.1.6 Effect of lifestyle and/or other exposures on risk (see question 6) .................................. 26

4.1.7 Effect of physiological state (see question 7) .............................................................................. 26

4.1.8 Search for a hereditary component in risk (see question 8) ................................................ 26

4.1.9 Non-targeted and systemic effects (see key question 9) ........................................................ 26

4.2 Studies on induction of non-cancer effects: research on mechanisms and epidemiological approaches, conclusions, perspectives ............................................................................ 30

4.2.1 Studies concerning the cardiovascular system and cardiovascular disease (CVD) .... 30

4.2.2 Radiation dose rate specific responses (see question 2) (CVD) .......................................... 30

4.2.3 Tissue specific responses (see question 3)(CVD) ...................................................................... 31

4.2.4 Modifications of risk by genetic and epigenetic factors and gender (see question 4) (CVD) 32

4.2.5 Effects of age (see question 5) ........................................................................................................... 33

4.2.6 Effects of lifestyle (see question 6) .................................................................................................. 33

4.2.7 Effects of physiological state (see question 7) ............................................................................ 33

4.2.8 Hereditary effects (question 8) ......................................................................................................... 33

4.2.9 Non-targeted and immunological effects (question 9) CVD ................................................. 33

4.3 Lens opacities ................................................................................................................................................ 35

4.4 Neurological effects of low and moderate doses of ionizing radiation ................................. 40

4.5 Development of Biomarkers (see Pernot E et al. 2012, 2014) .................................................. 45

4.6 Epidemiological studies ............................................................................................................................ 48

4.7 Molecular and mathematical modeling .............................................................................................. 51

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5 DoReMi outcomes ................................................................................................................................................. 53

5.1 Effect of radiation quality ......................................................................................................................... 53

5.2 Dose and dose rate effects........................................................................................................................ 53

5.3 Tissue sensitivity and tissue specific responses ............................................................................. 54

5.4 Internal emitters .......................................................................................................................................... 55

5.5 Cancer induction .......................................................................................................................................... 55

5.6 Induction of non-cancer effects ............................................................................................................. 56

5.7 Individual sensitivity .................................................................................................................................. 58

5.8 Non-targeted effects and immune system modulatory effects of IR ...................................... 59

5.9 Epidemiological studies – The way towards molecular epidemiology ................................. 59

6 Implications of DoReMi research for Radiation Protection ................................................................. 60

6.1 Shape of dose response for cancer and the application of Linear non threshold hypothesis ..................................................................................................................................................................... 60

6.2 Individual radiation sensitivity and variable predisposition for IR induced pathologies 63

6.3 Non-cancer effects and overall detriment by low dose radiations ......................................... 64

6.4 Applications for operational radiation protection - Improvement of pysical and biological dosimetry .................................................................................................................................................. 64

7 Recommendations for future lines of research based on DoReMi experience ............................ 65

7.1 Dose and dose rate dependence of cancer risk ............................................................................... 66

7.2 Non-cancer effects ....................................................................................................................................... 67

7.3 Individual radiation sensitivity .............................................................................................................. 68

7.4 Roadmap for future low dose risk research ..................................................................................... 70

8 Results obtained in the Operational WPs of DoReMi ............................................................................. 72

8.1 Infrastructures: operational progress, conclusions, possible future developments ....... 72

8.2 Recommendations for infrastructures (DoReMi) .......................................................................... 73

8.3 Education and Training: operational progress, conclusions, possible future developments .............................................................................................................................................................. 74

8.4 Recommendations for Education & Training and Infrastructures.......................................... 77

9 Concluding remarks (relationship DoReMi/MELODI)........................................................................... 77

References .......................................................................................................................................................................... 79

Annex 1: DoReMi 1st Version TRA derived Subquestions (2010) .......................................................... 101

Annex 2: DoReMi tasks and publications related to each task .................................................................. 106

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1 Foreword

Research on low dose and low dose rate effects of ionising radiation and the mechanisms involved with the aim to improve low dose health risk evaluations and radiation protection. The general population is regularly exposed to low radiation doses (<100 mGy) and doses rates (<10 mGy/min) due to current environmental, industrial and medical diagnostic exposures. Fortunately, exposures to higher doses and dose rates due to industrial, radiotherapeutic and nuclear accidents (such as are Chernobyl and Fukushima) are encountered much more rarely. Probably, as a consequence of the important nuclear accidents one finds in the general public a fairly widespread, considerable degree of anxiety towards radiation-induced health and environmental risks. The actual radioprotection system is quite well established for medium and high doses of ionizing radiation exposures (>100 mGy) but not yet as well for low dose exposures (<100 mGy). This is largely due to the fact that epidemiological studies are statistically very much limited for estimating low dose radiation health risks, and thus have to be backed up by mechanistic studies. In 2009, the High Level Expert Group (HLEG, www.hleg.de) convened by the EU was asked to pinpoint deficiencies in the current radioprotection system in Europe and to find ways to improve it. Based on the assessment of the present situation and the identification of remaining scientific uncertainties in radiation protection, the HLEG proposed several important actions and the establishment of a long-term strategy in low dose health risk research at the European level. Following these recommendations, the European Community favored the launching of the long term initiative MELODI - Multidisciplinary European Low Dose Risk Initiative (www.melodi-online.eu) in form of an independent association together with a supportive short term tool, the European research project and Network of Excellence DoReMi - Low Dose Research towards Multidisciplinary Integration (www.doremi-noe.net, 2010-2015) within the Euratom’s Seventh Framework Programme. DoReMi project included the structuring of MELODI, the establishment of suitable short and long term research agendas, i.e. (the short term DoReMi Transitional Research agenda (TRA) and the long term MELODI Strategic Research Agenda (SRA) launching most urgent pilot research studies as well as the maintenance and development of suitable infrastructures and education & training activities in the domain of low dose and low dose rate health risk research and radiation protection.

The DoReMi project ended in December 2015. This provides a good opportunity to give an overview on the achievements and strategic research developments within the DoReMi project in the actual context of low dose health risk research as well as on the remaining, still open research questions and prioritized issues that need a serious, well-sustained future follow-up at the European and international level.

2 Executive summary

The DoReMi - Low Dose Research towards Multidisciplinary Integration (www.doremi-noe.net) was funded as a Network of Excellence as part of the Euratom FP7 program, and ran from 2010 to2015. DoReMi had the following tasks:

1. To form a network of European institutions participating in research into the risks from low doses of ionizing radiation;

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2. To structure the set up of the Research Platform MELODI Multidisciplinary European Low Dose Risk Research Initiative, (www.melodi-online.eu);

3. To set priorities for research to be funded during the term of DoReMi (Transitional Research Agenda, TRA);

4. To carry out a programme of research projects and to launch further calls in accordance with the TRA to attract new partners to the NOE;

5. To formulate a long-term Strategic Research Agenda (SRA) to give guidance to MELODI and to the research to follow after the completion of DoReMi.

The research project aimed to respond to societal needs to reduce the uncertainties concerning the health effects of low dose exposures. Low doses were broadly defined as less than 100 mGy of low LET radiation and low dose rates as less than 10 mGy per minute of low-LET radiation. The general population is regularly exposed to low dose IR from natural, environmental (residual contamination from nuclear accidents and radioactive waste), industrial and medical (diagnostic (CT scans) and therapeutic (out of field exposures) sources. Since for statistical reasons, epidemiological studies to assess radiation risks at low dose levels (<100 mGy) are very limited, it was clear that risk estimations had to be backed up by a better mechanistic understanding of low dose radiation effects. However, fundamental studies on low doses were rare. They constituted a great scientific challenge because of the lack of reliable methods to detect and quantify relevant biological effects in the low dose range. Since DoReMi ended in December 2015, with this final TRA it is timely to highlight main results obtained and to give a future outlook. The process and methodology

Following the principles and recommendations of the High Level and Expert Group (HLEG) (see HLEG report 2009) focused on mechanistic and epidemiological studies concerning (1) low radiation dose-induced cancers, (2) individual radiation sensitivity and (3) low radiation dose-induced non cancer effects such as induction of cardiovascular disease, eye lens opacities and neurological effects. Radiation quality effects, tissue sensitivity and internal contamination were included as cross-cutting issues. The successive calls introduced new research disciplines and progressively contributed to this domain of research: new dosimetry approaches, molecular biology with ‘Omics’ (transcriptomics, proteomics, metabolomics, new generation sequencing etc.), molecular, gene wide analysis, epigenetics, Raman spectroscopy, mathematical modeling, systems biology and immunology. Concomitant activities such as the establishment of Education and Training activities and access to suitable infrastructures (irradiation facilities, data-and tissue banking, epidemiological cohorts and analysis platforms) for low dose radiation research were included. The TRA and SRA were regularly updated and adjusted to most recent research developments. Results obtained during DoReMi

Epidemiology: First attempts to go for molecular epidemiology (cancer, non cancer) Use of molecular markers for individual risk assessments

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Mechanisms: Involvement of non-targeted effects and immunological (inflammatory and anti-

inflammatory effects in cancer) Low dose signature of gene expression for AML in mice Non linear responses for the induction of senescence Involvement of p53 pathway in thyroid cancer development Involvement of telomeres in cancer development Genetic and epigenetic profile changes associated with individual sensitivity

Extension of research areas:

New biomarkers (IGFBP5.) for exposure (γH2AX, 53BP1, FDXR…) and disease (stress, 8oxoGua), Raman spectroscopy of proteins, micro RNAs…

Imaging of DNA repair proteins at work in living cells after IR exposure (microbeam irradiation, SNAKE, Munich)

Detection of individual sensitivity (γH2AX, telomeres) Biodosimetry: FISH-CM-TL assay for prognostic purposes of individual

sensitivity, cancer and premature ageing susceptibility, and use of γH2AX as biomarker in emergency situations

Dosimetry: detection of long-term retrospective radon (and thoron) exposure Measures of doses involved in the promotion of lens opacities in interventional

cardiologists New models for cancer induction (PARTRAC) New research work on internal emitters. New mathematical modeling of epidemiological findings for health risk

evaluation Increased work on low doses and low dose rates

Conclusion

In conclusion, the results obtained within DoReMi clearly contribute to a better understanding of low dose and low-dose rate effects related to human health risks. With the help of feasibility and pilot studies DoReMi has contributed to the improvement of knowledge of low-dose/ dose-rate radiation cancer risk in humans (in particular for leukemia, thyroid cancer, lung cancer and solid cancers….) and has also improved low dose/dose-rate risk projection models based on knowledge on the processes that drive carcinogenesis and the development of non cancer effects. Low-dose responses are shown to involve non-linear components and a multitude of parameters and factors that can modulate the pathological outcomes. The modeling and estimation of low dose health risks is therefore clearly more complex than the evaluation of risks from high doses.

3 The process and methodology for the development of short-term and long-term research agendas

3.1 The MELODI initiative by the High-Level and Expert Group (HLEG)

In 2009, the European High Level and Expert Group (HLEG) identified a series of key policy questions to be addressed by a strategic European research agenda (www.hleg.de). Although much is known about the quantitative effects of exposure to

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ionising radiation, considerable uncertainties and divergent views remain about the health effects at low doses. The over-arching policy questions addressed by the HLEG report are: How robust is the current system of radiation protection and risk assessment, given its uncertainties? How can it be improved for delivering intended level of protection for the population from occupational, environmental and medical exposures to ionising radiation. The policy questions are further illustrated in Figure 1.

Figure 1. The main issues where judgements are made in the current system of radiation

protection. The four blue boxes denote judgements that fall directly in the main ICRP dosimetric system, whereas the two boxes on the right include issues that are at present

included only to a relatively minor degree. The key policy questions identified by the High Level Expert Group (HLEG) are the shape/s of cancer dose-risk relationship/s, variation in risk between individuals, differences in tissue sensitivities for cancer, effects of radiation quality, risks from internal exposures and the risks of non-cancer effects. The complex and multidisciplinary nature of these issues is such that their resolution can be achieved only through the integration of research at a European, or even international level. The HLEG Report therefore proposed the establishment of a trans-national organisation capable of ensuring an appropriate governance of research in this field, and a scientific strategy capable of structuring future research in the most effective way, taking into account available resources. This resulted in the establishment of the European Research Platform MELODI (Multidisciplinary European Low Dose Research Initiative) to sustain the impetus and continue evolution of the research programme via the SRA. The MELODI initiative (Multidisciplinary European Low Dose Initiative) was a joint statement of funding bodies and research organisations to bring together, in a step by step approach and with a view to sustainability, their respective R&D programmes in the area of low dose health effects into an transnational program capable of addressing the challenges of low dose risks, in accordance with the strategy described in the HLEG report. Starting from Letters of Intent by five European organisations in 2009, the MELODI association was established in 2010 and gathered more than 30 members by 2015.

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3.2 DoReMi Network of Excellence – an operational tool for setting up the MELODI platform

To progress on the recommendations of HLEG and integration of low dose research in Europe, a Network of Excellence DoReMi - Low Dose Research towards Multidisciplinary Integration was funded by the Euratom Research and Training programme (Grant agreement No 662287) for a period of 6 years (2010-15). DoReMi provided an operational tool to continue the development of the MELODI platform (Multidisciplinary European Low Dose Risk Research Initiative) that represents the major national bodies and research programmes with a long-term commitment to low dose risk research in Europe. The High Level and Expert Group pointed out that many EU member states have lost key competences and are no longer capable of independently retaining their current research activities in radiation sciences, with implications for effectively fulfilling operational and policy needs and obligations. Up-to-date research and education and training activities carried out by DoReMi and MELODI are also needed to ensure the European competence in radiation sciences and radiation protection. To support the creation of sustainable program and operational structures, the Joint Programme of Activities (JPA) of DoReMi included:

(i) A Joint Programme of Research (JPR) covering the research priorities (key questions) outlined above and including the sharing and updating of existing infrastructures.

(ii) A Joint Programme of Integration (JPI) to promote sustainable integration between the key players in Europe.

(iii) A Joint Programme for the Spreading of Excellence (JPSE), covering in particular knowledge management, training & mobility and the communication of significant DoReMi findings to stakeholders and policymakers.

The DoReMi joint programme for research focused on the areas identified by the HLEG and MELODI as being the most promising in terms of addressing and resolving the key policy questions. DoReMi has acted as an operational tool for the sustained development of the MELODI platform during the first years, creating sustainable integration of European research on low dose risk and providing first answers to key policy questions. Strategic planning of DoReMi activities was carried out in close collaboration with MELODI. The long term Strategic Research Agenda (SRA) for European low dose radiation risk research has been developed by MELODI. DoReMi has formulated research priorities in a Transitional Research Agenda (TRA) that focuses on objectives that were feasible to achieve within the 6-year lifetime of the project and that were in areas where stimulus was needed in order to proceed with the longer-term strategic objectives of the SRA.

The Joint Programme of Research addressed three main topics: the shape of dose response curve for cancer, effects of individual susceptibilities and the risks of non-cancer effects. Radiation quality, internal exposures and tissue sensitivities are addressed as cross cutting themes within these main research areas (Figure 2). The research activities have taken a multi-disciplinary approach, including interfacing with

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the broader (i.e. non-radiation) biological, toxicological and epidemiological research communities. A substantial proportion of the activities of DoReMi were dedicated to the joint programme of research as DoReMi took the lead towards sustainable integration of low dose risk research in Europe. In the longer term this will aid the resolution of the key policy questions in radiation protection.

Figure 2. DoReMi Joint Programme of Research.

DoReMi specifically addressed the availability of suitable infrastructures for performing low dose risk research. Experimental radiation research is highly dependent on the availability of appropriate radiation sources that are reliable, capable of delivering a range of radiations, are robust and accurate. Low dose research also needs access to well-defined epidemiological cohorts, reliable databases and biobanks and as well the appropriate platforms for analysis. After the initial mapping of infrastructures and their availability, DoReMi has now provided access to several new infrastructures that will enhance the European capabilities in addressing scientific questions relevant for low dose risk. The scientific working groups and operational structure for the MELODI platform also emerged from DoReMi: the Working Groups for TRA/SRA, Infrastructures and Education & Training were formed from DoReMi Work Packages 2, 4 and 3, respectively, and they later became essential elements of joint planning for MELODI (Figure 3). The research program of DoReMi was structured under main topics: shape of dose response for cancer (WP5), individual sensitivities (WP6) and non-cancer effects (WP7).

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Figure 3. DoReMi Work Package structure

To ensure the way forward towards societally and scientifically driven improvements in radiation protection in Europe the DoReMi NoE and the MELODI association became the two major instruments. DoReMi took up the main scientific challenges by launching short term feasibility studies and pushing low dose and low dose rate research together with the implementation of suitable infrastructures and dissemination activities. It thus provided the scientific backbone and operational support for structuring MELODI. In this way, DoReMi helped in:

1. Setting-up the long term strategic research agendas (SRA) and statements, regular updating in view of latest scientific developments and in widening scientific concepts and approaches;

2. Structuring of MELODI Working groups such as the SRA WG, infrastructure WG, education & training WG;

3. Defining and assuring sustainability of infrastructures, dissemination activities (education & training and website);

4. Organizing specific Workshops exploring future research activities; 5. Definition and sustainability of infrastructures; 6. Preparing well-focused scientific calls; and 7. Preparing the organization of future and long term research activities together

with a Roadmap. So, DoReMi and MELODI activities were based on very close interaction. The independent status of the MELODI Association allowed that the short-term contributions of DoReMi could always be independently checked (by MELODI) for their relevance and suitability in terms of the scientific long-term research concepts for low dose health risk research and radiation protection. The DoReMi and MELODI low dose research programs differed from other well-known research initiatives (for example, DOE in USA etc.) in that they focused from the start on the most urgent scientific questions in radiation protection and the promotion of a corresponding, highly integrated multidisciplinary research, focusing on low doses and low dose rates defined as <100 mGy and <10mGy/h, respectively.

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3.3 Methodology for establishment of the DoReMi Transitional Research Agenda (TRA)

In structuring the DoReMi research agenda, there were several objectives. First of all, the agenda should address the uncertainties and need for multidisciplinary approaches highlighted by HLEG. Secondly, the agenda should serve two purposes: the shorter term research needs that could be addressed during the DoReMi lifetime, as well as the longer term research needs (> 20 years) that could be addressed by MELODI SRA. Thirdly, the agenda should serve as basis for research calls organized by DoReMi, thus there should a mechanism to prioritize topics for the calls and promote integration and multidisciplinarity. In line with the HLEG concepts, research activities of the DoReMi project were focused on the following uncertainties in radiation protection including: (1) the shape of dose response for cancer following (or not) the linear non threshold hypothesis (LNT), (2) the dose and dose rate effectiveness factor (DDREF), (3) effects radiation quality and energy and corresponding radiation weighing factors (WR), (4) tissue sensitivities and tissue weighing factors for different tissues and organs (WT), (5) internal emitter effects (their biokinetics and dosimetry), (6) effects of individual radiation sensitivities, genetic and epigenetic control, gender, age, lifestyle, metabolic status and other/mixed exposures, (7) non cancer effects including circulatory diseases, lens opacities and cognitive functions (8) transgenerational effects and (9) non targeted effects. To structure the research agenda, DoReMi identified nine key questions:

1. What is the dependence on energy deposition?

2. What is the dependence on dose rate?

3. What are the tissue sensitivities?

4. What is the modification of risk by genetic and epigenetic factors and gender?

5. What is the effect of age on risk?

6. What is the effect of lifestyle and/or other exposures on risk?

7. What is the effect of physiological state?

8. Is there a hereditary component in risk?

9. What is the role of non-targeted effects in health risk?

These key questions address both cancer and non-cancer diseases and the research should cover both mechanistic studies and epidemiology, ultimately aiming in multidisciplinary approaches. Each key question can be broken down to subquestions, guiding the formulation of hypotheses and helping the development of research approaches and projects. During the first six months of the project, the DoReMi MB developed a detailed process for the TRA updating and implementation. The first version of TRA was prepared at month 6 (June 2010) and four SRA statements were subsequently prepared in order to point out priorities for DoReMi calls and other Euratom calls. The implementation of the first TRA has taken place via DoReMi Joint Programme of Research (DoW), DoReMi Call Plan, initiatives for annual EC calls (jointly with MELODI) and initiatives for bi- and multilateral projects.

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Input for updating of TRA was foreseen by several ways: 1. Following on-going research 2. Arranging exploratory workshops and hearing meetings 3. Analyzing the outcome of surveys on on-going research, infrastructures and

training and education activities, 4. Developing a table of key questions and research areas/projects 5. Taking into account WP strategic reviews and outcome of feasibility studies 6. Participating in MELODI workshops 7. Consulting MELODI 8. Consulting External Advisory Board (EAB) 9. Consulting other relevant Euratom projects and scientific community

In retrospect, it can be concluded that most of these channels have indeed contributed to the development of the work programme, not only within DoReMi and MELODI but more broadly in the Euratom context. In our experience, the channels are complementary and serve slightly different purposes. Following on-going research (1) is something all scientists do, and it is driven by the individual interests. In our experience, arranging exploratory workshops (2) is a very powerful way for brainstorming on emerging topics and creating new multidisciplinary consortia; several Euratom low dose projects have been initially conceived in DoReMi workshops. Various surveys (3), however, have not been too effective, as the percentage of responders to questionnaires tends to be low. To be effective, the methodology for surveys needs to be carefully designed (attractive and easy for those who are consulted). Developing a table of key questions and research areas/projects (4) is a systematic approach supporting the identification of topics for internal and competitive calls, however, it suffers from the large number of subquestions and potential projects that could be initiated. Outcome of feasibility studies (5) has certainly been taken into account by the individual groups and WP leaders (Go/No-Go steps) and WP strategic reviews were part of the TRA update process (months 36 and 72). Participation to MELODI workshops (6) provided links to broader scientific and radiation protection communities and provides important feedback on the priorities in terms of relevance and potential impact. Consulting MELODI (7) has been particularly fruitful for the integration aspects and joint programming. Consulting External Advisory Board (8) was crucial for the evaluation of scientific and technological quality and feasibility of approaches (evaluation of calls for proposals). Consulting other relevant Euratom projects and scientific community (9) has been essential in finding synergies and avoiding duplication. As DoReMi was the first Euratom project launching competitive calls, this experience has been helpful for identifying best practices for MELODI, OPERRA and joint programming during Horizon 2020. The table of key questions and the criteria for evaluation of relevance, potential impact and feasibility of various approaches in short term, medium term and long term has been useful in the identification of topics for DoReMi internal and external calls. Relevance, potential impact and feasibility of approaches were evaluated both by Management Board (when deciding on call topics) and External Advisory Board (when evaluating the proposals). Most of the flexibility funding was reserved for competitive calls and therefore the need for additional competencies was a major driver in the identification of competitive call topics (according to EC rules, competitive calls can be organised when the existing consortium cannot perform a specific task). DoReMi was

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taking up research that is feasible in short term (initial program and calls) and prospecting for future approaches (exploratory workshops, feasibility studies). The initial work plan was of course very much dependent on the competencies of the partners available at that time. Consideration on feasibility of approaches remains quite essential for the identification of roadmaps for the short-term, medium term and long term research. In case the relevant expertise, methods, infrastructures, models or cohorts are readily available and costs are reasonable, the activity can be started readily and the topic is among the short term activities (-> launch internal or competitive call). In case the relevant expertise, methods, infrastructures, models or cohorts are there within 2-3 years or the costs are expected to go down, the activities are planned for medium term. Meanwhile, preparatory steps should be undertaken such as like brainstorming in exploratory workshops, feasibility testing, considering logistics and collection of data and samples. In case an approach is not considered feasible within 5-6 years, it is better to wait for better times, follow the progress of sciences and novel technologies potentially enabling the research, consult experts and get prepared by collecting samples and data and considering necessary infrastructures. Selection of topics from among the vast number of subquestions related to the nine key questions identified by DoReMi was a challenge. For making the selection, DoReMi listed criteria to assess the relevance and potential impact of proposed research. When assessing the relevance, the following criteria were considered:

Is the project in line with HLEG strategy and DoReMi TRA? Relevance of the topic for radiation protection Repeating old or creating new? Multidisciplinarity of approach

When considering the potential impact, the following criteria should be considered:

Prospects for improved protection of citizens, workers or patients Addressing needs of stakeholders Applicability of the knowledge to RP Change in paradigm? May change RP system? May change RP procedures?

The criteria for assessing the relevance are quite straightforward while the potential impact is more difficult to assess. The role of EAB evaluating the proposals is important and DoReMi was lucky to have several prominent RP experts to assess this point. Also MELODI and relevant stakeholder groups could be more actively consulted for the selection of priorities. Ideally, we should fund great science that fully meets the needs of RP stakeholders and society. In the mid-term of the project, the Management Board made a gap analysis of research funded so far. DoReMi project portfolio was reflected against the initial nine key questions set for cancer and non-cancer and epidemiological and mechanistic studies. This analysis showed that by mid-term, no tasks responded to three key questions: what is the effect of age on risk, what is the effect of lifestyle and/or other exposures to risk and is there a hereditary component in risk. The three research areas that were most

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frequently addressed where the dependence on dose rate, tissue sensitivities and modification of risk by genetic and epigenetic factors.

4 Results obtained during DoReMi

The research programme of DoReMi focused on getting answers to the questions and subquestions formulated at the beginning of the project. The nine key questions addressed both cancer and non-cancer diseases, and investigations on mechanisms as well as epidemiological studies (see the Table 1 below).

CANCER NON-CANCER

Mechanisms Epidemiology Mechanisms Epidemiology

Key question 1: What is the dependence on energy deposition?

Key question 2: What is the dependence on dose rate?

Key question 3: What are the tissue sensitivities?

Key question 4: What is the modification of risk by genetic and epigenetic factors and gender?

Key question 5: What is the effect of age on risk?

Key question 6: What is the effect of lifestyle and/or other exposures on risk?

Key question 7: What is the effect of physiological state?

Key question 8: Is there a hereditary component in risk?

Key question 9: What is the role of non-targeted effects in health risk?

Table 1: DoReMi key questions

As main research areas were addressed the induction of cancers (4.1) and non-cancers

(4.2), and individual radiation sensitivity.

The DoReMi project has been one of the first European research projects addressing the

individual radiosensitivity. The main aims here were to identify the causes and

contribution of heritable differences in the sensitivity of individuals to the carcinogenic

effects of low doses and low dose rates of IR and to identify the genetic and epigenetic

factors involved. Since individual radiation sensitivity may concern the induction of

cancers (chapter 4.1) as well as of non-cancer effects (chapter 4.2), the results obtained

within DoReMi on individual radiation sensitivity have been included in the two

chapters.

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Because of the limited time allotted to the project (6 years) mechanistic experimental studies could progress more rapidly than epidemiological studies. Most results could be already published before the end of DoReMi, however, quite a number are in preparation and will be published after DoReMi. In the following chapter, we describe the main results obtained based on published data. 4.1 Studies on cancer induction: research on mechanisms and

epidemiological approaches, conclusions, perspectives

Progress in mechanisms involved

Cancer is generally regarded as a metabolic disease due to internal or external stress leading to gene mutations in important tumor suppressor genes, metabolic dysfunction and genomic instability, with cancer initiating promoting and clonal expansion steps (Hanahan and Weinberg 2003, 2011). The uniqueness of the radiation-mutation model for cancer induction has been challenged by recent findings pointing to the involvement of (1) stem cells in cancer induction as cancer stem cells or as actors of repopulation and replacement of damaged parts of tissues and the involvement of cell-cell communication (Brown N et al. 2015), (2) the crucial involvement of the cell microenvironment and cell-cell interaction and communication (Campa A et al. 2013, 2015) and (3) the existence of a high genetic (mutational) heterogeneity and profound differences in the genetic constitution of cells in different organs throughout the body, for example, in the brain (Linnarsson, 2015).

4.1.1 Radiation quality specific responses (see question 1):

DNA fragmentation at high and low LET

DNA has been generally recognized as the most important cellular target for radiation-induced adverse long-term stochastic effects such as cancers but also for radiotoxic effects. Most critical lesions are DNA double-strand breaks and clustered DNA lesions. The quantity and quality of DNA damage is determined by the radiation dose and differ depending on radiation quality. Due to differences in radiation track structures high and low LET radiations induce different spectra and qualities of DNA lesions (Goodhead DT 1994). This is of biological importance because persistent (unrepaired or mis-repaired) DNA lesions are thought to be at the origin of radiotoxic and genotoxic effects (such as mutations and chromosome aberrations) leading to genetic instability and cancer (Hoeijmakers JH 2001). Although it has been shown previously that DNA damage, for example, double-strand breaks (DSB) are apparently strictly linearly induced (Rothkamm and Löbrich 2003) and that DNA repair is starting at fairly low doses 10 mGy (Rothkamm and Löbrich 2003) and chromosomal aberrations (translocations) and micronuclei are linearly induced down to 50 mGy and 10 mGy, respectively (Boei J et al. 2012), it has been now demonstrated in DoReMi that the radiation-induced uptake of DNA into chromosomes (integration) is non-linear in the dose range between 0.2 and 0.5-1 Gy. Moreover, a model system was developed to determine the impact of radiation exposure on the frequencies by which micronuclei are taken up and integrated in the genome of recipient cells. The TP53 status appears to affect uptake and integration of micronuclei, and the relationship of IR-induced MN and chromothrypsis has been explored. Chromothrypsis is a novel mechanism for radiation-induced chromosomal rearrangements that is present in most cancers (Mirishita M et al. 2016).

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Within DoReMi, the different capacities of low and high LET radiations (photons, as well as heavy ions and alpha particles, respectively) to induce fragmentation in cellular DNA as well as the effects of different specific energies have been clearly demonstrated using experimental biological data as well as molecular modeling (Monte Carlo simulations with the PARTRAC code) (Alloni D et al. 2011, 2012, 2013, 2015). PARTRAC models of initial damage were used to improve the prediction of RE/ radiation quality effects and their experimental validation. Concerning the dose deposition and DNA damage (measured as H2AX foci) by low-energy β-emitters the intracellular localization was found to be crucial (Alloni D et al. 2014). Effects of - and -irradiation on mitochondrial function were compared in biological and mathematical models. Different levels and qualities of DNA fragmentation also affect DNA repair dynamics as well as the induction of reactive oxygen species (ROS) and radicals associated with the release of pro-inflammatory cytokines such as IL-6 (Mariotti LG et al. 2012). Also, the induction of DSBs (in terms of -H2AX foci) following multiple radiation exposures were assessed. DNA repair kinetics were differently affected by different fractionated exposures (Mariotti et al. 2013). Using a newly built ion microbeam installation (SNAKE) live cell imaging allowed demonstrating the influence of high locally induced damage density on the recruitment of DNA damage response proteins (Drexler GA et al. 2015). Modeling also supported RBE values higher than 1 for high LET radiations. PARTRAC models have been found to be useful for simulating light ion track structures and biological effects of energies down to keV/u (relevant for an effective Hadron therapy). For example, unique DNA fragmentation patterns were found with low energy ions at 125-225 keVu-1 (DoReMi ad hoc study TREND) (Schmitt E et al. 2015). Furthermore, PARTRAC calculations were very useful for determining the biological effectiveness of neutrons as a function of secondary proton energy in the ANDANTE project (Baiocco G et al. 2015). As emphasized in a review paper, the dose-dependence of cell and tissue responses to radiation of different quality is highly influenced by cell-to-cell communication and NTE (Campa A et al. 2013, 2015). Previously, Portess D et al. (2007) and Abdelrazzak A.B. et al. (2011) had shown that low-dose irradiation (photons as well as alpha rays) of non-transformed cells stimulated the selective removal of precancerous cells via intercellular induction of apoptosis. Very importantly, within the DoReMi project, these results were confirmed and extended by specifying the release of signaling proteins and the transcriptional profiles of irradiated non-transformed cells on co-cultured non-irradiated transformed (malignant) cells (Babini G et al. 2015). Modeling of these cell-cell interactions showed that low dose radiation can modulate the signaling processes underlying the intercellular induction of apoptosis. Depending on system parameters, these perturbations can act in an anti-or pro- carcinogenic way (Kundrat P et al. 2011, Kundrat P and Friedland W 2015). Interestingly, in a mouse model of acute myeloid leukemia (AML) it could be demonstrated that the minimal deleted region on chromosome 2 indicating radiation-induced AML differed in size and complexity depending on radiation quality (X-rays (low LET) versus neutrons (high LET) (Brown N et al. 2015).

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For measurements of Radon-222 exposures in environmental conditions and homes a new, cheap and convenient method has been developed for retrospective radon measurements (Pressyanov D et al. 2015a,b, Mitev K et al. 2015). Obviously, these results are likely to have an important bearing on low dose health risk estimations and on the use of radiations of different quality in anti-cancer radiation therapy.

4.1.2 Radiation dose rate specific responses (see question 2):

The biological and health effects of low dose-rates have become in recent years a very controversial matter in radiation protection (Rühm W et al. 2015). The International Commission on Radiological Protection (ICRP) proposed the concept of extrapolating existing epidemiological data at high doses and dose rates down to low doses and low dose rates (ICRP 2007) and a so-called dose and dose-rate effectiveness factor (DDREF). The DoReMi project aimed to contribute to this general concept with some new data on low dose-rate effects at the molecular, cellular, animal and human level. Scientifically, this has been (and is still) not an easy task because there is considerable ambiguity about the definition of dose-rate effects. Does low dose rate exposure mean permanent chronic exposure to low doses (for example in contaminated areas of permanent radioactive background of soil or air pollution)? Or does it mean intermittent exposures to low doses such as fractionated daily exposures to (low) radiation doses (for example CT scans or multiple mammographic examinations in radiation diagnostics or in strict radiation therapy schedules) or protracted exposures (for example, irregular or regular spaced radiation exposures due to industrial exposures or to environmental contamination)? Unfortunately, these terms are not always handled very strictly (for example in epidemiological studies). In mechanistic terms, however, this makes a big difference for cells, tissues and organisms since different schedules may leave more or less time to recover from radiation insults and/or activate stem cell niches for repopulation of damaged tissues, and thus differ in biological outcomes. Although ICRP has proposed a DDREF factor of about 2, in the scientific literature the reduction factors obtained between high and low dose rates may vary quite substantially depending on the biological endpoint studied (Rühm et al. 2015). Nevertheless, the DoReMi project yielded interesting new finding on these subjects: Effects related to cancer induction or treatment Fractionated exposures

In radiotherapy (RT) aiming to control tumor growth and inactivate tumor cells effects the tumor microenvironment is of great importance. This can be affected by radiation-induced modulation of the immune system involving out-of-field reactions. Based on the activation of dendritic cells by released cytokines and the uptake of tumor peptides from irradiated cells combinations of RT and immune therapies (for immune checkpoint inhibition) could be proposed (Derer A et al. 2015). In the same line, preclinical studies demonstrated that combining RT with immune stimulation could induce anti-tumor immunity. In fact, after RT from dying tumor cells mature dendritic cells take up tumor antigen and mediate the induction of adaptive and

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innate anti-tumor immunity (Candeias S and US Gaipl 2015); Locally-triggered, systemic immune activation can also lead to spontaneous regression of tumors or metastases that are outside the radiation field (abscopal effects) (Frey B et al. 2012a). Thus, effects of IR on the immune system contribute to the success of anti-cancer therapy. Apparently, in RT or chemotherapy (CT) local hyperthermia can modulate the innate and adaptive immune system and has been shown to increase natural killer (NK) cell activity, lymphocyte infiltration and heat-shock protein mediated induction of immunogenic tumor cells in patients (Frey B et al. 2012b). Moreover, combination with CT with X-rays can induce in vitro cell death forms of colorectal tumors with immunogenic potential (Frey B et al. 2012c). Moreover, more recently, it could be shown that norm-and hypo-fractionated RT induces a fast human colorectal tumor-cell death with an immunogenic potential that triggers the maturation and activation of immunologically active dendritic cells in vitro (Kulzer L et al. 2014). Since building up immune reactions takes time, finding the most beneficial combination and chronology between distinct fractionation of radiation doses in RT and immune therapy is a big challenge for innovative antitumor therapies (Frey A et al. 2014 and 2015). In addition, RT and immunotherapy should also focus on abrogation of the suppression of regulatory T cells (Persa E et al. 2015). This statement is underlined by the experimental findings that (1) regulatory T cells (Treg) are intrinsically radioresistant because of reduced apoptosis and increased proliferation leading to their systemic and/or intratumoral enrichment, however, (2) in a tumorous environment Tregs acquire a highly suppressive phenotype that is increased by RT. In addition, the importance of the subtle interplay between targeted effects of IR (X-rays) on cancer cells (such as DNA damage and cell death) and non-targeted effects involving activation of immunogenic tumor responses exerted by the innate immune system (natural killer cells) as well as by T cells activated by dendritic cells (adaptive immune system) has been emphasized in the context of RT (Gaipl US et al. 2014). The general findings showing that RT can contribute to anti-tumor immunity (Rubner Y et al. 2012) have been also supported by the fact that fractionated RT stimulated the induction of glioblastoma death forms (with HSP70 release in p53 mutated glioblastoma cells) with immunogenic potential (Rubner Y et al. 2014). Still, the role of dendritic cells (as part of the innate immune system) depends on the treatment schedules (Manda K et al. 2012). In some conditions, protracted low dose IR may result in radioresistance, in others, chronic low dose IR may lead to immune suppressive effects including killing of certain cell types. Somewhat in line with this, in vitro hypofractionated irradiation increased the immunogenic potential of caspase-3 proficient breast cancer cells with basal low immunogenicity (Kötter B et al. 2015). Adaptive responses A mechanistic study on the DNA (DSB) repair dynamics of cells exposed to fractionated IR and subsequent modeling showed that induction of DSBs (as measured by the induction of H2AX foci) is highest by the first initial dose and decreased by subsequent exposures (Mariotti IG. et al. 2013). Thus, induction of DNA damage was non linear with split dose exposures (Mariotti LG et al. 2013). (This effect may be radiation quality and energy dependent, since results obtained with soft X-rays (radio-mammography) showed an increased (super-additive) H2AX level after a split dose 2

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mGy plus 2 mGy protocol (Colins C et al. 2012). Interestingly, cytokine secretion GCSF, IL-6, IL-8, TGFbeta) are also stimulated in conditions of adaptive responses but do not seem to underlie adaptive responses that clearly affect DNA repair kinetics of radiation-induced DSB (Dieriks B et al. 2011; Mariotti LG et al. 2013). Studies in mouse strains with different genetic repair capacities mice showed that fractionated low-dose radiation (100 mGy daily) increased DNA damage with cumulative doses and affected replication and apoptosis in the lung parenchyma, thus influencing lung function (Flockerzi E et al. 2014). Knowing that intensity-modulated RT (IMRT) for thoracic malignancies increases the exposure of surrounding healthy lung tissue to low-dose radiation these findings call for some caution when using such new RT modalities. Moreover, different cell populations in the lung parenchyma (bronchiolar and alveolar epithelial cells) showed varying vulnerabilities to IR. The number of DSBs induced (measured as H2AX foci) varied in different cell populations with highest levels in repair deficient (ATM, SCID) mice. Interestingly, however, mice showing heterozygocity for the ATM gene were able to deal with the DNA damage induced by the repetitive low-dose radiation suggesting that their DNA repair capacity was sufficient. This suggests that the individual DNA repair capacity is very important regarding low dose radiation health risks. Fractionated low-dose radiation exposure induced in mouse testis an increased several weeks persistent load of DNA damage (DSBs) in surviving spermatogonia stem cells suggesting severe consequences for the genomic integrity of the fertilizing sperm (Grewenig A et al. 2015). For humans, this may also indicate increased reproductive risk with potential treatment-related infertility from low scattered doses to testis from RT. Moderate dose Interestingly, in the mouse a non-linear response to medium dose IR could be demonstrated consisting of reduced secretion of the inflammatory cytokine IL-1 by stimulated macrophages of radiosensitive Balb/c mice after exposures to 0.5 and 0.7 Gy of IR. This effect was not seen in the less radiosensitive C57BL/6 mouse and human TNF- transgenic animals (Frischholz B et al. 2013).

4.1.3 Tissue specific responses (see question 3):

Cancer related effects

In papillary thyroid carcinoma cells low doses of X-rays affected cell cycle distribution, and very likely p53-dependent p16 activation but did not affect apoptosis. Clear, senescence was seen at higher doses involving the activation of p16 and 21 (Abou-El-Ardat K et al 2011). Low X-ray doses appear to significantly affect cell proliferation in normal thyroids but less in RET/PTC positive thyroids (Abou-El-Ardat K et al. 2012). Using wild type and RET/PTC3 mice it was shown that iodine deficiency induced a long lasting angiogenic phenotype in thyroid cancer cells (RET/PTC3) (Gerard AC et al. 2012) In DoReMi, differences in radiation responses were highlighted when comparing skin, lung, breast and endothelial 3-D models with current 2-D monolayer culture (Acheva A et al. 2014).

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Through a DoReMi workshop on stem cell research seeded the RISK-IR project on the importance of stem cells in IR–induced carcinogenesis (Raj K et al. 2012). The importance of stem cell radio-sensitivity for radiation-induced cancers and possibly non-cancers was stressed. Studies in animals (mice) showed clear mouse strain-dependent differences in the DNA damage and repair responses in bronchiolar and alveolar epithelial lung cells (Flockerzi E et al. 2014) In particular, differences in 53BP1 induction in bronchiolar and alveolar epithelial cells.: were seen. Besides the fact that fractionated exposures (100 mGy) are suspected to cause an increase in possible secondary malignancies, the genetically controlled DNA repair capacity was shown to determine the amount of cumulative DNA damage induced by repetitive low dose 6 MV photon radiation, lung, heart and brain cells and different susceptibilities to the induction of such DNA damage. Lymphoblastoid cells drawn from different individuals showed quite different radiation sensitivities (Gürtler A et al. 2014). In contrast to ATM homozygotes, ATM heterozygotes lymphoblast cells apparently were able to cope with DNA damage from repetitive low dose exposures (Schanz S et al. 2014) indicating that DNA repair of low dose radiation damage was sufficient in those cells. Moreover, experiments with strains of mice (C57BL/6) differing in genetic repair capacity (ATM, SCID) showed that after repetitive low dose radiation persistent DNA damage foci (mainly unrepaired DSBs) accumulated in heart cardiomyocytes, brain cortical neurons and in lung cells (bronchiolar and alveolar). Increases in DNA damage were highest in SCID (DNA-PKs deficient) mice and in brain, and lung cells (Schanz S et al. 2014). Repetitive low dose radiation gave rise to very significant levels of persisting DNA foci in cortical neurons in brain tissue of repair deficient mice (ATM-/-). The findings suggest that even low doses of radiation significantly can increase the health risk in individuals, particularly in those with compromised repair capacity. Fractionated low dose irradiation of testicular mouse tissue induced persistent DSB in spermatogonial stem cells affecting proliferation, differentiation and apoptosis of testicular germ cell populations and thus the process of spermatogenesis (Grewenig A et al. 2015). The clonogenic potential of tumor cells plays an important role in the treatment of breast cancer by RT. It could be shown that single exposures or hypofractionated irradiation reduced the clonogenicity in caspase 3 deficient p53 wildtype (MCF-7) cells but was less effective on caspase-3 intact, hormone receptor negative, p53 mutated MDA-MB231 breast cancer cells. This was accompanied by effects on the immune system (activation of dendritic cells) in the cancer cells (Kötter B et al. 2015). Also, norm- and hypofractionated RT induced human colorectal tumor-cell death with some immunogenic potential (Kulzer L et al. 2014). Because the interaction of dendritic cells and T lymphocytes in low dose IR immune reactions their respective radiation sensitivity needs particular attention (Manda K et al. 2012). Regarding RT, the immune responses induced are very important for the therapeutic results. Indeed, RT exerts contradictory effects on the antitumor response, some

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parameters may increase: (increased proliferation) leading to intratumoral enrichment. Irradiation does not enhance regulatory T cells (Treg cells). However, in a tumor environment Treg cells acquire a highly suppressive phenotype that is enhanced by RT. Thus, Treg targeted pre-clinical and clinical immunotherapy approaches together with RT may merit further exploration (Persa E et al. 2015). In a new approach to characterize irradiated cells and tissues after low dose exposure, for the first time, Raman spectroscopy was employed to measure the biochemical profile of healthy and diseased cells and tissues (Maguire A et al. 2015 a,b). Following low doses of 0.05 and a moderate dose of 0.5 Gy, in lymphocytes from different donors inter-individual variability of DNA damage (as measured by H2AX fluorescence) was observed that correlated well with spectral variability of biochemical profiles (Maguire A et al. 2015b). The methodology also allowed discriminating different peripheral blood mononuclear cell types (T-cell lymphocytes and myeloid cells) (Maguire A et al. 2015a). The radiosensitivity of lymphoblasts derived from the autosomal recessive human syndrome Schwachman–Diamond was investigated (Mavragani IV et al. 2016). Mutation (Lack) of the Schwachman-Bodian-Diamond syndrome gene leads to higher residual DNA damage (SSBs and DSBs) after IR exposures suggesting defaults in SSB and DSB repair (Morini J et al. 2015). Differential radiation sensitivity was observed between patient-derived cells and those derived from healthy donors. Analysis of primary fibroblasts derived from patients with the Cockayne syndrome conferring slight radiation sensitivity revealed an altered redox balance with increased-steady-state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of mitochondrial membrane potential and a decreased rate of basal oxidative phosphorylation which may explain at least in part their radiation sensitivity and clinical features (Pascucci B et al. 2012).

4.1.4 Modifications of risk by genetic and epigenetic factors (see question 4)

The role of genetic and epigenetic regulation of low dose responses leading to the induction of genetic instability and cancer were largely unknown when DoReMi started. Therefore, it has been the aim of the DoReMi project to better understand the contribution of these regulatory pathways to IR induced cancers and to the modulation of individual sensitivity. In recent years, molecular and cellular biology analyses revealed that all insults on cells and tissues elicit specific cellular reactions turning off or turning on common (general) or specific (for example, IR-responsive) metabolic pathways, transcription of specific genes, production and activation of proteins, modulating epigenetic control mechanisms (through modification of chromatin, activation or inactivation of regulatory elements (gene specific microRNAs), and the cellular release of intercellular messengers (such as radicals and cytokines). Depending on the organisatory level (cellules, tissue, whole body), different signaling systems are involved: In cells: activation of receptor proteins on membrane (activating MAP kinase cascades), DNA damage (DSBs) activating phospho 3 inositidyl kinases, activation of transcription factors leading to the transcription of genes involved in defenses (antioxidants, DNA repair, energy metabolism), activation of kinases that activate important effector proteins, production

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of cytokines, hormones…In tissues, intercellular signaling occurs through cell-cell communication involving intercellular release of oxidative radicals (lipids) and cytokines. In the body, signaling is activating immune-competent cells (for immunological defenses), distantly hormone receptive cells or nerve cells through activation of mediators. These signaling systems are crucial for cellular and tissue responses to genotoxic stress (including that of IR). Activation or modulation of them determines the cellular and tissue reactivity and the final pathological (cancer or non cancers) or non-pathological effects. Thus, not only the induction of damage is important for cellular responses but also the signaling of such damage in order to activate cellular defense systems (antioxidant enzymes, antiradical enzymes, DNA repair systems (for example, the repair of IR-induced DSB) and inflammatory or anti-inflammatory immune responses. DoReMi took an active part in studying these basic mechanisms involved in responses to ionizing radiation exposures, putting special emphasis on low dose responses. The results obtained in these mechanistic studies show the existence of non-linear responses for many biological endpoints in terms of dose and dose-rate. The dose dependent effects concern in particular gene expression (Kabacik S et al. 2015; Katsura M et al. 2016), inflammatory immune reactions (Rödel F et al. 2012,a, b, 2013) and anti-inflammatory responses (Frischholz B et a. 2013, Gaipl US et al. 2014, Lödermann 2012, Rödel F et al. 2012, 2013, Rubner Y et al. 2012, Wunderlich R et al. 2015), up-regulation of p16 at 62.5 mGy and induction of senescence in thyroid cancer cells (0.5 Gy) (Abou-El-Ardat K et al. 2011). Of particular interest have been the results showing that inflammatory immune reactions in endothelial (mononuclear and poly-nuclear) cells can be modulated at moderate doses (0.5-0.7 Gy) (Frischholz R et al. 2013, Rödel F et al. 2012). Furthermore, non-linear dose-rate dependent effects have been observed for the induction of senescence by gamma-ray exposures in human epithelial cells (Yentrapalli R et al. 2013b.). Pathway analyses have shown the preferential involvement of genes and proteins related to oxidative stress and oxidative metabolism and oxidative defense mechanisms after low doses and low dose rate exposures as compared to the involvement of DDR response and cell structural genes and proteins at higher doses (>100 mGy).

The analysis of telomere involvement in radiation responses suggests that the number of radiation-induced dysfunctional telomeres per cell is likely to determine the development of cancer (low level of dysfunctional telomeres per cell, &-4), whereas higher levels of dysfunctional telomeres (>5) will give rise to senescence (Shim G. et al. 2014). Directly cancer-related effects

Progress in research on gene expression following low dose exposures clearly revealed an important role for the transcription factor PU.1 in the initiation and development of IR-induced AML (Verbiest T et al. 2015). In fact, deletion of chromosome 2 (del2) and Sfri/1/ Pu.1 loss constitutes a molecular signature of radiation induced acute myeloid leukemia (AML) (Olme C-H et al. 2013a,b). GPF fluorescence expression under control of the Sfpi1 promoter in the rAML-susceptible CBA/H mouse strain allowed to monitor

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critical gene loss events, i.e. the initiation of leukemia and the disease progression in live leukemic cells (Olme C-H et al. 2013). The complexity of del2 in radiation-induced AML in mice showed a clear LET-dependence without impact upon the sfpi1/PU.1 deletion and/or point mutation frequency on the other chromosome 2. A minimal deleted region (5.5 Mb) (still including Sfp1/PU.1) on chromosome 2 (in which the tumor suppressor gene DSPi1 is located encoding the transcription factor) PU.1 could be defined independent of radiation quality (Brown N et al. 2015). Also, evidence has been obtained for the involvement of dose and time dependent miRNA expression in IR responses (Kabacik S et al. 2014). Indeed, micro RNAs such as miRNAs miR-34a-5p (a target of TP53 with strong pro-apoptotic and anti-proliferative properties), miR-182-5p (with likewise oncogene and tumor suppressor related features) were responsive in a time and dose dependent manner. Furthermore, a multi-omic analysis of mouse AML using integrated transcriptomic and proteomic analyses brought to light a number of possible new biomarkers that are associated with the development of radiation-induced AML (Olme CH et al. 2013a,b; Verbiest T et al. 2015). A 17 gene/protein signature was found to define AMLs and to distinguish AML cell lines (Verbiest T et al. 2015). Fluorescence reporter gene systems to monitor critical gene loss events and disease progression over time were developed as well. Downregulation of PU.1 occurs also in humans and may include heterozygous deletion of the SP1I1 locus and mutation of the -14kb SPI1 upstream regulatory element as well as p53 deletion, FLT3-ITD mutation and the recurrent AML1-ETO (t(8;21)) and PML-RARA t(15;17) translocations (Verbiest T et al. 2015). In a papillary thyroid carcinoma model (TPC-1 cells) low doses of 62.5 mGy induced a change in cell cycle distribution, possibly p53-dependent p16 activation but no significant apoptosis. After a moderate dose of 0.5 Gy signs of senescence appeared (Abou-El-Ardat K et al. 2011). Transcriptomic analysis of the thyroid cancer cell line TPC-1 (with a RET/PTC1 translocation) in comparison to wild-type mouse thyroids showed a considerable overlap of expressed genes at a high dose of 4 Gy but no common genes at 62.5 mGy. Furthermore, a certain radiation-responsive signature of miRNA was found in this cell line (Abou-El-Ardat K et al.2012). The importance of Rb1 gene expression in alpha-radiation induced osteosarcoma was demonstrated in mouse strains with different tumor susceptibilities. For the first time, a clear functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure could be established (Rosemann M et al. 2014). Modifications in telomere lengths may play an important role in the process of carcinogenesis (Pernot et al. 2012; Shim G et al. 2014). Also, the effect of the quantitative effects of exposure to chronic -radiation was studied in the well characterized ApcMin/+ mouse model to better understand IR–induced colon cancer development following chronic vs. acute -irradiation (CLOGIGAT, DoReMi Task 5.10). Individual radiation sensitivity (see question 4)

Studies in mice revealed the importance of RB1 expression in radiation-induced osteosarcoma. Indeed, alpha-ray induced sarcoma and different tumor susceptibility

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genes is found in different mouse strains (BALB/c sensitive versus CBA resistant to 227th injection): Reduced Rb1 expression by common variants in regulatory regions can modify the risk for malignant transformation of bone cells (osteoblasts) after IR exposure (Rosemann M et al. 2014). This could also be shown by the fact that conditioned loss of 1 copy of the Rb1 gene (haplo-type) in the osteoblast lineage resulted in radiation sensitivity and telomere loss could be restored by an Rb1 expressing lentivirus (Gonzalez-Vasconcellos et al. 2013). Rb1 is a tumor suppressor transcription factor that regulates the G1/S transit in the cell cycle. Loss of one copy of the Rb1 gene leads to sporadic genomic instability with increasing passage number of the cells in culture, together with increased telophase bridges, increased G1, increased polyploidy after IR. Apparently, the transcript of a telomeric long noncoding RNA is under the control of an Rb1 regulated promoter. Specific genetic polymorphisms in tumor suppressor genes (p53) play a role in breast and colorectal cancer susceptibility. A meta-analysis of cancer risk associated with a specific rS17878362 polymorphism of the TP53 tumor suppressor gene shows that cancer is increased for homozygous A2A2 carriers for breast and colorectal but not for lung cancer. Clearly, rs17878362 is associated with increased cancer risk with a population and tumor specific effect (Sagne C et al. 2013, 2014). Clearly, there is an impact of G-quadruplex structures and intronic polymorphisms Rs17878362 and rs1642785 on basal and IR-induced expression of alternative p53 transcripts (Perriaud L et al. 2014). The results underline that tumor suppressor gene modifications and polymorphisms are involved in IR-induced carcinogenesis.

It is well known that mutations of the ATM gene strongly affect individual radiation responses. The majority of protein-coding genes allowed discrimination of the AT from healthy donors 2 hours (but not at 24 h) after high dose IR exposure. Furthermore, it was demonstrated that in stimulated human T lymphocytes important genes (such as CDKN1A, SESN1, ATF3, MDM2, PUMA and GADD45A were upregulated by IR and a significant modification of miRNA expression (miR-34-5p and miR-182-5p) took place. MicroRNas such a miR-34a-5p and miRNA-182-5p were responsive in a dose-and time-dependent manner. Individual differences were noted in miRNA-182-5p expression in two different healthy donors. In addition, for the first time evidence was provided that also long noncoding RNAs such as FAS-AS1 lncRNA can be up-regulated by IR exposure in an ATM-dependent manner (Kabacik S et al. 2015). In primary fibroblasts obtained from patients carrying the genetic disease Cockayne syndrome predisposed to premature aging and slight radiation sensitivity, changes in oxidative and energy metabolism were observed (Pascucci B et al. 2012). Genetic studies on papillary thyroid carcinoma in the UKRAM cohort after the Chernobyl accident revealed CLIP2 expression status as a possibly very important biomarker for IR-induced papillary thyroid carcinoma (Selmansberger M et al. 2015). Also, laser Raman spectroscopy profiles were shown to be suitable for distinguishing low and high radiotoxicity susceptible patients and are able to monitor biochemical changes induced during RT (Maguire A et al. 2015a,b). The observed changes could be related to the level of persistence of DNA repair foci and to individual G2 scores.

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The RadSENS project in DoReMi extended previous work (Skiöld et al. Mut Res. 2013 and 2015) on the identification of pathways that differ in non-radiosensitive and highly radiosensitive subjects in RT. The release of oxidized bases (8-oxo-dG) from cells from irradiated and unirradiated patients was clearly reduced in radiosensitive patients, suggesting that 8-oxo-dG release can be used as biomarker for identifying radiosensitive patients. Knowing that miRNAs regulate more than 50% of cell protein coding genes and regulate important processes such as DNA repair, cell cycle control and apoptosis) it was of interest to test miRNAs expression in radiosensitive and non-radiosensitive patients. In leukocytes from patients, Skiöld S et al. Mut Res (2013) showed that miRNAs expression differed in radiosensitive and non-radio-sensitive breast cancer patients at background level and after ionizing irradiation. Proteome and miRNAs expression were negatively correlated, for example, mi RNA let-7f was upregulated, and the corresponding genes (caspase 3, proteome beta type 2 and Ras related protein 8B were downregulated. Let-7 miRNA was recognized as critical regulators of development, stem cell differentiation, glucose metabolism and tumorigenesis.miR-486-5p affected hematopoietic cell growth and survival (with the target genes FOX01, PTEN and PI3KP). Some miRNAs changed expression at low doses exposures. This was the case for miR-183 involved in regulation of apoptosis and cell invasiveness, and for miR-224 involved in the regulation of apoptosis inhibitor 5 (APII5) (Skiöld S et al. Mutat Res. 2015) (Also, miR381, mi86-5p, mi-490-3p mi-Let7f played a significant role since they were differently expressed after IR in sensitive patients).

Distinct IR induced miRNA and proteomic profile changes are associated with radiosensitivity of head and neck cancer patients (RAD-SENS).

Altogether, this clearly indicates that biomarkers will be very useful to both quantify the extent of susceptibility and to identify individuals at risk, a notion which will have an important bearing on personalized medicine (Chen R et al. 2012, Chen R and Snyder M 2012).

4.1.5 Effect of age (see question 5)

The effects of age on radiation-induced cancer has not specifically been addressed in DoReMi. However, studies addressing senescence were informative for age effects, and susceptibility to oxidative stress was studied using certain disease models. Modifications in telomere lengths may play an important role not only in aging but also in the process of carcinogenesis (Pernot et al. 2012; Shim G et al. 2014). In fact, a model is proposed that contributes to a better understanding of IR- induced processes such as carcinogenesis and senescence: 1 to 4 dysfunctional telomeres (as evidenced by the presence of telomere induced foci (TIFs)) in the presence of dysfunctional cell cycle checkpoints and/or DNA damage response mechanisms may trigger carcinogenesis due to a sufficient amount of genomic instability that drives progression of carcinogenesis involving limited cell death or induction of senescence. However, if there are more than five dysfunctional telomeres (TIFs) induced in normal human cell populations and tissues showing intact cell cycle checkpoints and DNA damage responses, genomic instability is overwhelming and the process of carcinogenesis is halted and processes such as senescence and apoptosis prevail that provoke diseases involving cell death such

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as vascular diseases (death in vascular cells and tissues) (M’Kacher R et al. 2015) and possibly others, such as eye lens opacities and neurological dysfunctions. Regarding the radiation sensitivity of the gastrula-embryo in lacZ transgenic mice, the capacity for DSB repair is of utmost importance. Gastrual embryos were radiation sensitive in the pUR288 shuttle vector assay for point mutations and small deletions after doses up to 2.5 Gy. They showed distinct radiation-sensitive profiles and some stage specific responses (Jacquet P et al. 2015).

4.1.6 Effect of lifestyle and/or other exposures on risk (see question 6)

The effect of lifestyle and /or other exposures on radiation –induced cancer risk has not been specifically addressed in DoReMi.

4.1.7 Effect of physiological state (see question 7)

The effect of physiological state has not been specifically addressed in DoReMi.

4.1.8 Search for a hereditary component in risk (see question 8)

The key question on hereditary and trans-generational effects was not worked on in DoReMi because of short term time constraints but needs to be considered for future long-term research. For example, three and more generation molecular epidemiological studies appear to be possible on some post-Chernobyl and Mayak cohorts.

4.1.9 Non-targeted and systemic effects (see key question 9)

Mechanisms that may facilitate or inhibit cancer development

In the last 30 years, the notion of the bystander effect and the induction of genomic instability has been quite disturbing for the traditional thinking of the biological effects of IR which implied that biological effects occur in irradiated cells with the DNA in the nucleus being the main target (Little JB 2006). In fact, evidence for so-called non-DNA targeted effects (NTE) arising in cells that did not receive radiation exposure to the nucleus has been accumulating (Morgan WF 2003). The observed NTE mainly involved radiation-induced genomic instability (characterized by the occurrence of increased frequencies of mutations and chromosomal aberrations in the distant descendants of irradiated cells) and the bystander phenomenon (characterized by the transmission of damage signals from irradiated to non-irradiated (bystander) cells modulating significantly the phenotype and microenvironment of the latter. In principle, this also may include radioadaptative and transgenerational responses (Averbeck D 2010). Very intriguingly, these NTE turned out to play generally a very significant role at low doses (<100 mGy) and also at high radiation doses (>1 Gy) but in a different manner (Morgan WF and Sowa MB 2015). This implies that low dose-induced non-targeted effects may well include non-linear phenomena. In fact, this was demonstrated early on by Nagasawa H and JB Little (1999) for the alpha particles-induced point mutations (HPRT) in mammalian cells (Little JB 2006). Bystander signaling: ROS and cytokine release

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Following the European projects RISK-RAD and NOTE (Salomaa S et al. 2010), the DoReMi project focused on low dose radiation research and included NTE as an important subject of research (Campa A et al. 2015; Hatz VI et al. 2015; Kadhim MA and MA Hill 2015; Kadhim MA 2015). In radiation-induced NTE, intracellular- as well intercellular signaling plays an important role. It affects radiation responses of cells (inactivation, mutation, genomic instability, cell transformation but also may elicit protective and adaptive effects) and tissues (induction of cancers and non cancers but also may elicit increased cell proliferation, anti-inflammatory and anti-cancer effects) and this even throughout the body (through so-called long distant, abscopal effects) (Averbeck D 2010; Hatzi VI et al. 2015, Morgan WF and Sowa MB 2015). It is a general feature of NTE that intracellular transduction pathways (leading to changes in genetic and epigenetic expression profiles, production of ROS and neoplastic transformation (Campa A et al. 2015, Hatzi VI et al. 2015) as well as intercellular communication between cells and tissues (mediated by long lived ROS (NOs) and cytokine, chemokine and growth factor release) lead to specific changes in non-targeted regions (out-of field effects) in the microenvironment of irradiated cells and tissues (Hei TK et al. 2008; Morgan WF and MB Sowa 2015). Different levels of intra- and intercellular signaling are involved (Campa et al. ‘2013’ 2015). Moreover, it has to be noted that the same ROS, cytokines and growth factors are also very important in inflammatory responses induced by various stressors including IR (Spitz DR et al. 2004; Hei T et al. 2011). From the intimate relationship between IR-induced intercellular mediators, inflammation and immunological responses, cancer and non cancer induction (as recently reviewed by:Rödel F et al. 2015; Campa A et al. 2015; Morgan WF, Sowa MB 2015;Hatzi VI et al. 2015; Kadhim MA and Hill MA 2015 and Candeias SM, Gaipl US 2016) a possible link between NTE and the immune system has become more and more evident in recent years. As a consequence, a DoReMi workshop in November 2013 in Budapest on IR-induced immunological modifications seeded specific studies on the involvement of cell-to-cell interaction and immunological processes in inflammatory and carcinogenic and non-carcinogenic effects (diseases). This opened a new dimension within DoReMi and in low dose radiation research and radiation protection. The experiments performed within DoReMi on bystander effects on dendritic cells (DC) and T cells exerted by activated macrophages that have been exposed to low and moderate doses of X‐radiation showed that in particular bystander effects on T cells are induced by irradiated macrophages. Low and intermediate doses of X-radiation reduced the expression of surface MHCII molecules on activated macrophages. When these macrophages are in contact with T cells they slightly reduce their proliferation starting at a radiation dose of 0.1Gy. By this, irradiated macrophages induce bystander effects in T cells. Further, supernatants (SN) of activated and irradiated macrophages slightly impacted on DC: a decreased surface expression of CD40 on DC was observed after contact with SN of macrophages that had been irradiated with 0.01Gy, 0.05Gy, 0.1Gy, 0.3Gy, 0.5Gy, 1.0Gy or 2.0Gy. However, no consecutive impact on T cell proliferation was detectable. This suggests that only mild bystander effects on DC had been induced by irradiated macrophages. Of further note is that low and intermediate doses of radiation did not impact on the functionality and viability of antigen-presenting immune cells such as macrophages (Wunderlich R et al. 2015). Modulation of carcinogenesis via systemic effects

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Interestingly, NTE could be observed also in vivo and can contribute to systemic effects because immunological effects via inflammatory (at high doses) and anti-inflammatory (low doses) responses could be seen (Rödel et al. 2015). However, while high doses of radiation foster infiltration of immune cells into tumors, low doses of radiation does not impact on outgrowth of tumors, but slightly modulate the infiltration of immune cells in existing tumors at later time points after radiation exposure (Frey et al, 2015). Acute inflammatory reactions include the accumulation of neutrophils. A recent study (Schauer C et al. 2014) shows that neutrophils are recruited to sites of inflammation undergoing oxidative burst and forming so-called neutrophil extracellular traps (NETs). These are thought to play an important role in the resolution of neutrophilic inflammation by degrading cytokines and chemokines, disrupting neutrophil recruitment and activation. During DoReMi, clear indications were obtained on the anti-inflammatory (and sometimes anti-carcinogenic defenses) that can be induced by IR below 0.5 Gy in contrast to pro-inflammatory processes that occur at higher IR doses (Rödel F et al. 2012). A non-linear interaction between inflammatory and signaling pathways in the dose range of 0.5-2 Gy was observed (Babini G et al. 2015). In mice, ionizing radiation was shown to contribute to the induction of anti-tumor immunity (Rubner Y et al. 2012). The secretion of IL-1β by activated macrophages from Balb/c mice was found reduced after LD-RT with moderate doses of 0.5 or 0.7 Gy of X-rays (Frischholz B et al. 2013). Protein levels of inflammatory signaling molecules were reduced in human monocytes differentiated to macrophages, with a pronounced reduction after X-ray treatment with 0.5 and 0.7 Gy . From this, it clearly appears, that distinct low and intermediate doses of X-rays can induce an anti-inflammatory phenotype of activated macrophages by lowering the amount of secreted IL-1b in a NF-kB-dependent manner (Lödermann B et al. 2012). Furthermore, IR exposure can affect the immunogenic potential of cancer cells: it was shown in vitro that hypo-fractionated irradiation leads to increased immunogenic potential of caspase-3 proficient breast cancer cells with originally low basal immunogenicity (Kötter B et al. 2015). Also, norm-and hypo-fractionated radiation therapy (high doses) induces fast human colorectal tumor-cell death with an immunogenic potential that triggers dendritic cell maturation and activation in vitro (Kulzer L et al. 2014). Interestingly, there is also some pre-clinical evidence for IR induced systemic anti-tumor responses that are likely to have an important bearing on ongoing clinical applications (Derer A et al. 2015). Regarding fractionated exposures in RT, it appears that RT can stimulate the induction of cell death and Hsp70 release in radio-resistant p53 mutated glioblastoma cell lines. In fact, the induced forms of glioblastoma cell death show immunogenic potential and may constitute a microenvironment favorable for immune therapies in a clinical context (Rubner Y. et al. 2014). As shown in a subsequent clinical study on primary glioblastoma multiform tumors and their recurrence, the expression of danger signals high mobility group box protein 1 (HMGB1), heat shock protein 70 (HSP70) and calreticulin (CRT) were measured in 9 patients. It could be shown that decreased expression of HMGB1 and increased expression of extracellular HSP70 and CRT in the relapse appeared to be beneficial for patient survival (Muth C et al. 2015). In DoReMi, a holistic approach has

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been proposed for IR-induced responses including DNA damage responses (DDR), death signal responses, apoptosis, pro- and anti-inflammatory responses and body wide systemic immune responses including NTE and abscopal effects (Mavragani IV et al. 2016). In particular, danger-signal mediated systemic immune effects of RT are thought to be of great importance in the clinical setting. In the context of combined radiation therapy (RT) and immunotherapy, also the effects of IR on regulatory T cells and the induction of a tumor suppressive phenotype were explored (Persa A et al. 2015). The modulatory action of low and high doses of IR and the implication in benign and malign diseases was examined (Frey B et al. 2015). This truly is likely to improve (high dose) RT protocols by inducing increased anti-tumor immunity. However, some aspects remain controversial, for example, some reports revealed that protracted low dose–IR can result in radio-resistance, others reported immune-suppressive effects of chronic low dose–IR associated with cell killing and sensitization of certain cell types. Apparently, the interaction of dendritic cells and T cells needed further studies (Manda A et al. 2012). Microvesicles and exosomes as cargo carrying messengers Reactive oxygen species (ROS) play an important role in NTE and affect low dose radiation responses involving ROS production and anti-oxidant defenses (such as SOD activity) (Mariotti et al. 2012). Also, reactive nitrogen species (Han W et al. 2007), cytokines (TNF-a etc.) (Schaue D et al. 2012), Cox -2, mitochondrial dysfunction (Spitz DR et al. 2004), intercellular communication through intercellular gap junctions (Azzam EJ et al. 2001), calcium ion flux changes (Campa A et al. 2015) as well as the release of microvesicles (exosomes) (Al-Mayah AH et al. 2012) from cells were shown to be involved in NTE. During DoReMi, research on mechanistic aspects of non-targeted effects was thus extended to research on small membrane microvesicles (exosomes) that are secreted by cells to the extracellular environment and can be taken up by neighboring recipient cells (Al-Mayah AH et al. 2015). In this way, exosomes are involved in modulating radiation-induced bystander signaling in human cells (Jella KK et al. 2014). The exosomes can carry as cargo RNA and proteins (Al-Mayah AH et al. 2015). In the DoReMi project, it could be shown that exosomes from irradiated cell media are relevant for the induction of chromosomal instability in vivo in a C57BL/6 mouse model. The presence of the TSG101 protein indicates the presence of microvesicles in unirradiated and irradiated mice. Moreover, extracellular vesicles (exosomes) derived from bone marrow of C57BL/6 mice could be labeled with technetium-99m isotope and their biodistribution be followed in mice in vivo. The microvesicles biodistribution analysis indicates the presence of the bystander microvesicles (after injection of microvesicles from irradiated mice) in the spleen and bone marrow of the injected mice. The uptake of such exosomes after IR by bystander cells is expected to cause cellular dysfunctions. Radiation increased the fraction of NK cells in the spleen in a dose dependent manner, a similar trend was observed in microvesicle treated bystander mice. The microvesicle cargos are in the process to be further characterized. Modeling of IR-induced cell-to-cell interactions leading to the induction of apoptosis and inhibition of proliferation of pre-transformed cells by the signaling from irradiated normal neighboring cells has shown that the tissue microenvironment can modify the pathogenic process (Kundrat et al. 2015, Babini G et al. 2015c).

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Apparently, NTE can also modulate low dose health risks since some epidemiological data sets are better explained by clonal expansion models that take into account bystander effects (for IR induced lung cancer) or genomic instability (for IR induced breast cancer) (Eidemüller et al. 2012, 2015).

4.2 Studies on induction of non-cancer effects: research on mechanisms and epidemiological approaches, conclusions, perspectives

4.2.1 Studies concerning the cardiovascular system and cardiovascular disease (CVD)

Radiation quality specific responses (see question 1) (CVD) Within DoReMi this question was not addressed.

4.2.2 Radiation dose rate specific responses (see question 2) (CVD)

Chronic exposures in vitro on CVD

Since epidemiological data suggested that cardiovascular diseases could be induced by ionizing radiation at lower exposure levels than previously thought DoReMi addressed the question of the effects of low dose rates on the cardiovascular system (Little MP et al. 2010; Shimizu Y et al. 2010). The endothelial cells covering the inner surface of the vascular system and the heart are considered to be critical targets in radiation-induced cardiovascular disease (Schultz-Hector S and Trott KR 2007). Using human umbilical vein endothelial cells, HUVEC and its immortalized derivative EA.hy926 cell line, it was shown that acute low doses of X-rays (50 mGy) induce DNA damage (DNA double-strand breaks, DSB) and apoptosis in endothelial cells (Rombouts C et al. 2013). Curiously, more DSB/Gy were formed after low dose irradiation, and a dose-dependent increase in apoptotic cells down 0.5 Gy in HUVEC and 0.1 Gy in EA.hy926 cells. Also, the phenomena of low-dose rate induced premature senescence in endothelial (HUVEC) cells was analyzed in some detail (Yentrapalli R et al. 2013b). The appearance of senescence-associated molecular markers such as -galactosidase and p21 was tested. Interestingly, at low dose-rate of 1.4 mGy/h no decrease in growth rate or increase of -galactosidase was observed in spite of increased expression of p21. However, at a slightly higher dose-rate (2.4 mGy/h) clear premature senescence was induced as shown by reduced growth rate and induction of -galactosidase. This clearly pointed to the existence of non-linear effects at low-dose rate exposures. Gene expression analysis and proteomic analysis (Yentrapalli R et al. 2013a,b) showed that senescence-related cellular pathways were influenced: cytoskeletal organization, cell-cell communication, adhesion and inflammatory pathways, together with an activation of the p53/p21 pathway reducing the replicative potential and promoting senescence. This was also accompanied by a reduction of the PI3K/Akt/mTOR pathway, reduced expression of cytoskeletal structures and EIF2 signaling. Endothelial cell senescence was also found age-dependent. Therefore, these results support the hypothesis that the increased rate of cardiovascular disease seen in populations chronically exposed to low-dose rate radiation may involve molecular processes that are quite similar to age-related endothelial senescence in humans.

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Striking differences were observed in these cells when comparing exposures to low dose-rates 4.1 mGy/h to 1.4 mGy/h exposures. At the same total dose (2 Gy), there was a clear increase in the release of pro-inflammatory cytokines IL-6 and MCP-1 at 4.1 Gy/h together with a decreased capacity for vascular network formation, which was absent at 1.4 mGy/h. This clearly revealed the existence of non-linear effects following low-dose rate exposures (Ebrahimian T et al. 2015).

Moreover, using the same cell system and dose-rates of 1.4 and 4.1 mGy/h, an early stress response (involving insulin-like growth factor binding protein 5 (IGFBP5)) was observed after exposure to 4.1 mGy/h likely thought to trigger the observed premature senescence at 4.1 mGy/h but not seen at 1.4 mGy/h (Rombouts C et al. 2014). This underscores the existence of non-linear effects at low dose-rate exposures in these endothelial cells. Mechanistically, it is of interest that also a non-linear induction of DSB (revealed by H2AX foci) induction) by X-irradiation (with a window of 0.5-0.7Gy) was observed in endothelial cell lines involving non-linear production of ROS and SOD activity (Large M et al. 2014). Low dose fractionated exposures in RT

Using repeated exposures to an even lower dose (10 mGy) persistent DNA damage (DSB) was found in irradiated mice in the lung (bronchiolar and alveolar cells), the heart (cardiomyocytes) and the brain (cortical neurons) (Schanz S et al. 2014). Interestingly, for some pathological conditions beneficial effects of fractionated moderate dose radiation exposures could be observed in clinical settings. In optimized clinical trials (although not statistically significant because of low patient numbers involved) Ott OJ et al. (2012a,b; 2013ab; 2014ab; 2015) showed that a moderate dose (0.5 Gy) fractionated RT (6 fractions in 3 weeks) is an effective treatment for benign elbow syndrome (Ott OJ et al. 2012ab, 2014b), benign calcaneodynia (Ott OJ et al. 2013b, 2014a), and achillodynia (Ott OJ et al. 2013a; 2015). 4.2.3 Tissue specific responses (see question 3)(CVD)

Clear evidence has been obtained within DoReMi that low-dose and low-dose rate IR can induce premature senescence in endothelial cells, which are part of the cardiovascular system. In fact, dysfunction of endothelial cells plays an important role in the development and progression of CVD, in particular atherosclerosis (Versari D. et al 2009). High dose exposures are known to affect endothelial cell function (Gaugler MH 2005), and low doses are suspected to induce anti-inflammatory effects leading to radiation-induced inhibition of leukocyte adhesion to endothelial cells in vitro (Hildebrandt et al. 2007). In DoReMI, acute low dose X-irradiation was shown to induce a dose dependent increase apoptosis in endothelial cells (HUVEC) down to 0.5 Gy as well as a non-linear dose response for the induction of DSB (Rombouts C et al. 2013). Furthermore, a non-linear low dose-rate effect was observed (with significant difference between 1.4 mGy/h and 4.1 mGy/h exposures to γ-rays) for the induction of premature senescence in those cells (Yentrapalli R et al. 2013a,b).

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It appears that the increased rate of cardiovascular diseases seen in chronically low dose exposed population is likely to involve processes that mimic age-related endothelial senescence in humans. (Yentrapalli R et al. 2013a,b). 4.2.4 Modifications of risk by genetic and epigenetic factors and gender (see

question 4) (CVD)

Gene expression studies revealed that inactivation of the p53/p21 pathway (affecting cell replication) and the phosphoinositidyl 3-kinase with the PI3K/Akt/mTOR pathway (Yentrapalli R et al. 2013a,b) as well as IGFBP5 gene expression (Rombouts C et al. 2014) are involved in the induction of premature senescence in endothelial (HUVEC) cells. Furthermore, telomere shortening (which indicates premature cell-aging) has been proposed as a new prognostic factor for IR induced cardiovascular diseases arising after RT of Hodgkin lymphoma (M’Kacher R et al. 2015). A recent proteomic study and microRNA analysis of endothelial replicative senescence showed that also several non-coding RNAs are involved in the regulation of metabolism, cell cycle progression and cytoskeletal organization during the process of endothelial senescence (Yentrapalli R et al. 2015). An extended study showed that high LET irradiation (accelerated nickel ions at 183 keV/mm) at moderate 0.5 Gy to high doses of 2 and 5 Gy induced persistent DNA damage response accompanied by the down regulation of some cell recycle control genes, and an upregulation of several genes affecting DDR, oxidative stress, apoptosis and intercellular signaling (Beck M et al. 2014). Further analysis of the gene sets suggested the involvement of important

-κB is triggered by ATM (activated by DSB), and danger signals are released from dying cells that activate NF-κB via Toll-like receptors. The resulting activation of the immune system can be either detrimental or anti-carcinogenic (Hellweg CE 2015). The NF-B essential modulator (NEMO) may act as a critical switch for regulating cellular senescence and apoptosis after exposure to IR (Dong X et al. 2015). Several lines of evidence also indicate the importance of mitochondrial functions in IR –induced effects on the cardiovascular system (Barjaktarovic Z et al. 2011, 2013, Dong CX et al. 2015) and proteome alterations in long term vascular dysfunction (Azimzadeh O et al. 2015).

Taking these results together (chapters 5.1.1-5.1.4), it appears that IR exposures at low and low dose rate may affect the normal functions of endothelial cells, an effect likely to be of importance in out-of-field radiation effects during RT as well as in hadron therapy or long term space mission exposures (Beck et al. 2014). Low dose IR effects were analysed in Fibulin-4 deficient mice mimicking human aneurysm, a primary cause of degeneration of the aortic wall, dilatation of the aorta, risk of rupture and death. The aortic diameter changes induced were assessed by using a special designed microCT equipment (Quantum FX mCT) for low dose irradiation of mice. Low dose CT exposures did not induce significant changes in aortic diameter in aneurysmal fibulin-4 mutant mice. In the frame of the ELDoREndo (2013-2015) project, the effects of low doses of X-rays on the structure and function of vascular endothelium in diabetes prone mice before

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and during the development of hyperglycemia were analysed knowing that diabetes is often associated with vascular complications. The animals were exposed to five whole body irradiations (WBI) at 0.002, 0.02, or 0.1 Gy of X-rays per day for five days, so that the total absorbed doses equaled to 0.01, 0.1 , or 0.5 Gy per mouse.

The results obtained in the ELDoREndo project so far suggest that: db/db mice display hyperglycemia associated with alterations in lipid profile

(elevated total cholesterol and triglycerides), inflammation (elevated TNF-α, decreased IL-4), endothelial dysfunction (impaired NO-dependent function, elevated endothelin-1, reduced thrombomodulin).

Whole body irradiation at the tested low doses of X-rays does not induce significant and consistent effects of any of the measured parameters in diabetic (db/db) mice.

If there is any beneficial effect of low doses of low-LET radiation, it can be related only to a total of 0.05 Gy and to the effects on early diabetes that showed some tendency for improvement that was, however, not consistent and in most cases not significant.

Still, the possible beneficial effects of low doses of low-LET radiation on endothelial dysfunction in diabetes need to be confirmed by further studies using a) considerably larger experimental groups (at least 25 animals per group) and b) different exposure schemes (including chronic irradiation for e.g., 4-8 weeks) performed at the facilities now available at some of the DoReMi partners’ institutions.

4.2.5 Effects of age (see question 5)

Within DoReMi this question was not explicitly addressed. 4.2.6 Effects of lifestyle (see question 6)

Within DoReMi this question was not explicitly addressed. 4.2.7 Effects of physiological state (see question 7)

Within DoReMi this question was not explicitly addressed.

4.2.8 Hereditary effects (question 8)

Within DoReMi this question was not explicitly addressed.

4.2.9 Non-targeted and immunological effects (question 9) CVD

Several studies revealed the importance of differences in the reactivity of immunological active cells following IR (Rödel F et al. 2012 a, b). Immunological processes such as leukocyte/endothelial cell adhesion, adhesion molecule and cytokine/chemokine expression, apoptosis induction, and mononuclear/polymorphonuclear cell metabolism and activity could be modulated (Rödel F et al. 2012).

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Of immunological importance is also the fact that low and moderate doses of ionizing radiation up to 2 Gy modulated transmigration and chemotaxis of activated macrophages producing anti-inflammatory reactions but had no impact on viability and phagocytic functions of the macrophages (Wunderlich R et al. 2015). Studies on contribution of low dose X-radiation in induction of anti-inflammation proved that X-radiation does modulate the inflammatory phenotype of immune cells such as activated macrophages and endothelial cells (EC) and attenuate inflammation in a non-linear dose response relationship. Generally speaking, this clearly indicates that modulation of inflammation by low and high doses of IR has important implications for benign as well as malign diseases (Frey B et al. 2012). Low dose X-radiation induced a NF-κB dependent decreased secretion of active IL-1β by activated macrophages with a discontinuous dose-dependency. This was most pronounced at the moderate doses of 0.5Gy and 0.7Gy (Frischholz B et al. 2013). Low dose X-radiation further modulated transmigration and chemotaxis of activated macrophages and provoked an anti-inflammatory cytokine milieu, even at 0.01Gy, and in particular, reduction of IL-1β release. Interestingly, this did not impact on macrophage viability and phagocytic function (Wunderlich et al. 2015). In particular, the reduced secretion of the pro-inflammatory cytokine IL-1β correlated with reduced nuclear translocation of NF-B p65 starting at exposures to 0.5 Gy of X-irradiation. Such moderate doses (0.5 and 0.7 Gy) in a non-linear dose response relationship (with IL-1 upregulated, TGF- down regulated) attenuate inflammation (Lödermann B et al. 2012). In this context, it is of interest that also locally restricted moderate dose exposure of TNF-alpha transgenic mice with 0.5 Gy reduced bone-loss and inflammation in vivo (Deloch L et al. 2014) indicating an abscopal effect. At high doses (5 Gy), however, gamma –ray induced inflammatory responses in human skin fibroblasts involving NF-B were shown to arise more from culturing conditions than from radiation exposures (Babini G et al. 2015). Regarding endothelial cells, a non-linear regulation of reactive oxygen (ROS) production and superoxide dismutase activity in EC following irradiation with doses <1Gy may contribute to a discontinuous dose-response relationship of residual γH2AX foci detection. Further, a biphasic regulation of the anti-oxidative system in EC cells was observed following low dose X-radiation and again especially the intermediate dose of 0.5Gy resulted in the most pronounced effects. These mechanisms might contribute to anti-inflammatory effects in stimulated EC (Large M et al. 2014). In a follow-up study, a moderate dose (0.5 Gy) of X-irradiation resulted in a non-linear expression and activity of the anti-oxidative factor glutathione peroxidase (GPx) and the transcription factor nuclear factor E2-related factor 2 (Nrf2) in human endothelial cells that might exert anti-inflammatory effects in stimulated endothelial cells (Large M et al. 2015). Thus, the anti-oxidative system plays an important role in the IR response of endothelial cells. In fact, a biphasic regulation pattern could be demonstrated (Large M et al. 2015). In line with this, also chronic internal exposures to low doses of 137Cs impacted positively on the stability of atherosclerotic plaques by reducing inflammation in atherosclerosis pre-disposed ApoE-/- mice (Le Gallic C et al. 2015). On the other hand, chronic γ-irradiation was shown to cause a dose-rate dependent pro-inflammatory response and associated loss of function in human umbilical vein endothelial cells. This

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indicates that the dose-rate of IR is an important parameter that can alter the inflammatory profile of endothelial cells in vitro (Ebrahimian T et al. 2015). Profile changes included expression levels of adhesion molecules (E-selectin, ICAM-1 and VCAM-1) as well as the release of pro-inflammatory cytokines such as MCP-1 and IL-6). This dose-rate effect is likely to have an important bearing when considering the induction of cardiovascular diseases by IR in humans. In examination of changes in the immune system regarding the mouse T cell repertoire by high throughput DNA sequencing after in vivo IR exposure, a greater and longer-lived reduction in the TCR repertoire was found after acute 0.1 Gy exposure than after 1 Gy exposure. This also clearly indicates a non-linear radiation response. Thus, the development and/or homeostasis of T lymphocytes are affected by low dose radiation. However, no effects in terms of genetic instability (detected by illegitimate TCR rearrangements were observed 1 month after acute 0.1 Gy or 1 Gy exposures and after chronic exposures 0.2Gy or 1 Gy/3 weeks exposure of mice in vivo (see DoReMi MIRACLE, extension in collaboration with DoReMi OSTINATO,) were observed by now. From this, the interesting question arises if TCR trans-arrangements could be used as biomarkers for IR exposures. One can conclude that low to moderate dose X-radiation does result in attenuation of inflammation, and these immune modulations can be considered as being in particular non-targeted irradiation effects. Low dose X-radiation thereby modulates endothelial cell and macrophage activity and consecutively many non-cancer inflammatory conditions and may also impact on carcinogenesis by modulating the inflammatory microenvironment. 4.3 Lens opacities

Soon after the discovery of ionizing radiation the risk for developing cataracts after exposure to radiation has been recognized (Chalupecky H. 1897, Markiewicz E et al. 2015). The lens of the eye is one of the most radiosensitive tissues in the human body. Also, mouse lens epithelial cells were found more sensitive to -irradiation than lymphocytes (Bannik K et al. 2013). Cataracts could be induced by acute doses of less than 2 Gy of low-LET radiation or less than 5 Gy of protracted radiation (Ainsbury EA et al. 2009). The induction of posterior subcapsular cataracts was classified as a typical deterministic effect (Edwards AA and Lloyd DC 1998) with a threshold of approximately 2Gy (Ainsbury EA et al. 2009). However, there is evidence from the Japanese atomic bomb survivors, Chernobyl liquidators, US astronauts and other occupational exposures (see review by Little MP 2013 and Hammer GP et al. 2013) that also cortical cataracts may be associated with IR exposure, whereas nuclear cataracts are considered non radiogenic. These data suggested a threshold for radiation-induced cataractogenesis of 0.5 Gy. The International Commission on Radiological Protection (ICRP) (Stewart FA et al. 2012) recommended an occupational equivalent dose limit of 0.02 Sv per year (averaged over 5 years, with no single year exceeding 50 mSv) to prevent IR induced cataracts. However, solid dose-response data and mechanistic data for low doses (<O.5 Gy) were still lacking, and there is a discussion whether or not cataractogenesis should be considered as a deterministic or stochastic phenomenon (Hammer GP et al. 2013, Hamada N et al. 2014 a, b). Dose effects on lens opacities (see question 1)

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In order to explore lens opacities among a small cohort of interventional radiologists and cardiologists, a study (O’CLOC, 2010-2012) on interventional radiologists vs. unexposed individuals was performed. For all participants, information regarding the risk factors for lens opacities was collected; for exposed people, information regarding the description of type and frequency of IC procedures and the radiation protection material used was also collected. In all participants, a slit lamp examination was performed to determine when diagnosed the LOCS III classification of the detected lens opacity. This study found that the interventional radiologists have significant higher risk of developing a posterior subcapsular lens opacity compared to the unexposed group: among 106 interventional cardiologists (and 99 unexposed individuals), 17% (versus 5%) had developed post subcapsular cataracts after mean accumulated doses of 423 mSv (ranging from 25 mSv to 1658 mSv) (Jacob S. et al. 2013b) This result wasn’t found for nuclear and cortical lens opacities. Eye lens opacities were thus shown to be an urgent matter in radiation protection knowing that about 20% of interventional cardiologists had received more than 500 mSv. As a follow-up of the O’CLOC study, a method has been developed within the ELDO project (2012-2013) to estimate cumulative eye lens doses for past practices based on personal dose equivalent values, Hp (10), determined above the lead apron at the collar, chest and waist levels (Farah J et al. 2013). Using anthropolymorphic phantoms it could be shown that eye lens doses correlate best with Hp (10) measured on the left side of the phantom at the level of the collar. In spite of a relatively high spread of the correlative dose estimates when using chest-left whole body dose measurements, the method appears to be very useful for first order of magnitude retrospective eye dose assessments in individuals exposed to high exposure levels. For estimating cumulative eye lens doses for past practices, the evolution of the X-ray systems and procedures and their effects on eye lens dose have to be taken into account. Thus, health professionals should benefit from wearing specific eye dosimeters for accurate dose assessments. Both methodologies increase the precision of epidemiological studies on cataract development in accidentally or professionally exposed individuals. The methodology developed in the ELDO project served the implementation of a pilot epidemiological study (EVAMET project, 2013-2015) of lens opacities within a group of 69 Polish interventional cardiologists and 23 controls was performed (Domienik J et al. 2016). Regarding the analysis of data from occupational questionnaire the results showed clearly which kind of data are already available and which kind of data are still lacking to evaluate as precisely as possible cumulative eye lens doses. A reasonable data concerning the frequency of various procedures, access type, geometry of X-ray tube and the use of radiation protective devices were obtained. In view of the above, in order to provide the credible estimation of cumulative eye lens doses extra information was needed. It concerned the information, still lacking in the literature, about the dose per procedure for less common, but possibly contributing significantly to the eye lens dose, procedures and not included in the previous studies analyzing the cumulated eye lens doses. Also the correction factors for the use of various protective devices (lead glasses and ceiling lead glass) and different geometries (X-ray tube configuration, the use of bi-plane systems) are important and necessary when the direct doses on eyes are not at our disposal. All information presented above allowed to plan extra measurements which help, first, in assessing the eye lens doses for new scenarios (new procedures,

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geometries, etc. for which a little amount of data or no data at all are available) and, then, in estimating the cumulative eye lens dose. The latter ranged up to 1.55 Sv and 0.4 Sv for left and right eye, respectively (Domienik J et al. 2016). The EVAMET project aimed also to validate the two approaches developed in the ELDO project for retrospective assessment of cumulative eye dose lens and evaluation of corresponding uncertainties. To achieve this objective, the thorough measurement program was set up in routine practice with recruited interventional cardiologists. Every physician was asked to wear two dosimeters during each procedure he/she has been performing for at least two months (one measurement period): one near the eye lens which is closer to the X-ray tube and another one on the chest also on the side which is closer to the X-ray tube. Concerning the approach based on the whole body measurements, in total the data (Hp(10) and Hp(3) doses) from 33 measurement periods were collected. For individual data set the equivalent eye lens doses Hp(3) were calculated based on the reading from Hp(10) dosimeter and ELDO coefficients specific for the position of Hp(3) and Hp(10) dosimeter during the measurements. The results were then compared with the Hp(3) dose measured in the same time period. The relative differences (RF) calculated as the difference between the measured and calculated Hp(3) values changed from +50% up to -190% of the measured dose. In 70% measurements the calculated dose overestimated the measured one. Concerning the approach based on the questionnaire and the dose data for single procedure, the validation of the second method based on the data from the questionnaire was performed for 25 physicians who worn the EYE-D dosimeter. The cumulative annual eye lens dose was calculated on the basis of occupational data from the questionnaire (concerning the mean number of procedures performed annually, the percentage of procedures performed with the use of ceiling suspended shield, position of the X-ray tube (above or below) and in the case of coronary interventions the access type (radial or femoral) and the input data. The latter included the doses per single procedure of different type collected by NIOM during ORAMED project and later and various correction factors derived in parallel project launched in Poland. In the first attempt the mean doses per procedure were used. In the next step the calculated doses were compared with the measured ones extrapolated to the same time period (one year). The maximum relative difference (the difference between the measured and calculated dose) were about -550% of the measured dose which means that in the most extreme case the calculated dose overestimates the measured one by about 6.5 times. Also, in DoReMi a study on the contribution of low dose X-radiation in induction of cataractogenesis and influencing genetic and cell communication factors was started (LDR-OPTI-GEN) in 2013, and a mouse in vivo study on low dose radiation-induced cataracts (RadCat) in 2014 . The objectives of the LDR-OPTI-GEN study were to determine the low dose responses and possible dose threshold of the induction of lens opacities in X-ray irradiated lens epithelial cells. Effects of dose rate on lens opacities (see question 2)

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In DoReMi the effects of dose rate on radiation-induced lens opacities (cataracts) was

not specifically addressed.

Effects of tissue sensitivity (see question 3) The LDR-OPTI-Gen study an ultrastructural analysis has been performed through transmission electron microscopy on several donor (1,2 and 4) cell lines. However no changes in morphology were observed in any of the irradiated groups, including nuclear integrity and organelle number. Also, in the three donor cells lines DNA damage response and repair kinetics were analyzed using -H2AX assay (Rogakou et al., 1998). The results showed that there is no significant DNA damage at doses below 0.02Gy, however there is a clear dose-dependent increase in DNA damage above this dose. Analysis of the repair kinetics in all three donors showed that the cells repair damage effectively especially, at the lower range of doses, 0.02Gy and 0.2Gy. Were further investigated telomere length differences in delayed samples, which means growing them for as many passages as possible. The results showed a dose dependent increase in telomere length in the delayed samples too. It was also seen that telomere length increases after cells have been incubated till confluence, i.e. next passage. Delayed samples showed longer telomeres compared to samples irradiated and analyzed 30mins after radiation. Effects of genetic and/or epigenetic factors (see question 4)

In the LDR-OPTI-GEN study it was thought important to follow the development and the delayed induction of radiation responses by analysing telomerase activity, telomere length and telomere functions down to doses as low as 1 mGy, and to determine the role of genetic predisposition in radiation-induced lens opacities. In the RadCat study, a lifetime study was implemented in groups of mice. Mice were sacrificed at the scheduled time points (4 hrs, 24 hrs, 12 month, 18 month and 24 month post-irradiation). The eyes were embedded in plastic for histological and immuno-histochemical analysis. Individual bone marrow samples from the different time-points were collected for chromosomal analysis and telomere length measurement. According to the preliminary results of the RadCat study, even at the highest dose of 0.5 Gy no differences were observed in lens density among the groups (irradiated vs. non-irradiated, females vs. males or wild types vs. heterozygous mutants). In contrast, the retinae of the irradiated (0.5 Gy) mutant mice were statistically significantly thinner than the non-irradiated controls; this effect was not seen in the wild-type mice. Cytogenetic analysis for all types of chromosomal aberrations were completed for the 4 hrs, 24 hrs, 12 month & 18 month time-points for all doses where possible (exception 18 month 0.063 Gy) and for both sexes and genotypes. For the majority of samples, 50 metaphases have been analysed after Giemsa staining. Lens epithelial cells were also cultured for micronuclei analysis. However, not enough cells could be scored to make

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any meaningful conclusions. The overall trend in results for chromosomal analysis showed an increase in aberrations particularly after 0.5 Gy 4 hours post irradiation. There were also increases in all delayed groups (12 and 18 months). A dose response was observed in the majority of samples (wild type and mutant mice) at 4 hrs following 0.5Gy irradiation. Moreover, the mutants (male & female) irradiated by 0.063 Gy demonstrated a higher number of chromosomal aberrations than expected from a linear dose-response relationship. However, by 24 hrs, damage levels had returned to near background levels in all groups with exception of female wild type. In contrast, at both 12 and 18 month time-points, the frequency of aberrations was again higher in all groups compared to the untreated control counterparts with exception of female wild type, with highest levels observed following 0.5 Gy irradiation. Telomere length using IQ-FISH was measured in bone marrow samples irradiated (0Gy, 0.063Gy, 0.125Gy and 0.5Gy) samples at time points; 4 hours, 24 hours, 12 months and 18 months. Both wildtype and mutant Ercc2 mutant mice, male and female samples were measured at the above-mentioned time points. The preliminary results showed telomere length differences between samples analysed 4 hours after irradiation and 24 hours after irradiation. The samples showed that telomere length decreases after 24 hours of irradiation in both female and male samples. When comparing the mutant and wild-type samples a decrease in telomere length was seen in samples 24 hours after irradiation.

Effects of age (see question 5) This was not explicitly studied in DoReMi,. However, some results obtained show that lens epithelial cells express the enzyme telomerase, and the activity of this enzyme is reduced after irradiation in a dose-dependent manner. Interestigly, these cells show an increase in telomere length with age, a trend that gets reversed after exposure of cells to doses above 0.02 Gy. Also, telomerase activity is present in HLE cells, and telomere length increases both dose dependently and after each culturing passage. HLE cells have effective repair ability, especially at lower doses of 0.02Gy and 0.2Gy. Effects of lifestyle (see question 6)

Within DoReMi this question was not explicitly addressed. Effects of physiological state (see question 7)

Within DoReMi this question was not explicitly addressed. Hereditary effects (see question 8)

Within DoReMi this question was not explicitly addressed. Effects of non-targeted and immunological effects (see question 9)

In the LDR-OPTI-GEN study, three human lens epithelial donor cell lines were characterised in terms of their response to the low dose ranges mentioned above. Media transfer bystander experiments involving HLE to HLE media transfer and HLE to fibroblast transfer were also established. This was also performed in the reverse

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manner, i.e. fibroblast to HLE transfer. Preliminary results indicated that 0.001 Gy promotes the secretion of cytotoxic signals that are active in fibroblasts as well as in HLE cells of the same genotype. Donors 2 and 3 appeared to be most sensitive to radiation for a number of endpoints:

Donor 2 showed delayed changes in CD49c expression, which has been associated with posterior subscapular cataracts at high doses (1 and 2 Gy).

Donor 3 showed delayed changes in CD49c expression although this occurred at lower doses.

Both donors also showed an increase in ROS present at the delayed time point (approximately 10 days post irradiation), this occurred mainly at higher doses although changes were still observed at a dose as low as 0.02 Gy, although no reduction in viability was associated with this.

In any case, the low dose radiation-induced lens opacity remains an important issue in radiation protection (Bouffler SD et al. 2015).

4.4 Neurological effects of low and moderate doses of ionizing radiation

Out of the 9 questions, within DoReMi, only a few were addressed because mostly involving long-term animal studies and low to high doses. Reference to the effects of radiation doses (see question 1).

High doses of ionising radiation are known to affect hippocampus neurogenesis and cognitive functions (learning and memory) (Kim JS et al. 2013). There are epidemiological data that support the notion that also low doses may induce damage to the brain and cognitive impairment, particularly in children (Pearce M et al. 2012, Bernier MV et al. 2012). In mice exposure to -rays during development gave rise to neurobehavioral effects (Eriksson P 2010). Indeed, there is a general concern about the possible long-term effects of low dose exposures to the brain (Kempf SJ et al. 2013). More recently, it has been shown that cognitive effects of neonatally irradiated mice are accompanied by changed plasticity, adult neurogenesis and neuroinflammation (Kempf SJ et al. 2014) and that low dose IR affects mitochondria and synaptic signaling pathways in the murine hippocampus and cortex (Kempf SJ et al. 2015). Verreet T et al. 2015 revealed short and long term effects of X-irradiation in terms of gene expression profiling and immunohistochemical alterations after IR exposure during early mouse brain development. When assessing the long term behavioral effects of 1 Gy X-ray exposures in mice, it appeared that the effects observed reflect initially induced DNA damage, apoptosis and inflammation. However, a direct link between early-induced apoptosis in the embryonic brain and structural and behavioral alterations in the adult could not be established. In the irradiated embryos total brain volume, cortical thickness and ventricle size were decreased 24 hours after exposure. In the adult mice (40 weeks after exposure a decrease in neural cell number and persistent neuroinflammation was seen. In the neurocampus; neurogenic cell proliferation was reduced (Verret T et al. 2015). In order to understand possible adverse effects of computer tomography (CT scans) experiments were performed using acute low (0.1 Gy) and moderate O.5 Gy) doses of IR and female mice on postnatal day 10 (Kempf SJ et al. 2015). The analysis focused on the

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cellular proteome, the transcriptome, and neurological development/disease and on miRNAome analysis of the hippocampus and cortex assed 24 h after irradiation. Signaling pathways related to mitochondrial and synaptic functions were altered. At the same time, dendric spines and neurite outgrowth was enhanced in the cortex at 0.1 and 0.5 Gy, but in the hippocampus only at 0.5 Gy. Furthermore, the expression of several neural mRNAs associated with neural plasticity was increased. It is thought that these early changes in signaling pathways related to memory formation may be associated with neurocognitive side effects observed in patients after brain RT. Within DoReMi, the response of the developing brain to ionising radiation was analysed after in utero irradiation of mice in some detail. In particular, it was shown that X-irradiation inhibited neurite outgrowth and impaired synaptogenesis and neurotransmission. In short, external in utero irradiation leads to neuronal death and affects neurite length in primary neuronal cultures. Prolongation of neurites is reduced following irradiation in early stages even at low doses (0.1 Gy). Irradiation at later stages leads to defects in the generation of axonal branches. Dose rate effects (see question 2)

Seeking a better understanding of the possible role of IR in the induction or promotion of neurological diseases, the influence of a chronic low dose and low dose rate exposure onto the development of Parkinson symptoms in genetically predisposed Pitx3-EYL/EYL Ogg1-/- mouse mutant was also studied (Graupner A et al. 2015) using the new FIGARO chronic irradiation infrastructure. The possible protecting effect of the antioxidant selenium was tested as well. In particular, the effects of low dose rate on blood from chronic low dose gamma irradiated mice C57BL/6 mice versus Ogg1-:- mice (deficient in oxidative lesions repair such as 8-oxo Gua repair). 1.4 mGy/h for 45 days with Se diet (0.01 ppm) +/- 45 days repair were assessed (Graupner A et al. 2015). The results showed that chronic γ-irradiation increases genotoxic effects (rates of micronuclei formation and phenotypic mutations (RBCCD24-) and chromosome fragmentation. Moreover, DNA lesions and Pig-a mutations induced were reduced in Ogg+/- mice heterozygous for Ogg1 (a gene coding for an enzyme (glycosylase) involved in the repair of oxidative DNA damage), induction of a protective response after chronic 45-day -irradiation. Non-irradiated mice on low Se diet showed more mutagenic effects than mice on normal Se diet. Apparently, Se diet was protective for irradiated mice. Similar effects were observed in irradiated Ogg+/- mice as in non-irradiated control mice. This was taken as a sign for hormesis (Graupner A et al. 2015). Interestingly, after 45 days of exposure of mice at 1.41 mGy/h (total dose of 1.48 Gy) there was an increase in mutation and MN but not in DNA single strand breaks (ssb/alkaline labile sites). Surprisingly, chronic exposure did not affect the Ogg-/- mice (homozygous for Ogg1, deficient for repair of oxidative damage) in terms of mutation and MN induction but in terms of induction of oxidative DNA damage (ssb/alkaline sensitive sites). In fact, ssb/als increased with time (45 days) in non-irradiated mice significantly but not in irradiated mice Ogg1-/- mice supporting a non-linear threshold model (Graupner A et al. 2015). The effects on tissue sensitivity (see question 3)

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The sensitivity of neural cells, and in particular, of immature neurons is an important question when trying to understand the induction of neurodegenerative diseases by external stressors such an ionizing radiation. Recently, it has been shown that low doses ranging from 0.2 to 0.6 Gy of X-rays elicits a clear apoptotic response that could be prevented by treatments with dizocilpine or calpain inhibitor. This demonstrated that N-methyl D-aspartate (NMDA) receptors are involved in IR-induced neuronal death causing impairment of the neuronal network and cognitive capacities (Samari N et al. 2013). Neural stem cells (NSCs) were found relatively resistant to oncogenesis from medium dose exposure (0.5 Gy) as compared to high dose exposure (3 Gy). Concerning neural stem cells they are regarded as highly susceptible to IR-induced DNA damage and cell death. Using the patched 1 heterozygous mouse model (Ptc11/2), highly susceptible for the induction of medulloblastoma, striking differences in DNA damage responses and apoptosis between NSCs and progenitor cells including lack of p21 in NSCs (Tanori M et al. 2013). Age at exposure was important pointing to differentiation-stage specific differences in DNA repair pathways used in NSCs and progenitor cells to assure genomic integrity during neurogenesis. This has an important bearing on radiation risk assessment for IR induced brain damage. A particular cell type, the pericytes constitute an important microvascular component of the brain. Within DoReMi, a study was launched on low dose induced molecular and functional alterations in this system. In particular, the effects of low and moderate doses on the phenotype of pericytes, and their role in brain inflammation (age and pre-inflammatory status dependency) were investigated. IR exposures of 10-day-old mice induced a decrease in differentiated phenotype and an increase in the expression of a mesenchymal stem-cell like phenotype. Apparently, the immunological characteristics of pericytes in acute, septic shock-type inflammation (induced by LPS) were not influenced by IR. However, IR decreased the expression of inflammatory markers and cytokine expression on pericytes of SJL mice (with a genetic background predisposing for chronic autoimmune inflammation). Concerning the effects of internal contamination quite a detailed study has been carried out on cell proliferation and cell death induction during prenatal and postnatal brain development in rat after uranium exposure (Legrand M et al. 2015). As shown previously in studies on rats, depleted uranium exhibits its heavy metal chemical toxicity not only in the kidneys but also in the brain (Lestaevel P et al. 2005). Moreover, it was shown that chronic oral exposure to enriched uranium (highly radioactive) affected the nervous system and led to increase in paradoxical sleep, reduction of spatial working memory and an increase in anxiety (Houpert P et al. 2005). Within DoReMi Legrand M et al. (2015) analysed the cellular changes after uranium contamination in rats at different stages of development in neurogenic zones (embryonic and postnatal ages) in vivo and in vitro. They found in vivo no major changes in CNS morphology or organogenesis deficits in the CNS after uranium contamination. However, cell proliferation appeared to be affected after U contamination at E13 (hippocampus neuroepithelium) and PND21 (Hippocampus). Experiments in vitro using the neurosphere and differentiation assay showed that stem cells from contaminated animals may retain their properties to form clones and their multipotential properties, but both cell proliferation and differentiation are clearly disturbed during brain

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development in neurogenic zones after uranium contamination (Legrand M et al. 2015). The effects of genetic and epigenetic factors (see question 4)

A detailed gene expression analysis (Quintens R et al. 2015) revealed induction of the P53 pathway dependent genes early after exposure and inhibition of synaptogenesis and cytoskeleton pathways lateron as well as a distinct gene signature that is highly enriched during embryonic and post-natal brain development and during in vitro neuronal differentiation. Several genes were transcriptionally upregulated after irradiation in a p53-dependent manner. The genes were involved in brain-related functions such as neurite outgrowth, focal adhesion dynamics, neuronal differentiation, calcium sensing, and synaptic transmission. (By the way, an automated method for analysing dense neurite network outgrowth (MorphoNeuroNet) has been developed as well (Pani G et al. 2014). The strong overlap between gene expression profiles and phenotypes of prenatally irradiated mice and some neurological disorders (such as the microcephalic Magoh Mos2/+ mice, Silver DL et al. 2010) suggests that the observed changes in gene expression are likely to affect normal brain functions after irradiation of embryonic mice. The radiation-induced changes in the p53 network of the embryonic brain thus appear to be of utmost importance for the developing brain (Quintens R et al. 2015) These alterations are in line with the persistent proteomic changes in synaptic plasticity related signaling in the mouse hippocampus and cortex observed in in utero irradiated mice (Kempf SJ et al. 2015a,b) that appear to involve also deregulation of mitochondrial protein functions. Concerning the dose responses a recent study is also of interest: cultured human neural progenitor cells derived from embryonic stem cells when exposed to low to moderate doses (ranging from 31 to 498 mGy) for radiation administered chronically for 72 hours, dose-dependent gene expression profile changes were observed (Katsura M et al. 2016). These involved at 31 mGy inflammatory pathways (interferon signaling and cell-cell junctions), at 124 mGy DNA repair and cell adhesion molecules, and at 498 mGy nDNA synthesis, apoptosis metabolism and neural differentiation were affected by radiation. Thus, neuronal progenitor cell development is affected at 496 mGy but gene expression is already altered at >100 Gy Katsura M et al. 2016). This is also in line with previously reported neuro-inflammatory responses observed in rats chronically exposed to (137) cesium (Lestaevel P et al. 2008). Also, the profiles of radiation induced proteins and epigenetic markers (miRNAs and long non coding RNAs) differed after low and high dose exposures concerning synaptic signaling pathways in the murine cortex and hippocampus (Kempf SJ et al. 2015a,b,c). In brain pericytes gene expression levels and epigenetic profiles were determined after low doses of IR exposure (0.1 and 2 Gy). Only moderate changes in miRNA expression after 6 and 24 hours were seen after irradiation. The evaluation of DNA methylation levels in pericytes at different post-irradiation times showed a dose-dependent reduction of overall methylation status of pericyte DNA. Also, the methylation pattern of specific genes changed. Interestingly, the repair of IR induced DSBs (as revealed by H2AX) was less efficient at low dose (100 mGy) than at high dose, and long term persistence of residual damage (not dose-dependent) was noticed in pericytes. This is in line with previous findings (Rothkamm and Löbrich 2003) that showed that DNA repair in some cells is less efficient at low than at high dose of IR.

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Regarding more general mechanistic studies on the induction of non cancer effects, the effects of low to higher dose X-radiation over the dose range of 0.01-2 Gy on the induction of anti-inflammation was investigated as well in order to better understand the mechanisms behind, and to describe systemic radiation-induced tissue and whole organism responses and confirm or rebut system biology approaches (see chapter above on immunological effects of low dose IR). In parallel to DoReMi, several other Euratom projects were launched addressing non-

cancer effects: ProCardio, CEREBRAD and Solo.

Effects of age (see question 5)

Within DoReMi this question was not explicitly addressed. Effects of lifestyle (see question 6) Within DoReMi this question was not explicitly addressed. Effects of physiological state (see question 7)

Within DoReMi this question was not explicitly addressed. Hereditary effects (see question 8)

Within DoReMi this question was not explicitly addressed.

Non-targeted and immunological effects (see question 9)

IR-induced modulation of the immune system is also likely to alter physiology and play a role in the pathogenesis of radiation-induced non-cancer effects.. In fact, modulation of inflammatory immune reactions could be induced by moderate doses of IR at 0.5-0.7 Gy in endothelial mononuclear and poly-nuclear cells. Non-linear biphasic responses were observed (Rödel F et al. 2012 a, b). Such immunomodulation and anti-inflammatory activities induced by doses below 1 Gy were also reported in a recent review together with the induction of harmful side effects and high dose induced anti-tumor responses (Rödel F et al. 2015). On the basis of clonogenic assays on different glioblastoma cell lines, it could be shown that fractionated RT stimulates the induction of glioblastoma cell death forms with immunogenic potential (Rubner Y 2014). It was also recognized that RT can also exert beneficial effects on non malignant diseases (Seegenschmiedt MH, Micke O. 2012) and (Ott OJ et al.2012 a,b, 2013 a,b, 2014 a,b,c) Indeed, several positive effects of low doses of IR were also reported in patients with benign painful elbow or shoulder syndrome, achillodynia, calcaneodynia, possibly due to anti-inflammatory and immune modulating IR effects (Ott OJ et al.2012 a,b, 2013 a,b, 2014 a,b,c). Indeed, as proven in these clinical studies, again and in particular the intermediate dose of 0.5Gy efficiently attenuated an existing diseased state in several painful and inflammatory human syndromes such as elbow and shoulder as well as calcaneodynia and achillodynia. All of

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these inflammatory diseases can be managed by local low dose radiotherapy with a single dose of 0.5Gy. 4.5 Development of Biomarkers (see Pernot E et al. 2012, 2014)

Within DoReMi considerable work has been conducted on the testing and validation of potential biomarkers, taking advantage of rapid technological developments and reduction in costs in particular in the field of ‘omics’ (transcriptomics, proteomics, metabolomics, next generation sequencing etc.). It has been one of the main tasks of the WP6 in DoReMi to check the availability of suitable biomarkers in conjunction with suitable cohorts for molecular epidemiological studies (Pernot E. et al. 2012). However, according to Pernot et al. 2012 at the beginning of DoReMi the task appeared to be quite difficult: “When considering suitable biomarkers of IR for use in large scale epidemiological studies, criteria such as sensitivity (in particular for low dose exposure), specificity to IR, persistence, availability of biological samples, technical applicability to large scale and cost should be taken into account. These requirements considerably reduce the number of possible candidates and explain why, currently, there is no ideal biomarker for assessing exposure, effect or susceptibility of low dose radiation exposure. There are some good validated biomarkers for acute radiation exposure to doses above 100 mGy (e.g. dicentrics) but not for lower doses, although some good candidates do exist”. As pointed out in the first DoReMi TRA, it is expected that “the identification and development of suitable biomarkers should facilitate the way toward molecular epidemiology” (see First Version of TRA). Scientifically speaking, it has been evident that mechanistic knowledge on low dose and low dose rate effects is needed to back up epidemiological studies for the sake of improved health risk evaluations at low dose exposures to ionizing radiation. In particular, an urgent need for biomarkers of radiation exposure, radiation susceptibility (early effects) and late and persistent effects has been recognized (Pernot E et al. 2012). The following categories of biomarkers were considered: cytogenetic biomarkers, biomarkers related to nucleotide pool and DNA damage, those related to germline inherited mutations and variants (SNP, CNV and CNA), those related to induced mutations, (such as HPRT, GYPA), those related to transcriptional and translational changes, those related to epigenomic modifications histine modifications, methylation, micro-RNAs, phospho-proteines) and other biomarkers such as metabolomic biomarkers, markers associated with cell cycle modifications, cell survival and death (apoptosis) and biophysical dosimetry by electron paramagnetic resonance (EPR). The recently developed Roadmap shows the different steps needed for the development of suitable biomarkers: identification of candidates in mechanistic, pre-clinical studies (biophysical, biochemical, molecular biology, genetic, epigenetic and general biological studies), development of reproducible assays (dose-effect relationships and distribution, suitable protocols for clinical and general use), validation (inter-laboratory, clinical reproducibility, performance in cohorts), qualification (validation of biomarker in prospective studies) and application (applicability in molecular pro-and /or retrospective epidemiological studies to improve understanding of low dose and dose rate exposure impacts on human health risks and the environment).

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The usefulness of a decent number of biomarkers has been specified during DoReMi (see List of suitable biomarkers in the annex: E. Pernot et al, 2012). These include relevant biomarkers from cytogenetic studies: The extended gold standard of classical analysis of chromosome aberrations dicentrics and rings to chromosome aberration analysis using prematurely condensed chromosomes (PCC) and telomere/centromere-fluorescence in situ hybridization (TC-FISH); quantification by the cytokinesis block micronuclei (CBMN) assay, ETV6/NTRK3 rearrangements in papillary thyroid carcinoma (PTC), quantification of micro-nucleated reticulocytes, DNA damage and nucleotide pool related studies (H2AX marker for the induction of DSBs down to a few mGy)(Rothkamm K, Löbrich M 2003, Rombouts C et al. 2013, Dieriks B et al. 2011, Schanz S et al. 2014, Flockerzi E et al. 2014, Large M et al. 2014, Mariotti LG et al. 2013, Maguire A et al. 2015b, Pottier G et al. 2013, Viau M et al. 2015)), detection of CtDNA, extracellular 8oxo-dG (as biomarker for individual radiation sensitivity; from transcriptional profiling (multigene signatures, FDXR (Kabacik S et al. 2015), CLIP2 mRNA amplification in PTC (Selmansberger M et al. 2015a, b, c), signature of DNA damage response (DDR) activation; from germline variants and radiation-induced mutations (deletions, LHO with multigene signatures, radiation specific mutation profiles (Brown N et al. 2015, Verbiest T et al. 2015). In DoReMi the sensitivity of these biomarkers and their feasibility of use has been assessed as well (Pernot E et al. 2012). Very promising new biomarkers and new ideas warranting further discovery include: the detection of specific DNA lesions, DNA Repair capacity assays (Dieriks B et al. 2011), biochemical profile alterations Maguire LG et al. 2013), identification of radiation induced proteomic markers and post-translational modifications and modifier profiles (epigenetic markers and epigenetic profiling). Those warranting development in human studies: biomarkers related to redox balance, metabolic biomarkers in urine/serum and saliva (Pernot E. et al. 2014), telomere length as marker for radiation sensitivity (Shim G et al. 2014, Haddy et al. 2014) and Long non coding RNAs (LnRNAs) as biomarkers of exposure and/or effect such as PAPPA-AS1 (Kabacik S et al. 2015) and PARTICLE (O’Leary et al. 2015). In fact, within DoReMi several new biomarkers, the suitability of H2AX and 53BP-1 (Grewenig A et al. 2015) for the detection of radiation-induced DSBs and related repair capacities, Chromosome 2 deletion, Pu.1 loss for radiation induced AML, several transcriptional (example: CDKN1A) and epigenetic signatures (for example: miR-34a-p5 and miR-182-5p) were found (Kabacik S et al. 2015) and await now further validation. Moreover, Raman spectroscopic analysis revealed characteristic donor specific changes in human lymphocytes after low doses of IR in parallel to changes in the extent of induced DNA damage (H2AX) (Maguire A et al. 2015 a,b). Newly developed molecular biomarkers had quite an impact on the low dose and mechanistically oriented research in DoReMi. Some clearly helped to better define IR-induced lesions in DNA after exposure to different radiation qualities (Drexler GA et al. 2015, SNAKE) and to further elucidate mechanisms and dynamics of repair (Mariotti LG et al.2013) as well as individual differences in biochemical profiles (Maguire A et al. 2015b). Furthermore, the involvement of specific polymorphisms in breast, colorectal and lung cancer were reported (Perriaud L et al. 2014; Sagne C et al. 2013, 2014). The fact that -ray exposure induces individual differences in proteomic profiles in human lymphoblastoid cell lines points to the possibility to develop proteomic biomarkers for individual IR responses (Gürtler A et al. 2014).

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A recent very exciting finding from Atkinson’s group in Munich is that among long non coding RNAs a biomarker for low dose IR responses (PARTICLE) could be established that regulates locus-specific methylation (activation of certain genes) (O’Leary VB et al. 2015). In MIRACLE (DoReMi Task 5.2 extension) and OSTINATO (DoReMi Task 7.10) the DoReMi project also raised the question whether T cell lymphocytes rearrangements (Treg cells) could be used as biomarkers for exposures. Considering low dose effects on the vascular cell system, it is important to note that the insulin-like growth factor binding protein 5 IGFB5 has been shown to be specifically involved in low dose rate induced premature senescence in human endothelial cells (Rombouts C et al. 2014), and may constitute a biomarker for vascular damage. It should be emphasized here that on the basis of the biomarker validation for low dose molecular studies in DoReMi the gap between biomarker development, validation and epidemiological use could be bridged, and several specific epidemiological studies could be set up and/or proposed: including RADMAD (integrative personal Omics profiling, iPOP), studies of genetic consequences over 3 generations after IR exposure, nested case-control studies in medical cohorts (see for example: new CT scan study in Spain, CREAL), genetically predisposed cohorts (such as AT heterozygotes in OPERRA). Several studies are already underway, including Mayak, Sellafield, Chernobyl liquidator studies (leukaemia, thyroid cancers, CVD and others), Techa river (several generation), childhood cancers (case control studies of CVD and cancers in ProCardio and CEREBRAD), Rapper (large European cohorts with long term follow-up, possibly allowing study of GWAS) as well as non-radiation cohorts studies (EPIC and 14C-birth cohorts studies in OPERRA). The special case ofH2AX as biomarker Since the discovery that DNA double-strand breaks lead to the phosphorylation of the histone variant H2AX on serine 139 by the phospho inositol 3 kinase ATM (ATM mutated) and the formation of -H2AX foci (cluster) at the sites of DNA DSBs detectable by specific fluorescent antibodies (Rogakou EP et al. 1998), with one focus corresponding to one DNA DSB (Sedelnikova OA et al. 2001, Pilch DR et al. 2003) there has been quite a discussion in the scientific community about the possibility to use -H2AX as a biomarker for radiation-induced DNA DSBs. In particular, the detectability of very low yields of DSBs induced by very low doses ionizing radiation (X-rays) by this assay down to 1.2 mGy and the possibility to determine the repair activity in cells by counting remaining DSBs (i.e. persistent foci) after a time of repair (Rothkamm K, Löbrich M 2003) has boosted the use of this assay in many radiation biology research centers. However, the validity and scientific meaning of these foci have been reviewed very critically because they may differ in size and arise from a variety of conditions (lagging strand replication, collapsed replication forks, meiotic recombination, gene conversion and rearrangements as well as from different genotoxic stresses (including IR) (Rosemann M, Atkinson M 2015, Rothkamm K et al. 2015). Persistent foci indicate unrepaired and misrepaired DNA (Suzuki M et al. 2006). Persistent DNA lesions and foci have been generally associated with the induction of genomic instability and carcinogenesis, however, as pointed out recently (Martin M et al. 2014, Rosemann M,

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Atkinson M 2015) in some conditions, they may also be indicative for unresolved DNA damage that may promote cellular senescence. Still, in spite of the fact that H2AX has not yet been taken up as a biomarker for IR-induced DNA damage and repair capacity in a generally applicable and standardized fashion (see RENEB) it has been found already very useful in small molecular epidemiology and clinical assays to detect patients with increased individual radiation susceptibility and sensitivity for the induction of secondary cancers by low radiation doses (see Löbrich M et al. 2005,. Beels L et al. 2012, Halm et al. 2014, 2015, Schuler N et al. 214, Haddy N et al. 2014, Vandevoorde C et al. 2015). Moreover, it was recently shown that using a flow cytometry high throughput methodology H2AX fluorescence measurements can be proposed as a triage tool for the rapid estimation of received radiation doses in the event of accidental radiation exposure. (Viau M et al. 2015). Very importantly, γH2AX labeling can also serve for the detection of IR-induced complex clustered DNA damage (Meyer B et al. 2013, Lorat et al. 2014.) Using micro-irradiation, Meyer B et al. (2013) showed that IR-induced clustered DNA damage (containing localized clustered DSBs) triggers a pan-nuclear H2AX phosphorylation by the kinases ataxia telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) reducing the accumulation of MDC1 at DSBs. The binding of 53BP1 is not affected by the pan-nuclear response that is transient even if DNA repair is impaired (Meyer B et al. 2013). Conclusions on biomarker studies The biomarker studies are indeed very promising for future research on low dose risk. Gene expression and epigenetic studies in DoReMi are expected to lead to the development of new molecular biomarkers for exposure, metabolic change and pathology onset (cancer, non cancers) as well as individual sensitivity and susceptibility. They now await validation for practical uses, for example in molecular epidemiological studies, medical diagnosis and therapy and preventive measures in radioprotection.

These Biomarkers can be considered to be extremely useful for defining IR exposure, metabolic changes and pathological changes (different types of cancers and non cancers).

Indeed, a number of pilot molecular epidemiological studies could be carried out within DoReMi and other EU projects, see for example: CURE (DoReMi) Laurent O/ Gomolka M; Int-THYR (DoReMi) E. Cardis; Thyroid cancer in Ukraine (EPIRADBIO) Zitzelsberger) H; Haemangioma studies (Epiradbio F. de Vathaire and Non cancer effects in Mayak workers (SOLO T. Azizova and others). 4.6 Epidemiological studies

Cancer

Epidemiological studies are of utmost importance for estimating radiation-induced health risks in radiation protection (ICRP, HLEG reports). However, they show severe statistical limitations regarding health risk estimates for low and very low dose exposures (HLEG). It has been the aim of the DoReMi project to help back up

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epidemiological risk evaluation through suitable mechanistic low dose and dose rate studies ensuring assessment of IR-induced individual doses, metabolic short and long term changes and pathologies (cancer and non cancers). In recent years, several large cohort studies have been conducted pointing to the importance of health risk evaluations in the low dose range: Among these, the epidemiological study by Darby S et al. (2005) suggested a clear effect on lung cancer induction from domestic exposures to radon and showed that the carcinogenic effect of chronic radon exposure (alpha rays) was very much increased when associated with smoking. Richardson et al (2014) indicated that low dose exposures of children to CT scans may lead to a significant increase in leukemia and brain cancer. The very large retrospective INWORKS study on chronic occupational exposure to ionizing radiation of workers in France, the United Kingdom and the USA indicated an excess risk for leukemia and solid cancers as a function of cumulative dose (Richardson et al. 2015, Leuraud et al. 2015) without taking into account smoking and other confounding factors. The association between protracted low dose radiation exposure and leukemia was particularly strong (Leuraud K et al. 2015). All these studies could be consolidated by smaller scale epidemiological studies associated with mechanistic molecular and biological studies. The DoReMi project explored the possibility to perform well-defined molecular epidemiological studies in order to further substantiate low dose health risk evaluation and improve actual radiation protection measures. Internal exposures

Based on a critical review of epidemiological studies of populations exposed to internal contaminations (Pernot et al. 2012, Laurier et al. 2012) several actions on populations exposed to uranium were launched by DoReMi: Intemitum (Internal emitters in uranium miners), AirDoseUK (Internal doses in UK AEA and AWE cohorts) and CURE (Occupational uranium exposure study planning). The feasibility to improve present knowledge through a large European research project was shown by (1) improving dose reconstruction of internal contamination (2) combining biology, molecular studies and epidemiology in the characterization of the association between dose and risk, (3) quantifying the uncertainties (confounding factors.) and their impact of the dose-relationships and (4) coordinating and standardizing efforts between existing cohorts. INTEMITUM In DoReMi important incidence data on leukaemia and non-melanoma skin cancer in existing cohorts of uranium miners were collected and individual doses calculated. In 42 cases, red bone marrow equivalent doses for leukaemia were found to be 35 mSv Rn, 46 mSv gamma, LLR 106 mSv with an ERR/Sv of 3.75 90%CI 0.92-11.0. For basal skin carcinoma from deposition on the skin in 42 cases the equivalent skin doses were 9.8Sv RN, LLR 1.9 Sv with an ERR/Sv of 0.125 90%CI 0.049-0.362. This allowed an improvement in compensation procedures. AIRDose UK The UKAEA and AWE nuclear worker cohorts have been important in examining the effects of low dose external radiation exposure (Beral et al, 1985, Beral et al, 1988, Atkinson et al, 2004). Within DoReMi relevant data from over 1 million sample records

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of internal dosimetry were made available in the SHIELD database. A method has been developed for automating the calculation of doses from plutonium, uranium and tritium exposures using the industry standard IMBA software. The internal doses are now available for future studies of the health effects of these nuclides. CURE This project aimed at developing a cross-disciplinary approach, involving epidemiology, biology/toxicology and dosimetry, to improve risk estimation from uranium contamination. A common research protocol was prepared (including pooled epidemiological analysis and molecular epidemiology approaches with harmonized dosimetric procedures, biomarkers, standardized operating procedures or biobanking, logistics etc.) to overcome the limits of previous studies. The feasibility of a molecular epidemiology study was investigated and uranium research programs (activities) outside Europe reviewed. If this study were to be implemented, it would address the important issues of the characterization of dose-response relationships for internal contamination (low doses/dose rates) and cancers and non-cancer effects (circulatory and cognitive effects). Other epidemiological studies within DoReMi: Cancer In a sub group of 4054 men of the German uranium minor cohort (1946-1989) in milling facilities an excess mortality from lung cancer due to radon exposure, and from solid cancers due to external gamma radiation was observed but not statistically significant. Apparently, at low absorbed organ doses uranium was not associated with any cause of death (Kreuzer et al. 2014). Likewise, no excess of kidney cancer induction by radon was found in the French and German cohort of uranium miners (Laurier D et al. 2012, Dubray D et al. 2014). Interestingly, in a subcohort of 26766 German uranium miners hired in 1960 or later, characterized by very low exposure levels (average 0.2 working level) over a long time with high quality of exposure assessment and availability of individual data on possible confounders a clear association between lung cancer mortality (3334 deaths) and cumulative exposure to radon in working level months was found (excess relative risk per WLM was 0.013 (95% confidence interval 0.007-0.021) (Kreuzer M et al. 2015). (Mathematical model analysis of the ELDORADO cohort concerning lung cancer in uranium miners clearly showed that the cancer risk increased with obtained age, time of exposure and exposure rate. However, in the low dose range large uncertainties remain (Eidemüller M et al. 2012, 2015). Application of different models revealed that the excess risk for lung cancer decreased with increasing attained age, increasing post irradiation time and increasing exposure rate. A critical overview on the remaining uncertainties concerning low dose protracted/fractioned exposures (as received in medicine, from environment and at occupational context) and a proposal for the most suitable European cohorts that should be able to deal with still open questions was provided by the DoReMi Working Group on Epidemiological cohorts. A total of 53 cohorts were identified including:

medical (diagnostic (CT scans, conventional X-rays.) RT and other treatments (haemangioma, Tinea capitis, interventional cardiologists.),

occupational (nuclear workers (Mayak, Sellafield, cleanup workers, U miners, Chernobyl liquidators,

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environmental (Chernobyl, Ozyorsk, Techa River, natural background IR), and in utero cohorts (Mayak/Ozyorsk, Chernobyl.).

The availability of suitable biosamples for molecular epidemiological studies was checked as well (see also contacts with STORE in DoReMi). Of particular interest are the cohorts from nuclear industry and miners (evaluation of effects of internal emitters on cancers and non cancers), Ukrainian liquidators cohort studies (evaluation of lens capacities), medical cohorts for studies on individual sensitivity and cohorts for studies of genetic and multigenerational effects (AT heterozygotes, Mayak, Techa river, Chernobyl liquidators.). It should be noted here that this work by DoReMi was largely extended and complemented by other European projects such as EpiRad Bio, RISK-IR, Dark-Risk, Andante, EPI-CT, RENEB, ProCardio, CEREBRAD, Rapper, followed up within OPERRA (EPIC, 14C-birth cohorts, AT heterozygotes). In the near future, CT exposure cohorts for evaluation of DNA repair capacities in children/adults, Hemangioma cohorts for evaluation of CDV risks, German U miner cohort for evaluating high and low dose risks of lung cancer could be available with links to molecular and biological assessments. These studies were initiated in the hope to identify radiosensitive sub-populations in suitable cohorts (Pernot E et al. 2012). Among the 53 cohorts identified as being useful for low dose research a few medical cohorts (CT scan etc.) with populations of patients (for example, Vandevoorde C et al. 2015), where continued contact is possible after the radiological procedures are very valuable for the study of individual radiation sensitivity. For example, recent studies evaluated the potential for cancer predisposing syndromes for modifying the association between CT doses and risk of cancers (for example, Vandevoorde C et al. 2015; manuscript by Cardis E and Bosch de Basea in press). Other epidemiological studies within DoReMi: non-cancer effects (cardiovascular disease (CVD)) In order to get a better understanding of the pathogenesis and the risk estimation of radiation-induced cardiovascular diseases at low doses several available key cohorts were analysed for their suitability for future informative molecular epidemiological studies (Kreuzer M et al. 2015). With this, a strategy has been worked out for molecular epidemiological studies in Europe concerning low dose ionizing radiation cardiovascular diseases (Kreuzer M et al. 2015).The future molecular epidemiological studies were designed with a clear definition of objectives for such studies and criteria for informative studies, the methodology of evaluation of potential study populations and the definition and identification of biomarkers of interest. (This again underlines the importance of associating molecular biomarker studies with epidemiological studies at low doses and low dose rates of ionizing radiation (Pernot E et al. 2012, 2014).

4.7 Molecular and mathematical modeling

From pathway analysis to systems biology and health risk evaluation Modeling approaches are of great importance to combine data from a range of sources. In DoReMi, lung cancer risk after radon exposure in the Eldorado uranium workers was

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analysed with mechanistic models of carcinogenesis and empirical excess risk models (Eidemüller et al. 2012). The two-stage clonal expansion (TSCE) model (assuming that a cancer cell arises from a healthy cell in two basic steps) indicated that the clonal expansion rate of intermediate lung cells is reduced at older ages. Furthermore, the radon decay products appear to act on the clonal expansion rate in a non-linear way: with a steep increase for low exposure rates and a linear relationship with a lower slope for higher exposures. In the following, model building has been extended in other parallel European projects such as ProCardio and EpiRadBio (Zöllner S et al. 2015, Simonetto C et al. 2015, Eidemüller M et al. 2015). For the analysis of breast cancer risk involving the effect of genomic instability in the Swedish hemangioma cohort (Eidemüller M et al. 2015) three risk models were used: the standard excess relative risk (ERR) model, the TSCE model and a multistage model with a separate path of genomic instability (known to manifest itself in the progeny of exposed cells several generations after initial exposure). The latter model involving genomic instability was found to be highly significant when applied to the evaluation of IR induced breast cancer risk in the hemangioma cohort. Moreover, multistage models were also applied to analyse lung cancer mortality after plutonium exposure in the Mayak worker cohort (Zöllner S et al. 2015). These models included three mutation stages with an increased IR-induced rate of clonal expansion. Such a dose response could be a consequence of disturbance of cell cycle control mediated by bystander signaling or repopulation from stem cells following cell killing. With respect to lung carcinogenesis in the Mayak workers and the radiation risk from plutonium exposure the dose relationships in the three-stage models were strongly non-linear with a marked increase above a critical dose level (Zöllner S. et al. 2015). Furthermore, when analyzing the incidence and mortality from cerebrovascular diseases and the incidence of stroke in workers of the Mayak Production Association a sublinear dose response was observed for low doses for the incidence of cerebrovascular disease (Simonetto C et al. 2015). Younger age of exposure was significantly related to higher radiation risk. Since the TSCE model showed some limitations (difficulties to include different IR-induced pathways, to account for individual or genetic susceptibility and/or for bystander effects and genomic instability), more complex multi-stage models were successfully applied to the Japanese a-bomb survivors’ cohort of colon cancer (Kaiser JC et al. 2014) and the Swedish hemangioma cohort (Eidemüller M et al. 2015). Measurements of molecular changes in papillary thyroid cancers of cohorts Genrisk-T and UkrAm provided a concept for the development of a two-path model for thyroid cancer (Selmansberger M et al. 2015 a, b, c). Further developments (within OPERRA) include other more complex types of models taking into account different molecular pathways that are involved in the development of cancer and non-cancer diseases. The establishment of links is foreseen to mechanistic and mathematical models of low dose biological IR effects (Kundrat P et al. 2012, Kundrat P and Friedland 2015, Alloni D et al. 2011, 2012, 2013 and 2015) and systems biology approaches. In fact, future priorities are multi-scale modeling, modeling of sporadic pathogenesis and integration of molecular data from radio-epidemiological cohorts in order to allow better interpretation of molecular, clinical and epidemiological data to estimate low dose human health risk.

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5 DoReMi outcomes

5.1 Effect of radiation quality

DoReMi confirmed High LET effects: certains ions in the low energy range (<6MeV u-1) have been

found to produce DNA fragmentation, and the complexity of DNA damage induced depended on the energy of heavy ions.

High LET exposures increased cell-to-cell communication and increased cytokine release (bystander effect).

X-irradiation and exposures to neutrons differed in the induction of Chromosome 2 deletions associated with acute myeloid leukemia (AML) in mice.

The higher effectiveness of high LET versus low LET, and could correlate this to differences in DNA fragmentation and repair dynamics.

o That Radiation weighting factors WR have to be taken with caution: for example with protons the contribution of neutrons can be quite important.

o PARTRAC modeling revealed that in some cases (Protons) the energy of neutrons produced would affect biological effectiveness. More generally, the energy of the IR is important for the biological outcome.

RBE values have to be carefully determined according to the biological endpoint considered

Dosimetry Physical and biological dosimetry has been very much improved: in particular,

for low dose and low dose rate exposures to and ionising radiation direct dosimetry measurements a well as retrospective dose evaluations using phantoms and Monte Carlo calculations were refined and important links to EURADOS established (see extension in MELODI). This has been very important for the dose assessments concerning interventional cardiologists and the relationship between chest dosimeters and eye lens doses, as well as for radiotherapeutic out-of-field dose estimations (including Hadron therapy) and those of exposures to internal emitters. Moreover, a laboratory infrastructure for retrospective radon and thoron measurements has been created (University of Sofia). The infrastructure includes a laboratory for processing detectors (e. g. CD/DVDs used as retrospective radon and thoron detectors) and calibration facility. The infrastructure can be used in epidemiological studies on radon risk or where the radon exposure should be taken into account as potential confounder. Biological dosimetry has been progressing due to the development of suitable molecular biomarkers that can be used in molecular epidemiological studies and/ or emergency situations.

5.2 Dose and dose rate effects

Moderate doses elicited a non-linear response (on inflammatory cytokine production) after 0.5 and 0.7 Gy X-irradiation in radiosensitive but not in more radio-resistant mice.

In line with this, the induction of DSBs by low and moderate X-irradiation was non-linear with a window at 0.5 and 0.7 Gy.

Fractionated exposures

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Induced persistent DNA damage in mouse spermatogonia stem cells and also in lung, breast and brain cells.

Increased toxicity in normal mouse lung tissue. Affected DNA repair kinetics (DSBs) Showed beneficial anti-inflammatory and pain relieving effects in some clinical

conditions (benign elbow syndropme, benign calacaneodynia, achillodynia) Hypofractionated radiation exposure increased the immunogenic potential of

some tumor cells. Low dose rate exposures were found to exhibit non-linear effects on the

induction of premature senescence in epithelial cells (cardiovascular system) involving non-linear changes in gene expression (genes of cytoskeletal organization, cell-to cell communication, adhesion and inflammatory signaling pathways).

5.3 Tissue sensitivity and tissue specific responses

Tissue weighting factors WT are to be used with cautions: DoReMi results point to the importance of cellular complexity of tissues made up of different cell types (progenitor, stem cells etc.) and different differentiation stages of different radiation sensitivity. Tissue specific regulatory epigenetic factors may determine radiation sensitivity of individual tissues (and individuals, see below). Moreover, individual differences in genetic background may differentially influence sensitivity of specific tissues to radiation (e.g. bone cells when different Rb1 alleles are present, bone marrow when PU1 or AML variants are present, or multiple tissues when alleles of p53 are present.

Regarding the radiosensitivity of breast cancer cells, RT single or hypofractionated irradiation was less effective on p53 mutated breast cancer than on p53 wildtype cells. This was correlated with the activation of dendritic (immune) cells. Also, transmigration and chemotaxis of activated macrophages involved in anti-inflammatory reactions could be modulated by low and moderate doses of IR. Thus, immunologically active immune cell responses to IR need particular attention in anti-tumor RT.

In the mouse, brain and immature cortical neurons were quite susceptible to low dose IR induced damage affecting differentiation and gene expression.

Neural progenitors were resistant to low doses oncogenesis (0.25 Gy) but sensitive to high doses (3 Gy) in a medulloblastoma mouse model.

Research on stem cell radiosensitivity and /or radioresistance remains an important issue to understand radiation-induced carcinogenesis and non cancer induction.

Lymphoblastoid mouse cells homozygous for ATM were radiosensitive, whereas ATM heterozygotes and wild-type lymphoblastoids were radioresistant and able to cope with DNA damage induced by repetitive low dose exposures.

Bronchial and alveolar epithelial lung cells and also heart and brain cells of mice clearly differed in DNA damage and repair responses.

Low X-Ray doses affected cell proliferation in normal thyroids but less in thyroid cancerous tissue (RET/PTC positive thyroids) in iodine-deficient mice.

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5.4 Internal emitters

Major changes in CNS morphology and organogenesis were observed after internal contamination with uranium in rats at different developmental stages in neurogenic zones. Cell proliferation was affected in the hippocampus neuroepithelium and in the hippocampus. Stem cells from contaminated animals retained their multipotential properties but cell proliferation and differentiation during brain development were altered.

An epidemiological study (INTEMITUM) revealed an increased incidence of leukemia and non melanoma skin cancers in a small cohort of uranium miners.

For future studies on health effects of radionuclide contamination (plutonium, uranium, tritium) a method was developed for automatisation of exposure dose calculations.

In an epidemiological cohort on German miners in milling facilities, low absorbed organ doses from uranium were not associated with any cause of death.

A concept for a two-path model for thyroid cancer development from radioactive iodine contamination (Chernobyl) has been developed including molecular biomarkers (CLIP2).

A collaborative research project was launched (CURE) to improve health risk estimation (cancers/ non cancers) from uranium contamination using an integrated molecular epidemiology approach.

5.5 Cancer induction

Dose as a surrogate of risk? The DoReMi project has provided further evidence for the existence of non-linear low dose radiation responses. Although the exact relevance of these findings for low dose induced health effects is not yet fully established, these results call for some caution using the LNT model for estimating low dose health risk by simple extrapolation from high down to low dose effects. Mechanistic advances (from pathway analysis)

Extended molecular pathway analysis (using ‘omics’ approaches together with genetic and epigenetic profiling) revealed several important genetically and epigenetically controlled pathways involved in the induction of specific cancers. However, these studies have still to be completed by systems biology analysis of the metabolic networks in order to identify the crucial factors associated with well-defined cancer types. The tissue specificity of cancer induction needs to be further explored.

Clear advances were obtained in the mechanistic understanding of the development of acute myeloid leukemia, thyroid and bone cancer.

The involvement of signaling processes at the cellular, tissue and whole body level (bystander, abscopal effects) associated with immunological responses to low dose exposures in radiation-induced pro- or anti-inflammatory and/or tumor or anti-tumor effects has been clearly brought to light.

These studies need to be extended to the benefit of effective RT anti-cancer treatments and the prevention of RT out-of field side effects.

Molecular and mathematical modeling

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Modeling of initial events and biological effects has been largely refined during DoReMi:

o through PARTRAC models applied to link track structures and energy deposition to DNA fragmentation and Radiation Biological Effectiveness

o through modeling of radiation-induced cell-to-cell interactions (bystander effects) to apoptosis and pro- or anti-carcinogenic processes.

Dose-rate effects have not yet been modeled Mathematical modeling using epidemiological clonal expansion models

(including bystander effects and genomic instability) has been successfully applied to epidemiological data sets of IR induced breast cancer and lung cancer, respectively.

Biomarkers

Several biomarkers (such as γH2AX and 53BP1) have been used to determine exposure, the induction and repair kinetics of DSBs. The use of γH2AX has been automatized and may be used for emergency purposes. These biomarkers were also useful for live cell imaging (SNAKE).

Metabolic pathway analysis revealed new biomarkers o for cancer induction (p21, Rb1, CLIP2) o for non cancer endothelial vascular damage (IGFBP5), and await further

validation. Raman spectroscopic profiles are able to distinguish variations in individual

radiation sensitivity (suitable for the detection of radiosensitivity in patients) Long non-coding RNAs have been shown to be useful for the detection of low

dose responses ( some microRNAs are likely to become useful tissue specific markers)

Proteomic biomarker profiles have been developed as well, for distinguishing and predicting individual patient radiation responses (and await further validation).

The detection of cytidine DNA, extracellular 8oxo-dG as biomarker of individual sensitivity, multi-gene signatures for specific radiation induced mutation profiles, proteomic and epigenetic profile markers for IR induced metabolic changes are under development and validation.

Telomere length frequencies in tissue are proposed as biomarkers for either induction of cancer or senescence by ionizing radiation

Distinct IR induced changes in mRNA and proteomic profiles are associated with the radiosensitivity in head and neck cancer patients (RAD-SENS)

A p21 reporter mouse model has been developed able to indicate low dose exposures (0.2 Gy).

some of these biomarkers are found very promising for the use in molecular epidemiology.

5.6 Induction of non-cancer effects

Dose as a surrogate of risk? Detection of non-linear dose and dose rate effects in endothelial cells

(cardiovascular system) exclude firm statements on the non-linear thresholded (possibly deterministic) effects or stochastic (dose-dependent and linear) effects of IR on the cardiovascular system.

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Effects on the cardiovascular system have been observed down to < 50 mGy. In mice susceptible for CDV disease, low IR doses did not induce changes in aortic

diameter or survival. Mechanistic advances (from pathway analysis)

The p53/p21 pathway (affecting replication), pI3K/AKT/mTOR and IGFBP5 expression are involved in low dose radiation induced senescence in endothelial cells (cardiovascular system)

In endothelial cells, non-linear induction of DSBs (X-ray dose window 0.5 and 0.7 mGy) and a non-linear production of ROS and SOD were observed.

Non coding RNAs (miRNAs) (regulating metabolism, cell cycle and cytosceletal structure) are also involved in non-cancer effects.

Exposure of diabetic mice to low doses (100 mGy) induced an increase in pro-inflammatory cytokine TNF-α as well as endothelial dysfunction.

Molecular and mathematical modeling

Within DoReMi, modeling has not yet been carried out because of the lack of sufficient molecular and mechanistic data for non-cancer effects.

Biomarkers for non-cancer effects For the detection of IR induced vascular damage IGFBP5 has been proposed as suitable biomarker in endothelial cells. This biomarker needs now more general validation. No specific biomarker for the detection of eye lens damage has been discovered so far. Therefore, there is continued need for further molecular and mechanistic studies to get a better understanding of the mechanisms involved in low dose induced eye lens damage. For the detection of neurological alterations have been proposed recently :

A distinct signature of upregulated genes dependent on the transcription factor p53 has been identified following low dose exposure: the genes concern brain-related functions (neurite outgrowth, focal adhesion, neuronal differentiation, calcium sensing and synaptic transmission). These possible biomarkers for early neurological damage await now further validation.

MicroRNA profiling indicate low differential expression in pericytes after low dose exposure

Low dose exposure of the developing brain in mice inhibited neurite outgrowth, impaired synaptogenesis and neurotransmission.

External in utero exposure resulted in neuronal death suggesting alterations in brain function.

Chronic neonatal low dose rate irradiation showed significant impairment of motor coordination in Parkinson predisposed mutant mice at a total dose of 0.2 Gy.

Brain pericytes are relative radioresistant cells, however, accumulate persistent radiation-induced DNA damage, a likely precursor of genomic instability.

Studies in mice showed that pericytes have an important role in brain inflammation: radiation exposure can modulate their response to inflammatory stimuli.

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5.7 Individual sensitivity

One of the big challenges of DoReMi was to find and establish potential biomarkers for the detection of individual sensitivity and to analyse the underlying mechanisms. Here are first highlights:

New potential biomarkers have been found that are promising to identify individual radiation sensitivity (among these are: telomere length in lymphocytes, microRNAs in circulating lymphocytes, Raman spectrospcopic signatures and custom gene expression arrays). Future large scale studies should further validate these biomarkers.

Individuals with normal and increased sensitivity could be distinguished by leukocyte telomere length and subsequent telomere shortening after in vitro irradiation (2Gy). An extended study using medium and low doses is needed to verify whether these biomarkers are fully valid at lower doses.

Differences in Raman spectroscopy profiles of leukocytes obtained from prostate cancer patients during radiotherapy correlated well with observed differences in clinical indices of radiotoxicity and in vitro assays of DNA damage, with high best-case-scenario of sensitivity (94%) and specificity (90%). The performance of the assay should be checked at low doses and in other clinical conditions.

The circulating and leukocyte microRNA profiles differed between radiation hypersensitive and normal breast cancer patients and allowed to classify patients according to the severity of their radiation response. A study at low doses is urgently needed.

A custom microarray assaying a set of radiation-responsive genes was established showing a direct correlation between leukocyte mRNA expression and in vitro radiation dose. This study should be urgently extended to test the applicabiliy to in vivo exposures, and the correlation between the mRNA response and individual susceptibility.

Concerning the potential mechanisms influencing individual sensitivity Studies performed within DoReMi brought to light a number of genetic as well as epigenetic and biochemical processes revealing a hitherto unknown complexity in the determination of susceptibility.

A number of non-coding sequence polymorphisms of the tumour suppressor gene p53 can influence the biological function of the gene that is likely playing a significant role for individual susceptibility. However, the correlation of these polymorphisms with individual susceptibility needs to be further established, in particular at low doses.

Transcriptomic and proteomic data point to the importance of energy metabolism and the role of mitochondria in radiation responses. The relevance for low dose effects and individual responses needs further studies.

Chronic (low dose rate) exposure elicited persistent damaged sites that remain long after completion of repair and are somewhat correlated with repair capacity. An association with individual sensitivity should be checked as a priority.

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5.8 Non-targeted effects and immune system modulatory effects of IR

The DoReMi project revealed close links between radiation-induced non targeted (bystander) effects (NTE) and effects on the immune system. Here are some of the highlights:

DoReMi confirmed the important role of low dose non-targeted effects in radiaton responses in vitro and in vivo, the involvement of signaling pathways and inter-cellular signaling, and the intimate relationship between IR-induced intercellular mediators, inflammation and immunological responses modulating cancer and non cancer induction.

Mechanistically, it could be shown that membrane microvesicles secreted by cells are involved in intercellular communication and carry important cargo RNA and proteins.

NTE were observed in vivo and contributed to systemic effects (pro-inflammatory and anti-inflammatory responses) as evidenced by the release of pro- or anti-inflammatory cytokines.

While pro-inflammatory proceses were observed at higher IR doses, low and medium doses (below 0.5 Gy) could elicit anti-inflammatory and sometimes even anti-carcinogenic defences.

Very importantly, the immunogenic potential of cancer cells could be influenced by different dose fractionation protocols (affecting dendritic cell maturation and activation of immune cells’ activity (macrophages, B and T lymphocytes)) . This appears to be important for combined RT and immunotherapy in the treatments of cancers.

In some clinical studies, moderate doses of IR (0.5 Gy) were found to exert beneficial effects on some inflammatory syndromes (benign painful elbow or shoulder syndrome, achillodynia and calcaneodynia).

Reduced secretion of pro-inflammatory cytokine (IL-1) by activated macrophages was observed non-linearly with dose (window: 0.5 Gy and 0.7 Gy). Thus, there is evidence for non-linear low dose radiation effects on the immune system.

In endothelial cells (cardiovascular system), a non –linear regulation of reactive oxygen species production and superoxide dismutase (SOD) activity and other anti-oxidant factors was observed after irradiation at doses below 1 Gy, probably contributing to anti-inflammatory effects seen in endothelial cells at 0.5 Gy.

Interestingly, chronic -irradiation caused a dose-rate dependent pro-inflammatory response and loss of function in human umbilical vein endothelial cells. Modifications of the inflammatory profile included changes in adhesion molecules and the release of pro-inflammatory cytokines. This is likely to have an important bearing for IR induced non-cancer effects (on the vascular system).

Experiments in mice showed that low dose radiation can affect T lymphocyte homeostasis (and the T cell repertoire).

5.9 Epidemiological studies – The way towards molecular epidemiology

5.9.1 Cancer

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Because DoReMi has been a short term project, the possibility to perform new epidemiological studies was limited. In spite of this, the outcome concerning epidemiological studies has been of importance, in particular for well-targeted low dose exposure. As an important step, epidemiological cohorts have been reviewed for their suitability and availability for low dose/low dose rate (chronic) studies. A total of 53 cohorts has been identified by the Working Group on Epidemiological cohorts including medical treatments, occupational environmental and background exposures as well as in utero exposures. Specific actions were launched within DoReMi on suitable cohorts to assess low dose health risks from internal contamination (Intemitum, Internal emitters in uranium miners; Air Dose UK, Internal doses in UK AEA and AWE cohorts; and CURE, Occupational uranium exposure study planning.

5.9.2 Non-cancers

A strategy and protocol has been worked out for molecular epidemiological studies on cardiovascular diseases in Europe.

5.9.3 Modeling: molecular, mathematical, systems biology approaches and health risk evaluation

Lung cancer risk after radon exposure in ELDORADO uranium workers was analysed using mechanistic models of carcionogenesis and empirical excess risk models.

The two-stage clonal expansion model evolved into complex multi-stage models by taking into account molecular IR response pathways, bystander effects, genomic instability and genetic susceptibility. These models were applied to existing cohorts: breast cancer risk in Swedish hemangioma cohort, colon cancer risk in A-bomb survivors, lung cancer risk in Mayak workers, cerebrovascular diseases in Mayak workers.

A two-path model for thyroid cancers was developed as well. Links were established between mechanistic and mathematical models of low

dose biological effects and systems biology. For the future are foreseen: Multi-scale modeling, modeling of sporadic pathogenesis and integrative molecular-epidemiological studies in order to improve health risk assessments.

6 Implications of DoReMi research for Radiation Protection

6.1 Shape of dose response for cancer and the application of Linear non threshold hypothesis

Quantification of biological and health effects (cancer, other diseases and cell damage) associated with exposure to ionizing radiation has been a mojor issue for the ICRP since its foundation in 1928. While there is plenty of information on the human health for whole body doses above approximately 100 mGy, the effects associated with doses below 100 mGy are still being investigated and debated intensively (Rühm et al, 2016). The current radiological protection approach, proposed by ICRP for workers and the public, is largely based on risks obtained from high-dose and high-dose-rate studies, such as Japanese Life Span Study on atomic bomb survivors. The risk coefficients obtained from these studies can be reduced by the dose and dose rate effectiveness

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factor (DDREF) to account for the assumed lower effectiveness of low dose and low dose rate exposures. The 2007 ICRP Recommendations continue to propose a value of 2 for DDREF, whereas other international organisations suggest either application of different values or abandonment of the factor. In this context, it should be recalled that the interaction of ionizing radiation with matter occurs through energy deposition linearly with dose. From this, it has been inferred that also the induction of damage to prominent cellular constituents such as DNA proceeds in a linear fashion. In other words, it is assumed that there is a direct proportionality between energy deposition in the target cell and biological effects (Brenner and Sachs 2006, NCRP2001). As it was subsequently shown that at high doses important biological endpoints such as mutations, chromosomal aberrations and to some extent even pathological effects such as cancers generally followed a linear dose-effect relationship, this gave rise to the linear non-threshold (LNT) hypothesis largely employed in radiation protection and epidemiology implying a linear induction for adverse biological effects and health risks. The concept is quite convenient and of practical importance since it implies that radiation responses differ only quantitatively, and not qualitatively, with dose (Ulsh BA 2010), and in many situations the physical dose can be considered as a surrogate of health risk. In fact, this appeared to be quite sensible (reasonable), especially during the years where there were very few solid data available on the biological effects and underlying mechanisms at low doses (<100 mGy). However, in recent years and in the light of growing knowledge, the general validity of the LNT concept has been questioned, particularly for low dose exposures (Tubiana M et al. 2006, Ulsh BA 2010, Averbeck D. 2010, Calabrese EJ 2015).

To complement the traditional line of radiation research addressing DNA damage and response that typically follow the linear dose response pattern, DoReMi has focused its investigations on various non-linear cellular and tissue responses and underlying mechanisms. Some general conclusions can be drawn from the DoReMi research program:

DoReMi results call for some caution in applying linear extrapolation from high dose results to estimates of low dose risk as nonlinearities in cell/ tissue responses exist

Cellular responses to low doses are complex. DoReMi has shown that they include a mixture of simple cellular transcriptional, proteomic and metabolic responses and more complex regulatory responses integrated across tissues (including stress responses, damage responses, and immunological responses and other non-targeted effects). Neither the simple nor complex responses can be assumed to follow linear dose response kinetics.

In fact, cells may respond differently to high and low doses and to dose-rate changes in the manner and scale by which different response pathways and distinct regulatory pathways are activated.

A genetic basis for differences in individual sensitivity to cancer induced by radiation has been demonstrated. This unequivocably establishes that genetic differences between exposed individuals contributes to differences in risk

Other results relevant for RP: o Evidence for non linearities in low dose and low dose rate responses: low

dose rate dependency of senescence, moderate dose window for anti-

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inflammatory effects (promising for radiotherapy of tumors, moderate doses for the reduction of pain in non cancer affections (such as painful elbow or shoulder syndrome, achillodynia and calcineodynia), low dose DNA repair responses, induction of apoptosis (non linear) and chromosomal effects (MN linear?) (does this challenge the LNT hypothesis?)

o Better understanding of the radiation induced AML, osteosarcoma and thyroid cancers and some progress in better understanding the pathways involved in the induction of cancers

o Identification of important metabolic pathways and their involvement in disease (first attempts in systems biology)

o Better understanding of the complexity of genetic and epigenetic control of radiation sensitivity and cancer susceptibility

o Better understanding of the different effectiveness of high and low LET exposures: involvement of clustered lesions

o Better understanding of the effects of low and moderate doses of radiation effects on the immune system: effects on cytokine release, bystander and non-targeted effects, DNA repair components

o Better understanding of genetic and epigenetic controls thanks to transcriptional gene expression, protein expression analysis and epigenetic profiling.

o Better understanding of the mechanisms involved in radiation induced non cancers: cardiovascular disease and the importance of endothelial cell responses

o Regarding tissue sensitivity: low dose effects on cell research have been started to be investigated

o Better understanding of low and moderate dose induced neurological (behavioral) effect involving p53 related gene sets (Quintens et al. 2015)

o Analysis of epidemiological data (uranium miners, uranium millers etc.) (Kreuzer M et al. 2015, Drubay et al. 2014).

o Preparation of new suitable cohorts (CURE: Laurier D, EpiCT: Cardis E. et al)

Evidence for linear gene expression responses in healthy and Ataxia telangiectasia donors (Kabacik S et al. 2015).

Radiation induced AML involves chromosome 2 and Sfp1/Pu1 loss (Olme CH et al. 2013 a,b) and the inscription actor PU.1 is downregulated (Verbiest T et al. 2015)

It is shown that cancer induction at low doses can be modulated by the effects on the immune system. The results demonstrate the existence of a possible window 0.5-0.7 Gy and clearly different responses after acute or fractionated exposures (Frischholz B et al. 2013, Rödel F et al. 2012 a, b).

Immunological effects include NTE effects involving the production of ROS and signaling molecule activation and the release of pro- or anti-inflammatory cytokines (Gaipl US et al. 2014, Candeias and Gaipl 2015).

Evidence for adaptive responses (Dieriks B et al. 2011) IR induced exosomes and vesicles may act as possible general biomarkers and

mediators for these effects. Mathematical modeling of cancer induction data from epidemiological and

mechanistic data has been well progressing in DoReMi. There is evidence that

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some epidemiological data can be better fitted when considering and including data from mechanistic studies and biological parameters (Eidemüller et al. 2012, Kundrat et al. 2012, Kundrat and Friedland 2015).

In a cohort of Uranium millers, there is no significant excess mortality from lung cancer (due to radon exposure) and from solid cancers (due to external gamma irradiation (Kreuzer M et al 2015). No excess of kidney cancers was found in a French and German cohort among uranium miners (Drubay D et al. 2014).

6.2 Individual radiation sensitivity and variable predisposition for IR induced pathologies

Dose limits applied in radiation protection have been set to protect an “average person”, based on studies of risks (mostly cancer) seen in large populations such as the A-bomb survivors in Japan. For cancer induction, it is well established that there are differences in radiation sensivity between individuals and population subgroups, depending on their gender, age, genetic make-up lifestyle such as smoking, and exposures to other agents. In general, however, even though these differences are recognized, they are not specifically accounted for in the setting of dose limits for planning purposes in radiation protection practice apart from very few situations. At present, there is insufficient information to establish how large these various differences in sensitivity may be between individuals or between groups of individuals and their consequent influence on risk estimates at low dose (HLEG, 2009). Differences in radiation sensitivity between individuals, or groups, raise the ethical and policy question as to whether some individuals, or groups, are adequately protected by the present system and regulations. In order to address these policy questions, it is necessary to obtain better scientific information on the extent of variations in the sensitivity of the population, both in the sizes of of the variations and also in the proportions of the population that are affected. Therefore, research is needed to identify the factors that affect individual sensitivity to radiation risk and to obtain realistic estimates of how large the differencies may be in extreme cases and also on the spread of sensitivities in average population groups. DoReMi has contributed to the knowledge basis on individual susceptibility in the following areas:

Research in DoReMi reveals that quite a number of specific genetic and epigenetic factors are involved in the observed high variability between individuals (patients). This is of great importance for personalized diagnostics (CT) and treatments of patients (RT).

Some biomarkers have been found that determine individual sensitivity: telomere length appears to be one of those affecting secondary cancer development and/or cardiovascular disease (M’Kacher R et al. 2015, Shim G et al. 2014) , other markers indicate predisposition for cancers (CLIP2, Rb1 etc.) (Selmannsberger t al. 2015, Rosemann M et al. 2014) or non cancers (IGFB5) (Rombouts C et al. 2014)

(Age, gender and life-style as well as other exposures from physical and chemical agents have not much been worked out yet in DoReMi). Because of difficulty to get access to suitable biological samples from suitable cohorts (see SOLO). Several pilot studies have been brought on their way (CURE, CDV etc.)

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Clear differences can be demonstrated in lymphocytes from irradiated individuals by Raman spectroscopy (Maguire A et al. 2015). Raman spectrum profiling can be considered as a new potent biomarker for individual (low dose) radiation responses.

Extensive transcriptional and epigenetic profiling revealed new markers for individual and specific pathological responses (cancer and non cancers). Also, the involvement of mitochondrial and immunological dysfunction could be demonstrated.

6.3 Non-cancer effects and overall detriment by low dose radiations

The current system of radiation protection is based primarily on the protection against risk of cancer from low doses of radiation. A small additional allowance is made for possible hereditary detriment. It is well established that moderate to high doses of radiation can increase the occurrence also of a variety of non-cancer effects in exposed individuals, but for radiation protection purposes it is assumed that there is a threshold of dose below which no significant non-cancer effects (apart from hereditary disease) can arise. Recent studies have, however, called into question this assumption, particularly in respect of circulatory diseases, effects on cognitive function following radiation exposure in infancy and occurrence of opacities of the eye (cataract). In each case, epidemiological studies have suggested the possibility that these effects may arise after exposure to much lower doses than previously thought and possibly within the range of doses encountered in the use of radiation in industry and diagnostic medicine. The mechanisms behind these non-cancer effects are not well understood, and they need to be investigated, including the potential role of non-targeted effects (HLEG, 2009). In DoReMi, non-cancer effects have been studied from different perspectives:

Advances in dosimetric evaluation (interventional cardiology, Farah J et al. 2013, 2015) (interventional cardiologist survey and ELDO project: Dosimetric assessments together with suitable cohort building)

Non-linear contribution of dose rate effects to the response of endothelial cells: premature senescence (evidence for specific changes in transcriptomic, proteomic and epigenetic profiles) (Yentrapalli 2015a,b, Ebrahimian T et al. 2015)

Specific biomarker: IGFB5 (Rombouts C et al. 2014). Evidence for specific changes after low dose IR in aorta and pericytes. Neurological changes after depleted uranium contamination (in rats) involving

p53 mediated pathways (Quintens et al. 2015). Evidence for anti- and proinflammatory processes induced by low-dose IR

(Rödel et al. 2012a,b, 2013) Low dose irradiation does apparently not promote Parkinson’s diseases in

predisposed mice.

6.4 Applications for operational radiation protection - Improvement of pysical and biological dosimetry

Physical dosimetry

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convenient and cheap measure of retrospective radon and thoron exposure (alpha rays) in homes

Improvement of retrospective dose assessments: (ex: lens opacities risk estimation in interventional cardiologists)

Biological dosimetry

In addition to classical cytogenetic methods (dicentrics and rings) and MN, biomarkers (H2AX) for detecting IR exposure, telomere shortening can be used for the detection of IR induced senescence, cancer pre-conditions such as genomic instability and individual radiation sensitivity.

Analysis of telomere length and centromere loss by FISH-CT analysis.(applicable in RENEB): useful for emergencies

Raman spectroscopy can identify differences in lymphocyte populations of different individuals.

By showing that in addition to classical cytogenetic methods (dicentrics and rings) and MN, biomarkers (H2AX) for detecting IR exposure, telomere shortening can be used for the detection of IR, senescence, cancer pre-conditions such as genomic instability and individual radiation sensitivity.

Detection of non-linear responses concerning dose and dose rate effects

In, WP5, it has not been possible to define low dose induction curves for individual cancer types.

However, non-linear responses were observed concerning the induction and repair of IR induced damage (DNA DSBs), the induction of senescence and anti-inflammatory (Large M et al. 2014, Frischholz B et al. 2013) and anti-tumor responses. (in line with findings in the literature)

7 Recommendations for future lines of research based on DoReMi experience

The objective of DoReMi has been to establish a short term research agenda and to evaluate the feasibility of various lines of research on low dose risk. Most of the research conducted during DoReMi has addressed basic mechanisms relevant for induction of cancer and non-cancer effects as well as individual susceptibility to cancer. The approach for the network of excellence was to cover a variety of mechanisms and carry out feasibility studies in order to identify the most promising lines of research capable of observing effects at low doses and dose rates. It should be recognized, however, that such an approach cannot go very deeply into any single question and, despite a fairly broad coverage, several key questions were not addressed at all during the project. For example, the impact of age, sex or other exposures as risk modifiers or hereditary effects were not studied. Furthermore, the epidemiology part mostly consisted of preparation of strategies for future studies rather than conducting actual research. Therefore, gaps in knowledge still exist and long term commitment is needed to address all issues that are relevant for the low dose health risk evaluation. The extended research strategies emerging from these studies related to the long term initiative MELODI (Aerts A et al. 2014, Belli M et al. 2011, 2015) have been summarized by S. Salomaa et al. 2013, 2014a, b and are specified in the DoReMi TRAs

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and DoReMi statements as well as in MELODI SRA and statements (see corresponding websites). Here we provide an update at the end of DoReMi (2015). 7.1 Dose and dose rate dependence of cancer risk

The DoReMi research programme on shape of dose response for cancer had two overarching objectives: (i) to improve knowledge of low dose/dose rate radiation cancer risk in humans, and (ii) to improve low dose/dose rate risk projection models based on knowledge of the processes that drive carcinogenesis. Dose and dose-rate response relationships of cancer-related processes were examined and special attention was paid in cellular and tissue processes that could modify the cancer outcome (in particular, non-linear responses), such as senescence (aging), non-targeted and systemic effects and effects on immune system. Different steps in radiation carcinogenesis were addressed, ranging from initial events, stem cells and molecular pathways to preneoplastic changes and cancer among radiation worker cohorts exposed to uranium. The determination of retrospective or actual radiation doses and proper dosimetry remains a very important matter. However, because of the evidence for many non-linearities in biological responses the ‘blind’ use of radiation dose as a surrogate for radiation induced effects and health risk is questionable and needs to take into consideration the particular population and individual life and exposure conditions, in particular, for prospective radiation protection uses. The specific susceptibility of some cell types, tissues and organs to IR induced cancers and the shape of dose response curves at low doses and dose-rates continues to be an important challenge. Although it is clear that generally the risk of cancer is lower at low doses and dose rates than at high doses and high dose rates, there is great variability across organs. Cancers are mainly driven by mutations (and IR induced loss of heterozygosity) of important cancer suppressing genes in proliferative tissues leading to clonal expansion. DNA damaging agents such as ionizing radiation are acting as a switch that turn on the initiation and the promotion of carcinogenesis. After low dose exposures, numerous confounding factors very strongly interfere and influence the final outcome. As for future lines of research addressing dose and dose rate dependence of cancer risk, DoReMi concludes the following:

Increase further understanding of mechanisms in order to improve radiation protection

WR and WT values need still some refinement in the light of increasing uses of radiations of different quality and energy (X-rays, photons, protons, neutrons, heavy ions) and the increased knowledge on cell and tissue types and their interaction, respectively.

The concept of DDREF is questionable because of the existence of non-linear responses. Also, clear distinctions should be made between chronic (continuous) low dose exposures and protracted (slightly intermittent) and clearly fractionated (as in RT) exposures in mechanistic as well as in epidemiological studies.

Interactions between the genetic and epigenetic control systems of metabolic pathways affected by IR should be studied and this effects on long term reprogramming of differentiated tissues and stem cell systems. It has become clear that the induction of damage is important but for the final biological

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outcome of the radiation insult most important are the regulatory systems that activate or inactivate certain metabolic pathways involved in the IR response (example: the DDR ‘DNA Damage Response and other (regulatory) pathways) which are fully integrated in the IR cellular metabolic response network). The results are the basis of systems biology approaches and contribute to more precise modeling and improved low dose radiation health risk estimations.

IR responsive metabolic pathways should be further studied by available most recent techniques (omics, genetic and epigenetic profiling and new generation sequencing). This should give rise to the further identification of specific biomarkers. Taken up by solid bioinformatics systems biology analysis will help to understand the complex molecular (biochemical) networks and dysfunction of important metabolic nodes induced by low dose ionizing radiation.

Use systems biology analyses to relate biomarkers and cellular networks in a given biological model to functional disturbance. This may constitute an important supportive instrument for mathematical modeling and low dose health risk evaluations.

The development of specific biomarker studies is warranted: there is a need for suitable molecular biomarkers (for example: specific miRNAs involved in regulation) and specific for cell and tissue and pathology that can be used in molecular epidemiological studies.

Identify radiation responsive biological networks and perform metabolic and regulatory pathway analysis together with systems biology analysis.

Establish models of carcinogenesis based on improved understanding of underlying mechanisms (allowing the use of molecular biomarkers) linking IR-induced changes in metabolic pathways (associated with specific pathologies) to a systems biology approach and to suitable epidemiological cohorts to estimate low dose radiation health risks.

Classical epidemiological studies may be supported by molecular studies in order to reduce uncertainties in the low dose range. Such studies may include studies on genetic (transcriptional) and epigenetic profile changes (using data-and tissue bank materials such as STORE)

7.2 Non-cancer effects

In a context of a lack of epidemiological evidence for the non-cancer effects at low-dose exposures, and recognizing that (i) multi targeted biological effects observed in chronically exposed experimental models with internal emitters are quite puzzling; and (ii) no convincing mechanistic explanations are available that can account for the findings observed, the overarching strategic objective DoReMi for research on non-cancer effects was to implement a long-term, integrated approach involving several disciplines, namely, epidemiology, radiobiology, immunology and toxicology, for the purpose of risk evaluation for radiation-induced non-cancer effects. The induction of non-cancers at low doses and low dose rates is more difficult to assess than cancer. Results obtained in DoReMi suggest that indeed even doses lower than 0.5 Gy may induce cardiovascular, eye lens opacities and neurological effects, however, sufficiently sensitive biomarkers for the detection of these effects at very low doses have not yet been identified. The reason for that may be that normal cardiovascular, normal eye and neurological (brain) functions are based on the interaction of very different cell

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types of relatively low renewal rate and/or in particular cellular microenvironments that make them relatively resistant to external stresses and radiation insults although permanently submitted to accumulation of stresses during lifetime. In their functional context, apparently IR induced mutations are less important than the IR-induced and long term stress production of not fully functional cells and their elimination by apoptosis which deprives organs from proper functioning. Indeed, apoptosis in cells is usually induced at higher doses than mutations. For all non-cancer health effect outcomes, the are uncertainties and concerns about possible health effects at low doses, which would have important implications for radiation protection. More emphasis has to be put on this type of research in order to get an understanding of the mechanisms involved and their dose and dose-rate dependency, low and medium dose and dose rate responses (especially important, in medical applications), definition of the metabolic pathways affected and their regulation, the radio-sensitivity of the different cell types involved and their mode of interaction. The recommendations for future lines of research from DoReMi are:

to design well-controlled molecular epidemiology studies having in prospect the identification of markers of the initial steps of low-dose radiation-induced non-cancer health effects, the record of biological non-radiation “risk factors” connected to diseases under study, and the monitoring of pertinent blood biomarkers of biological radiation effects;

to promote, in the field of experimental radiobiology and radiotoxicology, high-throughput technologies (e.g. “omics”) and systems biology approaches that would be expected to better describe the complexity of low-dose radiation-induced tissue level responses;

to challenge the classical molecular DNA strand break paradigm in search for the mechanisms behind non-cancer effects, and in this way to promote research activities in the field of cell physiology (i.e. cell senescence, long term cell phenotypic changes), immunology and the radiobiology of intercellular communications and signaling;

to differentiate radiation-induced tissue and cell responses due to adaptation to the radiation stress, from true adverse alterations, involved in pathological processes;

to support research on mechanisms of radiation action, adopting a multi-scale/system biology approach and putting the emphasis on the relationship between initial stochastic track structures of low and high LET radiations, early chemical/biological processes and long term pathophysiological effects such as inflammatory response; and finally

to assess whether there are scientific arguments for replacing the classical “threshold” paradigm for non-cancer effects with the “non–threshold” paradigm.

7.3 Individual radiation sensitivity

An important objective for DoReMi has been to provide a scientific basis for decision-making on the inclusion of individual sensitivity as a modifier of risk at low doses. Current risk models use LNT to extrapolate from high to low doses. By convention this assumes an equal risk distribution amongst all members of an exposed population. Consequently, the risk assessments delivered by the models can only represent the average risk across the population. Despite inbuilt safety margins the present state-of-

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the-art knowledge does not allow us to assume that exposed individuals with a greater natural predisposition due to age, gender, genetic background or interactions of these variables with lifestyle and/or environment are effectively protected. Consequently, we have striven to understand how, and to what extent, individual susceptibility influences the carcinogenic effects of low doses and low dose rates. Research within DoReMi has confirmed that individual sensitivity is an important parameter response to low dose radiodiagnostic or radiotherapeutic exposures and that many very personal parameters (genetic, epigenetic, metabolic and physiological, immunological status, age, gender, lifestyle, specific multi-exposure conditions…) may affect individual low dose radiation responses. At low radiation doses and dose rates, it appears that cells and tissues (organs) show a large variety of responses depending on radiation energy and quality, cell and tissue type, genetic and epigenetic regulatory functions, degree of differentiation, metabolic and physiological state, immunological status, age, gender i.e. , (individual sensitivity and susceptibility) …but also on external confounding factors lifestyle, nutritional state, and exposures to other damaging (toxic) (physical and/or chemical) agents. Thus, the responses at low doses are the result of multifactorial integration and very complex interactions that can be highly and differentially modulated by all these parameters. In this condition, the responses are very complex, cells and tissues always try to do their best to survive and to keep as much as possible genomic integrity in spite of all internal (metabolic) and external stresses. This explains why low dose responses are generally much more variable than high dose responses. Indeed, much more parameters have to be taken into account. DoReMi project clearly brought to light and confirms the complexity of low dose and low dose rate responses. DoReMi demonstrated that indeed many metabolic pathways and factors are activated and can modulate the ultimate (final) radiation response. Mechanistic studies in DoReMi provided evidence for the existence of non-linear responses at low doses and dose rates. This leads DoReMi to call for a great deal of caution in the evaluation of low dose radiation health risks. Clearly, ‘blind’ extrapolations from high dose effects to low dose effects are not fully applicable. Instead, as much as possible specific exposure conditions, the individual situation and environmental contexts have to be taken into account and are crucial for human health. For the future studies, DoReMi recommends:

To develop systems model on the short-term and long-term responses to low doses of radiation so that differences in the response pathways can be detected and used to predict outcome

Cell type and tissue (organ) specific research should be fostered knowing the complexity of genetic and epigenetic make-up and the importance of cell-cell interactions and the microenvironment (for cancers and non cancers). Genetic and epigenetic profiling should help.

To identify biomarkers of susceptibility to radiation associated cancer and non-cancer effects that can be applied in molecular epidemiological studies

To investigate mechanisms by which age, sex and lifestyle and co-exposures to other agents may impact radiation risk

Use of well-defined epidemiological cohorts (sound dosimetric and definition of pathologic conditions and confounding factors and multi- or mixed exposure conditions) in conjunction with relevant molecular studies that feed into low

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dose health risk evaluations. Medical cohorts (therapy and diagnostics) and industrial cohorts should be preferable to follow because of good dosimetry and health assessment. Medical cohorts are in most cases easier accessible in terms of available biomaterial and ethical considerations.

7.4 Roadmap for future low dose risk research

Looking into the major trends in radiation research, the early and mid 1900´s was the era of basic discoveries such as X-rays, radioactive decay and development of radiation and nuclear physics and health physics. The discovery of DNA structure and DNA repair in 1950´s and 1960´s boosted the studies on DNA damage and DNA damage response that remained in the center of radiation research for a long time. Later in the last century, oncogenes and tumor suppressor genes were discovered which also increased our understanding on radiation carcinogenesis. More recently, there has been a rapid development of molecular biological methods. The milestone of sequencing the whole human genome was achieved early this century. Soon it was discovered that there are small variations in nucleotide sequences between individuals and single nucleotide polymorphisms (SNPs) may account for some of the differences in radiation sensitivity between individuals. The DNA paradigm of radiation effects was amended by discovery of non-targeted effects (radiation-induced genomic instability and bystander effects) some years ago. Since then, molecular level studies on intra- and intercellular signaling have boosted mechanistic investigations on tissue responses. This is becoming increasingly important along with the accumulating evidence on various non-cancer diseases being associated with fairly low doses of radiation and the need to consider non-DNA damage responses as possible mechanistic explanations. Currently, omics technologies together with bioinformatics are capable of handling large amounts of biological information. The rapid development of molecular biological methods and informatics is now enabling whole new fields, such as systems biology and molecular imaging at organism level. The long term vision of integration of radiobiology and epidemiology is becoming a reality. Molecular epidemiology is also part of the vision of DoReMi and MELODI. At the end of the project and in the light of the scientific progress achieved in DoReMi we now propose a Roadmap for future studies to be further developed by MELODI and others (Figure 4). The roadmap presents scientific and technological developments as a basis that enables research on mechanistic understanding of radiation effects. Uncovering mechanisms related to tissue damage, alterations in physiology, modification of signaling and metabolic networks and systems biological changes induced by radiation support the development of biomarkers that can be used for health risk assessment. Such biomarkers should cover biomarkers of exposure and metabolic and pathological changes and they need to be validated for molecular epidemiological studies. In parallel, they will be used in the development of risk models that are based not only on physics and chemistry but also radiobiology. The ultimate objective is to improve the robustness of the current system of radiation protection by solid science. The impact on society is via better and more individualized protection of people. Roadmaps developed for the DoReMi TRA and DoReMi statements (and also for the MELODI SRA and MELODI statements) turned out to be very useful for short- and long

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term planning and practical performance of prioritized research projects concerning low dose health risk research in Europe. Although scientific projects can be precisely defined in their actual duration by financial constraints the scientific evolution of the project is less foreseeable, and rarely ends with the official end of the project. Especially, in low dose health risk research scientific programming is difficult, because many parameters (some of them still unknown) may interfere and by definition, effects from chronic low dose exposures or contaminations as well as pathological outcomes are very long term mechanistic and epidemiological research issues. DoReMi has been able to perform important feasibility and pilot studies, now other projects are bound to follow these up if society really wants get scientifically well-founded answers to the most burning and still open questions concerning low dose and low dose rate radiation induced health risks in humans and the improvement of radiation protection of the general public, industrial and medical workers, patients and individuals.

Figure 4: Roadmap for future low dose health risk research and radiation protection

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8 Results obtained in the Operational WPs of DoReMi

8.1 Infrastructures: operational progress, conclusions, possible future developments

One Work package WS of DoReMi (WP4) was dedicated to addressing the basic practical needs of low dose research in terms of relevant infrastructures (see Sisko’s talk 7th MELODI WS on DoReMi, L. Sabatier)) Irradiation facilities

A large selection of facilities exists, but some limiting factors were noted: irradiation facilities were not always adapted to low doses, open access and support for handling biological samples not always ensured.

Generally, there was a shortage of low dose/dose rate facilities in Europe In particular, facilities were lacking to address radiation quality issues (Below

20MeV/u and above 100 MeV/u, microbeams) Facilities to work on internal contamination were rare, difficult to assess and not

clearly defined to allow optimal usage.

Epidemiological cohorts - Databases and biobanks In dedicated DoReMi WS, a large number of available cohorts were assessed and

the most suitable for low dose health risk research prioritized (see Pernod et al. 2011).

Main challenges were: to cope with variable dosimetry quality, to assure the availability of biological material (only available in about 25% of the cases) and to overcome the difficulties to obtain biological samples for adjacent molecular studies and access to the samples.

It was noted that National birth cohorts could provide interesting opportunities for future research activities. Of course, for molecular and mechanistic studies associated with epidemiological studies the establishment and use of data-and biobanks was considered to be essential. In this respect, it noted that the Data and Biobank STORE joined the DoReMi project identified as a new, potentially interesting resource. In the future, it appears to be of great interest to establish collaboration with BBMRl and to assess the feasibility for possible low dose research projects in the near future..

Analytical platforms National hubs ESFRI platforms

Progress achieved within the DoReMi project Following the 1st competitive call of DoReMi in 2011 were obtained:

Open access to the FIGARO low dose rate facility 1st competitive call (2011)

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o Upgrading of existing facility for the irradiation of fish for mouse studies at the Norwegian University of Life Sciences

o Validation of dosimetry (NRPA), Acquisition of authorisation for animal studies (NIPH)

o Access to DoReMi members for pilot experiments, and through internal calls resulting in two projects (OSTINATO: Parkinson Disease, CLOGIGAT: Gastrointestinal tumors)

Low dose/dose rate radiation effects in brain cancer risk 1st competitive

call (2011) o Collaboration between the ENEA (Italy) and IES (Japan) o Ptch1+/- model transferred to IES for low dose rate exposure, samples

harvested and sent back to the ENEA for analysis o Effects on CNS tumorogenesis (rate, pathophysiology, DNA methylation)

Low dose/dose rate gamma irradiation facility for in vitro biological

systems 1st internal (2011) o Construction of a low dose gamma irradiation facility for in vitro studies o Located at the ISS in Rome

8.2 Recommendations for infrastructures (DoReMi)

Most of these recommendations are already worked on by the OPERRA project (2013-2015) and the new wide-ranging project CONCERT in the framework of the European Joint Project (EJP) in HORIZON2020. Infrastructures (DoReMi recommendations)

Promote the accessibility and use of available infrastructures among researchers (through dissemination activities such as Education& Training, Website information and workshops)

Promote efforts for harmonization and integrative research through the use of available infrastructure (through dissemination activities such as Education& Training, Website information and workshops)

Identify as yet missing infrastructures that need to be developed Improve reproducibility by supporting infrastructures that meet essential quality

criteria (example: ‘Omics’) Improve sustainability of rare but essential infrastructures (example: internal

contamination ability for radon etc.) Improving access by focusing on infrastructures and facilities with recommended

criteria Rationalizing the use of existing infrastructures and available financial resources Attract and train scientists to use the recommended infrastructures

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Figure 5: Roadmap for infrastructures

8.3 Education and Training: operational progress, conclusions, possible future developments

Already the HLEG in 2009 recognized that there is an urgent need to keep and develop competences in radiation research and radiation protection at the European level in order to cope with future challenges in this domain of research and societal needs. Therefore, the DoReMi NoE included a specific work package (WP3) to provide and coordinate high-level training for research scientists and a career structure that will attract and retain top-level graduates within the research discipline. During the last 6 years, DoReMi has taken several new ground-breaking initiatives. Several meetings, in conjunction with the yearly MELODI workshops defined the problems and proposed solutions. First, the creation of an Integrated Training and Education Network (ITEN) was initiated. The initial concept was of a ‘virtual Institute’ that would act as a coordinating hub to construct courses and programmes out of existing E & T activities, and possibly become an independent legal entity and a spin-off enterprise. The format finally adopted was of a network formed from the members of DoReMi under the management of the Training and Education Committee (TEC). It was recognised that the MSc level was key to attracting students from a science Batchelor’s degree into the low-dose risk research area, possibly steering the most capable students into PhD research then career positions. One of the objectives of WP3 was to facilitate the formation of a Masters course in radiobiology, to replace the European Masters course that was set up and run by Klaus Trott at UCL, London, from 1992 to 2012. Many of the scientists now working in the field received their introduction to radiobiology during the 20 years that the course ran. Accordingly, a

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Masters course in radiobiology was developed by DoReMi partners at the Technical University of Munich and held the first enrolment of students in 2015. A regular DoReMi E&T Forum held in conjunction with the annual MELODI Workshop was set up to keep up with most recent developments, seek new opportunities, launch new activities and courses through effective integration of different DoReMi partners and to work on long term sustainability. The Forum served (1) to bring together key persons from key institutions and assure proper networking, (2) to define the E&T needs of the low dose research community, (3) To get mutual recognition of partners and resolve practical issues (4) review and discuss current and future activities (5) prepare new E&T iniatives (course modules, summer schools in networked programmes (6) establish links to other platforms. DoReMi WP3 launched a wide range of 1-3 week courses and yearly training events provided and organized by DoReMi partners.. Five series of DoReMi course free for students were held during 2010-2015 attracting around 500 students in total. The courses covered a great number of pertinent topics:

Human radiation genetics Molecular radiation carcinogenesis Radiation epidemiology and radioecology Cellular effects of low doses and low dose-rates with focus on DNA damage and

stress response Interdisciplinary radiation research focussing on radiation protection Modelling radiation effects from initial physical events Non-cancer effects of low dose radiation Environmental Radiobiology Inter-individual responses to low dose ionizing radiation: from damage

formation to biomarkers Data interpretation and uncertainty analysis

The courses are related to the DoReMi key questions (see main achievements here below) as follows: Response to key question 1: What is the dependence on energy deposition?

Course: Modelling radiation effects from initial physical events, UNIPV, Pavia, Italy.

Response to key question 2: What is the dependence on dose rate? Course: Cellular effects of low doses and low dose-rates with focus on DNA

damage and stress response. SU, Stockholm, Sweden. Course: Molecular consequences of low dose and low dose-rate exposures:

impact of individual susceptibility on outcome and biomarker development. IC/CEA, Paris, France

Response to key question 3: What are the tissue sensitivities? Course: Radiation‐induced effects with particular emphasis on genetics,

development, teratology, cognition as well as space‐related health issues. SCK-CEN, Mol, Belgium.

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Course: TIETO Non-cancer effects of low dose radiation. HMGU, Neuherburg Germany

Response to key question 4: What is the modification of risk by genetic and epigenetic factors and gender?

Radiation‐induced effects with particular emphasis on genetics, development, teratology, cognition as well as space‐related health issues. SCK-CEN, Mol, Belgium.

Molecular radiation carcinogenesis. HMGU, Neuherberg, Germany. Molecular Consequences of low dose and low dose-rate exposures: impact of

individual susceptibility on outcome and biomarker development. IC/CEA, Paris, France

Response to key question 5: What is the effect of age on risk? No specifically dedicated course

Response to key question 6: What is the effect of lifestyle and/or other exposures on risk?

No specifically dedicated course Response to key question7: What is the effect of physiological state?

No specifically dedicated course Response to key question 8: Is there a hereditary component in risk?

Course: Molecular Consequences of low dose and low dose-rate exposures: impact of individual susceptibility on outcome and biomarker development. IC/CEA, Paris, France

Course: Molecular radiation carcinogenesis. HMGU, Neuherberg, Germany

Response to key question 9: What is the role of non-targeted effects in health risk? No specifically dedicated course

Since E&T is a “public good” it heavily relies on funding support. As part of the DoReMi TRA and the dissemination programme the DoReMi WP2 helped with some initial funding for inviting experts in E&T, however, E&T activities need long term sustainability allowing proper managing, organisation and funding. For this, DoReMi was instrumental in the formation of the MELODI E&T Working Group as a continuing body responsible for promoting E&T. The MELODI Board of Directors formally set up the MELODI E&T WG in February 2014 with the task of producing a strategy for management and funding. As such, the E&T WG in contributing to the MELODI SRA and devolping its own E&T SRA the E&T WG puts into place an actively managed programme of workshops, seminars, summer schools, courses etc. which are fully integrated in the design and funding structure of all research activities. In particular, it promotes support for students and young scientists, dissemination and knowledge management as well as coordination and collaboration of E&T providers in permanent dialogue to other platforms. As strategy and recommendation for funding, the support from stakeholders, from the EC through RTD projects, and possible national and individual funding have been considered. At present, most of the recommendations have been successfully implemented in the CONCERT EJP in workpackage 7 for the next five years. WP7 is

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setting priorities for calls in radiation protection research and integration activities including all areas covered by MELODI, ALLIANCE, NERIS, EURADOS, Medical associations and social sciences and humanities. With this, E&T activities will continue to be an integrated part of on-going radiation and radiation protection research activities and future funded European projects.

8.4 Recommendations for Education & Training and Infrastructures

Although these activities have already been taken up by the OPERRA project and the future, wide ranging project CONCERT, we are listing here some aspects that should be reinforced taking into account the experiences from DoReMi:

Incorporation of E&T activities and development of sustainability through MELODI, OPERRA and CONCERT in the framework of Horizon 2020.

Adaptation of courses to the next generation of researchers Dissemination of new E&T work to the research community (Further

development of Website support) Inclusion of new areas of expertise, technologies, disciplines (omics,

bioinformatics, . . .) in E&T activities Development of awareness and use of infrastructures (Irradiation facilities,

epidemiological cohorts,

Develop access and increase the visibility of available infrastructure

9 Concluding remarks (relationship DoReMi/MELODI)

DoReMi WP2 set out to ‘structure’ MELODI. From the start in 2010 DoReMi WP2 pursued two objectives, (1) to get DoReMi organisatory and scientifically organised, and (2) to get MELODI structured organisatory and scientifically. The focus was as follows:

1. Launching the Joint Programme of Interaction (JPI) (survey of interested scientists and organisation interested in low dose research, attracting these scientists and integrating them around the joint scientific research project, i.e. the NoE DoReMi. This turned out to provide a solid basis for the development of MELODI.

2. Establishing a Research Agenda for the Joint Programme of Research (JPR) (establishing the TRA for DoReMi and at the same time the SRA for MELODI). In this way, scientific objectives could be regularly adjusted to scientific developments and results put forward within DoReMi. Regular updating of SRAs and cross talks between SRAs turned out to be very important to keep up with most recent research developments and were essential for the scientific consolidation of MELODI.

3. Establishing the Joint Programme of Spreading Excellence (JPSE) (promotion of brainstorming, consensus meetings, think tanks, workshops and dedicated meetings (Training & Education, Infrastructures; setting up the DoReMi Website and in parallel helping to set up the MELODI Website. Both turned out to be very important tools for dissemination and very useful for the joint functioning and networking of DoReMi and MELODI.

4. Seeking and providing support from stakeholders and experts (by inviting them to important workshops and dedicated meetings). This has been an important contribution because this ensured an internationally founded re-adjustment of initial scientific questioning, enlargement of multidisciplinarity and inclusion of

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new research aspects into the DoReMi and MELODI framework of low dose radiation and radiation protection research in Europe.

From the start, MELODI (long-term) tried to overcome DoReMi (short-term). In the first years, DoReMi was very welcome to help in organisatory matters (establishing working procedures, the MELODI SRAs and derived short scientific statements for the preparation of new calls at the EU level). In fact, DoReMi always sent also its own DoReMi TRA to MELODI for information and approval. After 3 years of DoReMi, MELODI started to drastically speed up its own development by defining more and more an own independent way (always in connection with DoReMi), this probably happened because of the discrete omen of the forthcoming, very extensive enlargement of European research activities in Radiation protection in the European Joint programme to be boosted in the framework of HORIZON2020 (giving a quick rise to OPERRRA 2014 and CONCERT 2015). Meanwhile, MELODI took care to set up its own working group for establishing its own SRA and SRA statements though WP2 was still accepted as a contributing guest visitor in the SRA WG. Also, other working groups (T&E and infrastructures) were set up. Also, in the newly built MELODI scientific Committee (set up earlier on with the help of DoReMi WP2) WP2 was only called upon as an invited contributor. Only lately, in May 2015, WP2 became a full member of the MELODI SC, probably to keep up with the scientific links with DoReMi. From this, it is clear that MELODI underwent a very fast development, in the first period in parallel to DoReMi, in the later period by trying to overcome DoReMi and at the same time, by largely widening the scope of European research activities far above the initial concept of low dose research and radiation protection. Now there is some hope that MELODI will be able to translate some of the messages from DoReMi into sound future long-term projects. However, with DoReMi the low dose health risk research is not yet finished. DoReMi studies opened the way with the aim to improve radiation protection through intense, well-focused low dose research concerning individual sensitivity, immunological studies, mixed exposures following mechanistic, systems biology and molecular epidemiological approaches allowing to decrease existing uncertainties and to improve radiation health risk evaluations.

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Annex 1: DoReMi 1st Version TRA derived Subquestions (2010)

WP5 WP5.1 :

are processes underlying or contributing to radiation-induced carcinogenesis uniform over the entire dose range

are there non-linear responses? are the responses dependent on LET and radiation quality?

WP5.2:

are there non-targeted and systemic (inflammation, immune function) processes involved?

(see also WP7) WP5.3:

what are the key events in neoplastic transformation (normal- tumor cells) and in mouse leukaemogenesis

are there suitable (molecular) biomarkers to indicate radiation-induced tumoorigenesis (see also WP6)

WP5.4:

can suitable models for carcinogenesis be developed that integrate mechanistic and epidemiological studies? (example: lung cancer?)

WP5.5:

what is the health risk from internal emitters (contamination)? can be a best suitable study design proposed?

WP5.1-WP5.5: overriding questions:

What is the involvement of stem cells? What is the role of radiation quality? (Dose and dose-rate?) What is the role of DNA damage, intra-and intercellular signaling and repair? What is the involvement of genomic instability? What is the role of genetic predisposition? Can be more basic knowledge provided concerning the mechanisms driving

radio-carcinogenesis and allowing low dose/low dose rate risk modeling? Priority outcomes:

High standard dosimetry related data Validation of signatures and strengthening of attributability (radiation- induced

cancers) Proper conservation of biosamples for further studies (WP3) New biomarkers from molecular and new generation sequencing data Understanding of stem cell involvement in the carcinogenic process Progress in in vivo follow up (genetic and epigenetic imprinting) and real time

imaging of radiation carcinogenesis Integration of systems biology approaches in models on radiation-induced

carcinogenesis and epidemiological studies to facilitate health risk assessments

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WP6

Does individual variability significantly affect low dose radiation and radiation quality responses such as the induction of cancer and non-cancer diseases?

Are there molecular approaches that can be validated by in vivo animal studies and epidemiological studies

Is there a dependence of individual radiation sensitivity on dose and dose rate and radiation quality?

What are the influences of gender, age, genetic and epigenetic factors, lifestyle (smoking, alcohol consumption..) and co-exposures and other confounders on individual sensitivity?

Are also physiological parameters (hypertension, obesity…), reproductive (hormonal) factors (and immunological factors) play a role?

Are there suitable biomarkers to determine different types of exposures (radiations, chemicals….?

With the help of (genetic and epigenetic ) biomarkers can one define radiation sensitive sub-populations?

WP6.1:

Are there suitable cohorts for supporting joint with molecular studies an epidemiological approach to individual radiation sensitivity? (mammography, children CT scans), uranium miners, Mayak workers, nuclear workers)

WP6.2:

Is there a specific molecular signature for the sensitivity of children to Chernobyl accident, radiation induced thyroid cancers?

what is the contribution of individual genetic variability on cancer development taking into account different cell types and tissues, age effects and radiation quality)? WP6.3:

Can specific modifier or susceptibility genes (and or variants) be identified that are associated with varying degrees of IR sensitivity and that can be validated in animal models?

WP6.4:

Are there specific modifier genes in humans that are relevant for susceptibility to radiation-induced osteosarkomagenesis, mammary tumors or medulloblastoma that can be validated in animal models using high or low dose rate exposure?

WP6.5

Can epigenetic (chromatin related) factors be identified contributing to individual IR induced cancer susceptibility?

WP6.6: Can a pilot study be performed to answer the question whether specific genetic factors influence individual susceptibility to low dose radiation-induced cancers? Does an individual susceptibility exist for IR induced non-cancer diseases (cataract, neurological and cardiovascular disorders) (in collaboration with WP7)?

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Can the familial predisposition (cancer among parents and siblings) be assessed? WP7 Non-cancer effects

What is the biological impact of different radiation qualities and radiation dose levels in terms of perturbing homeostasis and induction of pathological non-cancer effects (cardio- and cerebro-vascular diseases, neurological and cognitive effects, lens opacities)?

What is the importance of acute versus chronic r fractionated radiation exposures for non-cancer effects?

What is the molecular basis to expect that low dose radiation can cause or modulate pathological non-cancer effects?

Can molecular alterations of cellular homeostasis, redox potential and energy metabolism induced by low dose radiation induce or promote non-cancerous diseases?

Additional overriding questions:

What do we know about the radiation sensitivity of disease relevant cell types (epithelial cells, cells of central nervous system, stem cells..)?

To what extent oxidative and genetic damage contributes to non-cancer effects? Can relevant biomarkers be developed from transcriptomic, proteomic and

metabolic analyses and physiological markers? Can relevant biomarkers be identified (from proteomics and gene expression

data) that may be useful for molecular epidemiological studies? Can a systems biology approach be used to explain specific non-cancer

responses? Are low dose exposures relevant for the induction of non-cancer effects

(diseases)? Can the dose-responses be modeled? Play genetic variations, effects of age and radiation quality (dose and dose rate,

mixed fields) a role in individual radiation sensitivity in terms of the induction of non-cancer effects?

WP7.1

Can priorities be set for exploring IR-induced vascular effects, cognitive effects and lens capacities?

WP7.2

Can a molecular epidemiological study be conducted on low dose IR induced vascular damages (circulatory diseases)?

WP7.3 (cardiovascular)

Is there a threshold for induction? What is the contribution of radiation dose, inflammatory (immunological) effects,

cellular signaling, cellular senescence? What is the role of endothelial, smooth muscle cells, bon marrow progenitor and

stem cells?

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Are vascular effects induced that can be revealed by epidemiological children CT studies?

Can high throughput technology (proteomics) be used to get mechanistic insights into the radiation response of the endothelium after acute and chronic exposure including internal contamination with radio-nuclides ?

WP7.4 (lens opacities)

Can a threshold dose for the induction of lens opacities (posterior sub capsular) be determined?

Can one define the risk of cataract induction by IR? Can lens opacities can be detected in a cohort of interventional cardiologists

chronically exposed to IR <150 mSv? What is the involvement of DNA damage, protein cross-linking, disruption of

membrane channels and ion pumps as well as genetic factors such as Rad9 and ATM in IR-induced cataractogenesis?

WP7.5 (cognitive and neurological effects)

Can we increase our knowledge on the molecular mechanisms of cognitive effects induced by acute and chronic radiation exposures?

What are the effects of internal contamination? What is the contribution of oxidative stress to changes induced in

neurotransmission and neuromodulation? Questions derived from TRA versions 1 and 2: WP3 (Education & Training) WP3.1:

Can a sustainable Integrated Training and Education Network (ITN) be developed?

WP3.2: Can a specific low dose risk ITEN be set up promoting (MSc or PhD level)

university courses and radiation protection related training events (Bologna-accredited) as well as in particular topics (ad-hoc workshops and one-off specialist courses)?

WP3.3: Can sustainable funding for such courses be developed (in collaboration with

WP2)? WP3.4:

Can a special FORUM be organized for E&T coordination and integration in collaboration with MELODI?

WP3.5: Can new funding of training activities in ITEN developed in collaboration with

MELODI and in the framework of the EC-funded project OPERRA? WP4 Infrastructures

Can external (low dose, low dose rate, neutron charged particle beams, microbeams) and internal radiation facilities assessed and their access and use promoted?

Can suitable data and biobanks be assessed, made accessible and promoted for optimized radiation research?

105

Can suitable cohorts be identified and/or set up for well-defined epidemiological (classic and molecular) studies?

Can suitable platforms for high throughput analysis be assessed and their access and use promoted?

106

Annex 2: DoReMi tasks and publications related to each task

Table 1: WP3 Education and Training program

Task Work Starting

3.1 Preliminary investigative work prior to identify the format of the ITEN

2010

3.2 Set up a new low-dose risk ITEN following the recommendations from the previous task

2010

3.3 Develop sustainable funding in collaboration with WP2 2010

3.4 Oversee management of ITEN during the period of transition to sustainable funding

2010

3.5 Funding training activities 2010

The four tables below show the enlargement of the WP’s: Table 2: WP4 Infrastructures program enlargement

Task Work Starting

4.1 Survey of existing facilities for low dose risk research 2010

4.2 Characterization of infrastructure needs and roadmap of implementation

2010

4.3 Implementation of DoReMi support activities for shared infrastructures

2010

4.4 Development and implementation of access to Infrastructure 2010

4.5 Open Access to the UMB low dose irradiation facility (FIGARO)

2011

4.6 Dose/Dose-rate Radiation Effects in Brain Cancer Risk (DDRE-BrainCancer)

Tanori M et al. 2013 4.6 Developmental and oncogenic

radiation effects on neural cells and their differentiating

progeny in the mouse cerebellum. Irradiation of newborn

Ptc11/2 mice dramatically increases the frequency and

shortens the latency of MB.

2011

4.7 Low dose/dose rate gamma irradiation facility for in vitro biological systems (LIBIS)

2012

4.8 Integration of STORE into DoReMi as a trustable and viable database and/or pointer to biobanks and ascertain sustainability

2012

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4.9 Provision of ion microbeam irradiation facility SNAKE (MicroRAD)

Drexler GA et al. 2015 4.9 Live cell imaging and damage to

subcellular structures

2013

4.10 Laboratory infrastructure for retrospective radon and thoron dosimetry (RETRODOS)

Dimitrova I et al. 2016 4.10 Influence of the type of CD

case on the track density distribution in CDs exposed to

thoron

Georgiev S et al. 2016 4.10 and 5.5 Retrospective Rn-220

measurements by compact discs.

Pressyanov D et al. 2015 4.10 and 5.5 Optimization of

etching conditions for CD’s/DVDs used as detectors for

222Rn

2014

Table 3: WP5 Shape of dose response program enlargement

Task Work Starting

5.1 Phase – shifts in responses and processes at high/low doses and dose rates

Kabacik S et al. 2015 5.1 and 5.2 ATM status dependent

miRNA expression after IR. Different expression of

protein-coding genes in health and AT donors at 2h, but

not 24h after IR (2-5 Gy). There is a linear dose response

of the genes at 24 h. Some miRNAs are responsive to IR in

a dose and time dependent manner. There is upregulation

of FAS-AS1 lncRNA by IR in an ATM dependent manner.

Large M et al. 2014 5.1 Non linear regulation of ROS

production and SOD activity in endothelial cells (EA.hy926

HUVEC derived cells) contribute to non-linear

discontinuous dose-response relationship of gH2AX (DSB)

foci detection.

M’kacher R et al. 2015 5.1 and 6.2 Telomere shortening

may be taken as a prognostoc factor for cardiovascular

disease post-radiation exposure.

M'kacher R et al. 2014 5.1 New tool for biological

dosimetry: reevaluation and automation of the gold

standard method following telomere and centromere

staining.

M'kacher R et al. 2015 5.1 Detection and automated

scoring of dicentric chromosomes in nonstimulated

2010

108

lymphocyte prematurely condensed chromosomes after

telomere and centromere staining.

Shim G et al. 2014 5.1 and 6.2 Review : Cross-talk

between telomere maintenance and radiation effects.

Telomers are key players in the process of IR-induced

carcinogenesis.

5.1.1 Low dose Gene Expression signature (LoGiC) 2011

5.2 Assessing the relative contribution of targeted (DNA), non-targeted and systemic processes to radiation carcinogenesis

Acheva A et al. 2014 5.2 3-D models are relevant for

cancer, non cancer, tissue sensitivity studies and modeling

purposes

Babini G et al. 2015 5.2 Investigation of radiation-induced

multilayered signalling response of the inflammatory

pathway.

Babini G et al 2015 5.2 In vitro γ-ray-induced

inflammatory response is dominated by culturing

conditions rather than radiation exposures.

Campa A et al. 2013 5.2 Cancer model focusing on cell

communication and non targeted effects

Kabacik S et al. 2015 5.1 and 5.2 ATM status dependent

miRNA expression after IR. Different expression of

protein-coding genes in health and AT donors at 2h, but

not 24h after IR (2-5 Gy). There is a linear dose response

of the genes at 24 h. Some miRNAs are responsive to IR in

a dose and time dependent manner. There is upregulation

of FAS-AS1 lncRNA by IR in an ATM dependent manner.

Morini J et al. 2015 5.2 Radiosensitivity in lymphoblastoid

cell lines derived from Schwachman-Diamond Syndrome

patients.

Rödel F et al. 2012a 5.2, 5.2.1 and 7.6 Modulation of

inflammatory immune reactions by low doses of IR 0.5-0.7

Gy in endothelial, mononuclear and polynuclear cells. Non

linear biphasic responses are seen.

2010

5.2.1 Modulation of Inflammation by low and moderate dose Ionising Radiation (ModInIR)

Candeias SM, Gaipl US 2016 5.2.1 The immune system in

cancer prevention, development and therapy.

Derer A et al. 2015 5.2.1 and 7.6 Radio-immunotherapy-

induced immunogenic cancer cells as basis for induction

2011

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of systemic anti-tumor immune responses-pre-clinical

evidence and ongoing clinical applications.

Derer A et al. 2015 5.2.1 Immuno-modulating properties

of ionizing-radiation: ratyionale for the treatment of

cancer by combination radiotherapy and immune

checkpoint inhibitors.

Dieriks B et al. 2011 5.2.1 Adaptive responses on DSB

induction and cytokine secretion after 0 .1- 0.5 Gy and 24h

show upregulation of cytokines GM-CSF, IL6, IL8, TGF

beta. However, IL6 and TGFbeta are not responsible for

adaptive response. The adaptive response alters the

gH2AX spot size and DSB repair kinetics.

Frey B et al. 2012 5.2.1. Abscopal antitumor immunity and

immunogenic tumor cell death in cancer therapy.

Frey B et al. 2012 5.2.1 Modulation of RT effects by

hyperthermia activation of natural killer cells and

phagocytes.

Frey B et al. 2012 5.2.1 Induction of immunogenic

potential in colorectal tumors by CT and RT.

Frey B et al. 2014 5.2.1 Induction of immunogenic cell

death by IR. IR induced systemic antitumor responses can

be boosted by additional immune therapy.

Frey B et al. 2015 5.2.1 and 7.6 Inflammation can be

modulated by high and low high doses of ionizing

radiation with implications in benign and malign diseases.

Frischholz B et l. 2013 5.2.1 and 7.6 Low dose RT (0.5 or

07 Gy) in mice reduces the inflammatory phenotype in the

more radiosensitive macrophages of Balb/c mice.

Gaipl US et al. 2014 5.2.1 RT induced non targeted effects,

induction of immune modulating danger signals (hsp70,

ATP, HMGB1) by X-rays. Antitumor and immunogenic

effects are induced by IR.

Kötter B et al. 2015 5.2.1. Hypofractionated irradiation

(RT)stimulates cell death and subsequent dedritic cell

activation in caspase-3 proficient (p53 wt) breast cancer

cells with low immunogenicity.

Kulzer L. et al. 2014 5.2.1. RT induces immunostimulatory

forms of tumor cell death. The supernatant of fractionated

RT irradiated tumor cells resulted in a significant

increased secretion of immune activating cytokines

(IL12p70, IL8, IL6, TNFalpha) in comparison to single

dose treated tumor cells. Norm and fractionated RT

110

induces fast human colorectal tumor cell death with

immunogenic potential (triggering DC maturation and

activation in vitro). All this may help to improve RT.

Manda K et al. 2012 5.2.1 Protracted low dose IR can

result in radio-resistance, but immunosuppressive effects

of chronic low dose IR are also reported, with

sensitization of certain cell types (see IR effects on the

interaction of DC and T cells).

Mavragani IV et al. 2015 5.2.1 and 7.6 Laskaratou DA,

Frey B, Candeias SM, Gaipl US, Lumniczky K, Georgaklilas

AG. Key mechanisms involved in ionizing radiation-

induced systemic effects. A current review.

Muth C et al. 2015 5.2.1 Primary glioblastoma

muoltiforme tumors and recurrence. Comparative

analysis of the danger suignals HMGB1, HSP70 and

calreticulin.

Rödel F et al. 2012a 5.2.1, 5.2 and 7.6 Modulation of

inflammatory immune reactions by low doses of IR 0.5-0.7

Gy in endothelial, mononuclear and polynuclear cells. Non

linear biphasic responses are seen.

Rödel F et al. 2012b 5.2.1 and 7.6 Review on the

immunomodulatory properties of low dose RT, with a

maximum effect at 0.5 -0 .7 Gy.

Rödel F et al. 2013 5.2.1 and 7.6 Review on anti-

inflammatory activities at <1Gy, and induction of harmful

side effects, IR induced immune modulation or induction

of anti-tumor immune responses at higher doses.

Rubner Y et al. 2012 5.2.1 and 7.6 IR contributes to the

induction of anti-tumor immunity

Rubner Y et al. 2014 5.2.1. Fractionated RT induces

immunogenic cell death and Hsp70 release in p53

mutated glioblastoma cell lines.

Schauer C et al. 2014 5.2.1 Neutrophils recruited to sites

of inflammation undergo oxidative burst and form

neutrophil extracellular traps (NNETs). NETs promote the

resolution of neutrophilic inflammation by degrading

cytokine and chemokines and disrupting neutrophil

recruitment and activation.

Wunderlich R et al. 2014 5.2.1. Low and moderate doses

of IR up to 2 Gy modulate transmigration and chemotaxis

of activated macrophages, produces an anti-inflammatory

cytokine milieu, but does not affect viability and

111

phagocytic function of mouse macrophages.

5.3 The dynamics of pre-neoplastic change and clonal development Abou-El-Ardat K et al. 2011 5.3 In thyroid cancer cells

62.5 mGy upregulate p16. 0.5 mGy cause senescence

without involvement of p16, p21

Abou-El-Ardat K et al. 2012 5.3 Thyroid cancer cells are

radiation responsive and dose responsive. There is a

signature of miRNAs. Low doses induce proliferation in

normal thyroid cells

BrownN et al. 2015 5.3 Radiation quality influences the

induction of mouse chromosome 2 deletions in radiation-

induced acute myeloid leukemia.

Olme CH et al. 2013a 5.3 Radiation induced acute myeloid

leukaemia (AML) involves chromosome 2 deletion and

Sfpi1/Pu1 loss.

Olme CH 2013b 5.3 There is a signature for IR induced

AML : the chromosome 2 deletion detected in half of the

irradiated mice after one year does not provide an

advantage for growth and in vivo repopulation in the bone

marrow of the mice.

Pascucci B et al. 2012 5.3 Primay fibroblasts from

Cockayne syndrome (CS) patients share high levels of

oxidative stress, perturbed oxidative energy metabolism

and mitochondrial functions. Oxidative DNA damage is

causative for the observed pathophysiology (CS cells are

slightly X-ray sensitive).

Raj K et al. 2012 5.3 Workshop report on Stem cells and

Radiation sensitivity.

Verbiest T et al. 2015 5.3 The transcription factor PU.1 is

down regulated in IR induce AML. PU.1 plays a role in the

initiation and development of IR induced AML.

2010

5.4 Mathematical models to link experimental findings and epidemiological data

Eidemüller M et al. 2012 5.4 Lung cancer in uranium

miners. In the two stage expansion model (TSCE) the risk

increased with obtained age, time of exposure and

exposure rate. Large uncertainties remain for small

exposures.

Kundrat P et al. 2011 (2012) 5.4 Mechanistic model of

triggering apoptosis in transformed cells under coculture

2010

112

conditions. The model predicts that intercellular induction

of apoptosis is balancing the proliferation of transformed

cells

Kundrat P and Friedland W 2015 5.4 Low dose radiation can modulate the signaling processes underlying intercellar induction of apoptosis, an important anti-carcinogenic mechanism, depending on system parameters.

5.5 Assessing the risk from internal exposures Drubay D et al 2014 5.5 Kidney cancer induction by

radon? No excess of such cancers is found in the French

and German cohort among uranium miners.

Georgiev S et al. 2016 4.10 and 5.5 Retrospective Rn-220

measurements by compact discs.

Kreuzer M et al. 2015 5.5 and 5.8 In a cohort of Uranium

millers, there is no statistically significant excess mortality

from lung cancer due to radon exposure and from solid

cancers due to external gamma radiation (at low organ

doses).

Laurier D et al. 5.5 Review : Cancer risks from internal

contamination research opportunities.

Pressyanov D et al. 2015 4.10 and 5.5 Optimization of

etching conditions for CD’s/DVDs used as detectors for

222Rn

2010

5.5.1 Internal Emitters in Uranium Miners (INTEMITUM) 2013

5.5.2 Assembly of internal radiation dose for UKAEA and AWE epidemiology cohorts (AIRDoseUK)

2013

5.6 Track structures and initial events: an integrated approach to assess the issue of radiation quality dependence (INITIUM)

Alloni D. et al. 2012 5.6 Monte Carlo codes, PARTRAC

code for radiation-induced DNA damage.The RBE is >1 for

high LET IR.

Alloni D et al. 2011 5.6 PARTRAC code models DNA

fragmentation in diploid human fibroblasts at different

LETs and reveals the importance of small DNA fragments

Alloni D et al. 2013 5.6 PARTRAC code model: DNA

fragmentation after gamma and nitrogen heavy ions:

indication of an RBE of > 1 and a higher amount of short

DNA fragments induced for high LET nitrogen ions.

Alloni D et al. 2015 5.6 DNA double-strand breaks induced

2012

113

by carbon ions and alpha rays depend on the energy (>1

LMeV /u).

Mariotti LG et al. 2012 5.6 Crucial role of ROS in

transducing the effect of initial IR and subsequent release

of IL-6. The effect is LET dependent.

Mariotti LG et al. 2013 5.6 DNA repair dynamiccs after

low and high LET exposures. Full recovery is observed

after 12 hours. Split dose experimental results show that

initial IR exposure induces more gH2AX foci than

subsequent exposures

Schmitt et al. 2015 5.6 PARTRAC modelling can simulate

light ion track structures and biological effects at energies

down to keV/u.

5.7 Induction and facilitation of chromothripsis by low dose ionizing radiation (In-FaCT-IR)

2013

5.8 Concerted Action for an Integrated (biology-dosimetry-epidemiology) Research project on Occupational Uranium Exposure (CURE)

Kreuzer M et al. 2015 5.5 and 5.8 In a cohort of Uranium

millers, there is no statistically significant excess mortality

from lung cancer due to radon exposure and from solid

cancers due to external gamma radiation (at low organ

doses).

2013

5.9 Low dose radiation-induced non-targeter effects in vivo: the role of microvesicles in signal transduction (Rad-Mvivo)

2014

5.10 Effects of Chronic LOw-dose Gamma Irradiation on GAstrointestinal Tumorigenesis (CLOGICAT)

2014

Table 4: WP6 Individual sensitivities program enlargement

Task Work Starting

6.1 Molecular epidemiological studies to address the role of individual genetic variation in determining susceptibility to low doses

Flockerzi E et al. 2014 6.1 and 6.10 DNA repair and repair

capacity in lung in different mouse strains : differences in

53BP1 induction in bronchiolar and alveolar epithelial cells.

Fractionated doses 100 mGy daily increase toxicity and

probability to induce secondary malignancies.

Grewenig A. et al. 2015 6.1 and 6.10 Persistent DSBs in

2010

114

spermatogonial stem cells following fractionated low dose

irradiation of testicular mouse tissue.

Schanz S et al. 2014 6.1 and 6.10 Genetically-defined DNA

repair capacity determines the extent of DNA damage

accumulation in healthy mouse tissues (lung, heart, brain)

after repetitive low doses of ioniziung radiation. ATM +/-

mice can cope with such damage.

6.2 Identification of genetic modifiers of individual cancer susceptibility and their mechanisms of action

Gürtler A et al. 2014 6.2 10 Gy of gamma rays induced

individual differences in proteomic profiles are low

compared with inter-individual differences seen in

lymphoblastoids cell lines.

M’kacher R et al. 2015 5.1 and 6.2 Telomere shortening

may be taken as a prognostoc factor for cardiovascular

disease post-radiation exposure.

Pottier G et al 2013 6.2 Lead exposure induces telomere

instability in human cells by perturbing telomere

replication on the lagging strand. This is important in brain

development and neurotoxicity.

Rosemann M et al. 2014 6.2. Alpha-ray induced

osteosarcoma and different tumor susceptibility genes in

mouse strains : Reduced Rb1 expression by common

variants in regulatory regions can modify the risk for

malignant transformation of bone cells after IR exposure.

Shim G et al. 2014 5.1 and 6.2 Review : Cross-talk between

telomere maintenance and radiation effects. Telomeres are

key players in the process of IR-induced carcinogenesis.

2010

6.3 Modelling of the effects on risk prediction models due to changes in biological processes influenced by genetic variability

Selmansberger M et al. 2015 6.3 CLIP2 as radiation

biomarker in papillary thyroid carcinoma.

2010

6.4 The effect of genetic modifiers on carcinogenesis following low dose rate exposure

Perriaud L et al. 2014 6.4 Intronic TP53 polymorphisms

affect G4 formation and expression of isoform specific

transcripts of the TP53 gene.

Sagne C et al. 2013 6.4 Meta-analysis of cancer risk

2010

115

associated with a specific rS17878362 polymorphism of the

TP53 tumor suppressor gene. The cancer is increased for

homozygous A2A2 carriers for breast and colorectal but not

for lung cancer. rs17878362 is associated with increased

cancer risk with a population and tumor specific effects.

Sagne C et al. 2014 6.4 Age effect on cancer onset in Li-

Fraumeni/Li-Fraumeni-like syndrome. Dependency on G4

polymorphisms in haplotypes of the WT TP53 allele.

6.5 Contribution of genetic and epigenetic mechanisms that indirectly influence susceptibility to radiation-induced cancer

2010

6.6 Implementation of the DoReMi strategy for a large scale molecular epidemiological study to quantify genetic contribution to individual susceptibility

Pernot E. et al. 2012 6.6 Biomarkers suitable for

epidemiological studies

Pernot E et al. 2014 6.6 Saliva samples are potentially

useful for epidemiological studies with biomaker,

particularly in children.

2010

6.7 Planning expansion of research portfolio 2010

6.8 Predicting individual radiation sensitivity with Raman microspectroscopy (PRISM)

Maguire A et al.2015a 6.8 Classification of leukocyte

subtypes by Raman spectroscopy.

Maguire A et al. 2015b 6.8 Detection of IR induced damage

in lymphocytes after doses of 0.05 and 0.5 Gy by Raman

spectroscopy in parallel to DNA damage (gH2AX).

Differences are observed with different donors.

2011

6.9 Integrating radiation biomarker into epidemiology of post-Chernobyl thyroid cancer from Belarus (INT-Thyr)

2012

6.10 Characterization of DNA lesions in the nuclear ultrastructure of differentiated and tissue-specific stem cells after protracted low-dose radiation (Zif-TEM)

Flockerzi E et al. 2014 6.1 and 6.10 DNA repair and repair

capacity in lung in different mouse strains : differences in

53BP1 induction in bronchiolar and alveolar epithelial

cells. Fractionated doses 100 mGy daily increase toxicity

and probability to induce secondary malignancies.

Grewenig A. et al. 2015 6.1 and 6.10 Persistent DSBs in

spermatogonial stem cells following fractionated low dose

2013

116

irradiation of testicular mouse tissue.

Jacquet P et al. 2015 6.10 The radiation sensitivity of the

gastrula-embryonic stage involves DNA double-strand

break repair in mice.

Schanz S et al. 2014 6.1 and 6.10 Genetically-defined DNA

repair capacity determines the extent of DNA damage

accumulation in healthy mouse tissues (lung, heart, brain)

after repetitive low doses of ioniziung radiation. ATM +/-

mice can cope with such damage.

6.11 Mechanism of low dose response to ionizing radiation and its significance in radiation protection (RADSENS)

2013

Table 5: WP7 Non-cancer effects program enlargement

Task Work Starting

7.1 Structuring the research effort on non-cancer effects according to the HLEG roadmap: organisation of consultation/exploratory meetings and funding integrative RTD projects

2010

7.2 Preparation of a pilot study to conduct molecular epidemiology studies in vascular radiation damage

Kreuzer M at al. 2015 7.2 Low-dose ionising radiation and

cardiovascular diseases – Strategies for molecular

epidemiological studies in Europe

2010

7.3 Feasibility study towards a systems biology approach of radiation response of the endothelium

Ebrahimian T. et al. 2015 7.3 Chronic gamma-irradiation

induces a dose-rate-dependent pro-inflammatory response

and associated loss of function in human umbilical vein

endothelial cells.

Gerard A.C et al. 2012 7.3 Iodine deficiency induces a long

lasting angiogenic phenotype in thyroid cancer cells leading

to uncontrolled growth.

Rombouts C et al. 2013 7.3 Low dose X-rays induce DNA

damage and apoptosis in endothelial cells (HUVEC and

EA.hy926).

Rombouts C et al. 2014 7.3 Low dose rate (4.1 mGy/h)

results in premature senescence in endothelial cells. Gene

expression analysis shows that the insulin-like growth factor

binding protein 5 IGFBP5 is involved.

Yentrapalli R et al. 2013a 7.3 Induction of premature

2010

117

senescence in human umbelical endothelial cells exposed to

chronic low dose rate gamma ray exposure (4.1 mGy/h). Such

chronic exposure results in induction of the p53/p21

pathway in HUVEC cells affecting the replicative potential of

these cells and leading to premature senescence.

Yentrapalli R et al. 2013b 7.3 A non linear response to dose

rate is observed. Responses at 1.4 mGy/h differ from those at

4.1 mGy/h.

7.4 Pilot epidemiological study of lens opacities among a cohort of interventional

Farah J et al. 2013 7.4 Estimation of cumulative eye lens

doses in intervention settings of cardiologists radiologists

and cardiologists

Farah J et al. 2015 7.4 For interventional cardiologists, the

eye lens doses received were correlated to whole body doses

by application of the ELDO approach, and the cumulative eye

lens doses were estimated.

2010

7.4.1 Lens opacities: Methodology implementation (ELDO) 2012

7.5 Pilot study of external irradiation versus internal contamination effects on neurogenesis

Legrand M et al. 2015 7.5 After internal contamination with

depleted uranium (DU) does not accumulate up to gestation

(day 18) in rat fetuses and DU exposure during gestation and

lactation affected neurogenesis during prenatal and postnatal

brain development but without major morphological changes

in the brain.

Quintens R et al. 2015 7.5 New knowledge on radiation-

induced p53 network of thhe embryonic brain and provide

moe evidence concerning te importance of p53 and its

transcriptional targets during mouse brain development.

Samari N et al. 2013 7.5 IR is a source of stress for immature

neurons. N-methyl D-aspartate (NMDA) receptors are

involved in RI induced neural death by apoptosis.

2010

7.6 Study on contribution of low dose X-radiation in induction of anti-inflammation

Derer A et al. 2015 5.2.1 and 7.6 Radio-immunotherapy-

induced immunogenic cancer cells as basis for induction of

systemic anti-tumor immune responses-pre-clinical evidence

and ongoing clinical applications.

2011

118

Frey B et al. 2015 5.2.1 and 7.6 Inflammation can be

modulated by high and low high doses of ionizing radiation

with implications in benign and malign diseases.

Frischholz B et l. 2013 5.2.1 and 7.6 Low dose RT (0.5 or 07

Gy) in mice reduces the inflammatory phenotype in the more

radiosensitive macrophages of Balb/c mice.

Lödermann B et al. 2012 7.6 Modulation of IL-1beta

activated macrophages by low X-ray doses : Doses of 0.5 and

0.7 Gy in mice produce an anti-inflammatory phenotype of

activated macrophages by reducing NFkB dependent IL-1beta

secretion in mice.

Mavragani IV et al. 2015 5.2.1 and 7.6 Laskaratou DA, Frey B,

Candeias SM, Gaipl US, Lumniczky K, Georgaklilas AG. Key

mechanisms involved in ionizing radiation-induced systemic

effects. A current review.

Ott OJ et al. 2012 7.6 Management of benign painful elbow

syndrome by < 3Gy RT.

Ott OJ et al. 2012 7.6 Management of benign painful shoulder

syndrome by low dose RT 0.5-1 Gy single dose, not exceeding

3-6 Gy in total.

Ott OJ et al. 2013 7.6 Treatment of achillodynia is possible

with 0.5 or 1 Gy not exceeding 3-6 Gy in total.

Ott OJ et al. 2013 7.6 RT and calcaneodynia management.

Ott OJ et al. 2014 7.6 RT for benign calcaneodynia : long term

results.

Ott OJ et al. 2014 7.6 Optimization of low dose RT of benign

painful elbow syndrome

Ott OJ et al 2014 7.6 Optimization of low dose RT of benign

painful elbow syndrome : no significant differences between

0.5 and 1 Gy dose schedules.

Ott OJ et al. 2015 7.6 Radiotherapy for benign achillodynia.

Long term results of the Erlangen Dose Optimization trial.

Persa E et al. 2015 7.6 T regulatory cells (Tregs) are

responsible for the immune suppressive phenotype of cancer

patients. Combined RT and immunotherapy may abrogate

Treg suppression. In a tumorous environment,Tregs acquire

a highly suppressive phenotype that is increased by RT. The

exact role of Treg cells in radio-and immunotherapy needs to

be explored further.

Rödel F et al. 2012a 5.2, 5.2.1 and 7.6 Modulation of

inflammatory immune reactions by low doses of IR 0.5-0.7 Gy

in endothelial, mononuclear and polynuclear cells. Non linear

119

biphasic responses are seen

Rödel F et al. 2012b 5.2.1 and 7.6 Review on the

immunomodulatory properties of low dose RT, with a

maximum effect at 0.5 -0 .7 Gy.

Rödel F et al. 2013 5.2.1 and 7.6 Review on anti-

inflammatory activities at <1Gy, and induction of harmful

side effects, IR induced immune modulation or induction of

anti-tumor immune responses at higher doses.

Rubner Y et al. 2012 5.2.1 and 7.6 IR contributes to the

induction of anti-tumor immunity.

7.7 Low dose Gene Expression signature and its impact on Cardiovascular disease (LoGiC)

Te Riet L et al. 2016 7.7 AT1-receptor blockade, but not renin

inhibition, reduces aneurysm growth and cardiac failure in

fibulin-4 mice.

2011

7.8 Study on contribution of low dose X-radiation in induction of cataractogenesis and influencing genetic and cell communication factors (LDR-OPTI-GEN)

2013

7.9 Low and moderate dose radiation effects on brain microvascular pericytes: epigenetic mechanisms and functional consequences (PERIRAD)

2013

7.10 Influence of a chronic LD and LDR exposure onto the development of Parkinson symptoms in genetically predisposed Pitx3-EYL/EYL Ogg1-/- mouse mutant (OSTINATO)

Graupner A et al. 2015 7.10 The genotoxic effects of

continuous low dose rate gamma irradiation and selenium

deficiency in mice blood cells.

2013

7.11 Epidemiological pilot study on radiation-induced cataract in interventional cardiology (EVAMET)

2014

7.12 Effect of low doses of low-LET radiation on impaired vascular endothelium (ELDORENDO)

2014

7.13 Low-dose ionizing radiation-induced cataracts in the mouse: in vivo and studies (RadCat)

2014